Pengxiang Zhao

Na Li

Didier Astruc
email: d.astruc@ism.u-bordeaux1.fr

Rachel De Oliveira

Claudio Luiz

De Santa Maria

Juliette Vergnaud

Jaime Ruiz

Gillian Barratt

State of the art in gold nanoparticle synthesis

Keywords: Gold Nanoparticle Cluster Seed-growth Core@shell Thiol Oxide Dendrimer Polymer anisotropy, gold nanoparticles, nanocatalysis, nanomedicine, plasmon

Chapter 1: Review of the state of the Art in the synthesis of AuNPs ………………...……3 Chapter 2: Carborane -functionalized AuNPs for potential boron neutron capter therapy applications………………………………………………………………….

Acknowledgement

           First and foremost I would like to thank my supervisor Prof. Dr. Didier Astruc. It has been a great honor to be his Ph.D. student. He has taught me, both consciously and un-consciously, how a good chemist is done. I appreciate all his contributions of time and ideas, making my Ph.D. experience productive and stimulating. The motivation, enthusiasm and immense knowledge he has was contagious and motivational for my study. I also appreciate for the excellent example he has provided as a successful chemist and professor.

          

I would like to warmly thank Dr. Azzedine Bousseksou, a prestigious scientist, for his precious administrative and scientific help that have greatly facilitated this thesis.

          

I would especially like to thank my reading committee members: Angela Marinetti and Noël Lugnan and the other three external members of my PhD defense committee, Marie-Hélène Delville, Jacques Robert and Lionel Salmon, for their time, advice and insightful questions.

          

I am grateful for collaborations with Marí a Echeverrí a, Dr. Pengxiang Zhao, Dr. Lionel Salmon and Dr. Roberto Ciganda, all of you have been there to efficiently and kindly support me when I collected data for my Ph.D. thesis.

          

I am grateful to our group's engineer Jaime Ruiz who kept us organized and was always ready to help. The members of the Astruc group, Amalia, Yanlan, Christophe, Changlong, Haibin, Dong, and Sylvain have contributed immensely to my personal and professional time at University of Bordeaux. You are going to be the best memories of my Ph.D. experience.

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I gratefully acknowledge the funding support from the China Scholarship Council for my 3 years of Ph.D. The University of Bordeaux, the CNRS and L'Oréal are gratefully acknowledged.

Introduction

Gold nanoparticles (AuNPs) have emerged as a key field in nanoscience and nanotechnology because of their potential applications in catalysis, [1] materials science, optical biosensors, [2] as well as nanomedical diagnostics and therapeutics [3] due to their quantum-related and supramolecular properties. [4] The Huisgen-type Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction ("click" reaction) is the most efficient strategy to assemble 1,2,3-triazole ring linking two molecular fragments together because of its atom economy, regioselectivity, wide substrate scope and mild reaction conditions. [5] Moreover, the triazole group is completely bio-compatible. The "Click" modification of AuNPs has been favorable in the past few years. [6] This thesis includes the design and synthesis of ligands based upon "click" reactions, the preparation, stabilization and modification of gold nanoparticles (AuNPs), as well as the characterizations and applications of functional AuNPs in view of these applications. AuNPs were stabilized by either linear or dendritic thiolate ligand, triazolyl neutral ligands or triazolyl dendrimers. Various useful functional groups such as carborane, ferrocene, coumarin, cyclodextrin, and (or) polyethylene glycol (PEG) and many others were involved and were anticipated to potentially undergo various applications based upon their diagnostics, electrochemical, optical, supramolecular, encapsulating and catalytic properties. PEG species were appreciated as one of the triazole substituents owing to not only its excellent biocompatibility and enhanced permeability and retention (EPR) effect, [7] but also it facilitatation of the solubilization of AuNPs in water. A series of water-soluble AuNPs were prepared with narrow dispersity and long-term stability. The size and optical properties of AuNPs were controlled to provide catalytic, optical (sensing) and potentially biomedical applications (the latter ones in collaboration).

The first chapter of this thesis provides a review of the literature on the state of the art in AuNP synthesis which systematically illustrates the preparation and stabilization of AuNPs via different approaches, and the recent development of AuNPs.

The second chapter describes the functionalization of AuNPs by PEG and carborane via "click" reactions. The water-soluble AuNPs that were obtained may have potential applications in boron neutron capture therapy (BNCT) thanks to the exis tence of high boron content materials, carborane, on the AuNP surface in wide-scale.

The third chapter displays the stabilization of AuNPs with "clicked" triazole neutral ligands in aqueous solution or in organic solution. These AuNPs provide various applications in catalysis, sensing and ligand-substitution according to the specific properties of AuNPs and the functional groups. This chapter is further divided into three subsections.

The forth chapter includes two parts. The first part concerns the stabilization of AuNPs by PEGylated nona-branched dendrimers in aqueous solution. The size of AuNPs appears to be tunable. The dendritic structure influences both the morphology and the catalytic performance of AuNPs. In the second part, the catalytic activities of various AuNPs are compared via kinetic studies of 4-nitrophenol reduction.

The fifth chapter is a bibliographical review of the synthesis, applications and toxicity of anisotropic gold nanoparticles including nanorod s, nanowires, nanopolyhedros, nanoplates, nanostars and nanoshells. The specific optical properties of these AuNPs are also detailed.

Anisotropic AuNPs have significant importance in theranostics (therapeutic + diagnostic) and optical devices. They are at variance with spherical AuNPs studied in the previous chapters and open the route to further possibilities concerning biomedical and sensing applications.

The last section of this thesis is the "conclusion and perspectives" summarizing the research works in this thesis and the potential applications of functional AuNPs.

In the ANNEX of this thesis, a paper on the construction of a targeted anticancer drug delivery system involving biocompatible AuNPs was presented.

Introduction

This introduction consists in a review paper containing over 420 references disclosing the recent developments in the synthesis of gold nanoparticle. The synthesis of AuNPs is introduced by different methods based on various ligands and AuNP structures. Publications on AuNPs are abundant. Review papers on AuNPs that were published in recent years mainly focused on the construction and applications of AuNPs, since AuNPs became a popular material in nanomedicine and material science. Very few review papers summarized the synthesis of AuNPs in the last decades. However, strategies on the preparations of AuNPs developed fast, and thus it was necessary to disclose the state of the art of the preparation of AuNPs. This review was organized in collaboration with Dr. Pengxiang Zhao, a former group member who defended his PhD thesis in the University of Bordeaux in 2012.

Introduction

Metal nanoparticles (NPs) have long been considered to exhibit unique physical and chemical properties different from those of the bulk state or atoms, due to the quantum size effect resulting in specific electronic structures [1-7]. Gold nanoparticles (AuNPs) are probably the most remarkable members of the metal NP groups [8-11] and have attracted considerable interest and driven a variety of potential applications in catalysis [12-21], biology [22-26], and optics [27-29]. Indeed, more than 70,000 publications have appeared on AuNPs to date. Here we are specifically focusing on the principles and most recent improvements disclosed in the literature on the synthesis of AuNPs of various types.

After the seminal report by Faraday in 1857 [8] of the reduction of a tetrachloroaurate solution by phosphorus in carbon disulfide (a biphasic reaction), the preparation of AuNPs with controlled sizes and shapes has raised increased attention during the second half of the XXth century. The breakthroughs have been those by Turkevich in 1951 with the citrate method improved by Frens in 1973, then in 1981 with Schmid's report of a Au 55 -phosphine cluster and the notion of quantum dot, then by Mulvaney and Giersing in 1993 with the first synthesis and stabilization of AuNPs by thiolates, and finally by the Schiffrin group in 1994 with the report of the illustrious and most convenient Brust-Schiffrin biphasic method of thiolate-stabilized AuNPs [9]. Sophistications of these methods during the last decade, especially the seed-growth synthesis, have now led to promising applications.

AuNPs can be prepared by both "top down" and "bottom up" approaches. For "top down" procedures, a bulk state Au is systematically broken down to generate AuNPs of desired dimensions. In this case, particle assembly and formation is controlled by a pattern or matrix. However, the "top down "method is limited concerning the control of the size and shape of particles as well as further functionalization [30]. In contrast, in the "bottom up" strategy, the Abbreviations:

9-BBN, 9-borabicyclo[3.3.1] nonane; AFM, atomic force microscopy; ATO, sodium bis(2-ethylhexyl) sulfosuccinate; AuNP, gold nanoparticle; AuNR, gold nanorod; CNT, carbon nanotube; CPA, cyclic phenylazomethine; CTAB, cetyltrimethylammonium bromide; DENs, dendrimer-encapsulated nanoparticless; DLS, dynamic light scattering; DSNs, dendrimer-stabilized nanoparticle; DMAP, 4-(N,N-dimethylamino) pyridine; EDX, energy-dispersive X-ray; GMA, glycidyl methacrylate; GUVs, giant unilamellar vesicles; HADDF-STEM, highangle annular dark-field scanning transmission electron microscopy; HEPES, N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid; HR-TEM, high-resolution transmission electron microscopy; LDI, laser desorption ionization; MALDI, matrixassistedlaser desorption ionization; MOCVD, metal organic vapor deposition; MOF, molecular organic framework; MRI, magnetic resonance imaging; NICISS, neutral impact collision ion scattering spectroscopy; NIPM, poly(N-isopropylacrylamide); NIR, near infrared; NP, nanoparticle; PAAPHA, poly(acryloylaminophenylarsonic acid); PAMAM, poly(aminoamide) dendrimer; PCL, poly(caprolactone); PEG, poly(ethylene glycol); PEI, poly(ethylenimene); PEO, poly(ethylene oxide); PPO, poly(propylene oxide); PS, polystyrene; PVCL, poly(N-vinyl caprolactam); PVP, poly(vinyl pyridine); SDBS, benzenesulfonate; TAOB, tetra(octylammonium) bromide; TEM, transmission electron microscopy; TEG, tetra(ethylene glycol); THPC, tetrakis(hydroxymethyl) phosphonium chloride; TGA, thermogravimetric analysis; XRD, X-ray diffraction.

formation of AuNPs originates from individual molecules, because it involves a chemical or biological reduction [31]. This chemical reduction method involves two steps: nucleation and successive growth. When the nucleation and successive growth are completed in the same process, it is called in situ synthesis; otherwise it is called seed-growth method. For the in situ synthesis method, we will focus on the preparation of spherical or quasi-spherical AuNPs. For the seed-growth method, we will concentrate on the preparation of AuNPs having various sizes and shapes. In addition, we will discuss methods of AuNP functionalization. This review is not comprehensive, but it is limited to the essential and most useful preparation methods of AuNPs and their improvements, in particular the most recent development.

In situ synthesis

In general, the preparation of AuNPs by chemical reduction contains two major parts: (i) reduction using agents such as borohydrides, aminoboranes, hydrazine, formaldehyde, hydroxylamine, saturated and unsaturated alcohols, citric and oxalic acids, polyols, sugars, hydrogen peroxide, sulfites, carbon monoxide, hydrogen, acetylene, and monoelectronic reducing agents including electron-rich transition-metal sandwich complexes; (ii) stabilization by agents such as trisodium citrate dihydrate, sulfur ligands (in particular thiolates), phosphorus ligands, nitrogen-based ligands (including heterocycles), oxygen-based ligands, dendrimers, polymers and surfactant (in particicular cetyltrimethylammonium bromide abbreviated CTAB). The in situ synthesized AuNPs are also used for the seedgrowth or further functionalization. In this section, we will review the various in situ methods and their improvements.

2.1. AuNPs stabilized by simple molecules 2.1.1. Turkevich method 2.1.1.1. Citrate as both stabilizing and reducing agent. Among all the in situ syntheses of AuNPs by reduction of HAuCl 4 , citratestabilized AuNPs has been regarded as the most popular ones for a long time, since their introduction by Turkevich in 1951 [32]. The HAuCl 4 solution is boiled, and the trisodium citrate dihydrate is then quickly added under vigorous stirring. After a few minutes, the wine-red colloidal suspension is obtained, and the AuNP size is about 20 nm. In 1973, Frens [33] published an improvement, i.e. a broad size range of AuNPs (from 15 to 150 nm) was obtained by controlling the trisodium citrate to Au ratio. However, particles larger than 20 nm were always polydispersed. The histogram of size distribution could be readily determined by transmission electron microscopy (TEM).

Recently, several research groups have improved the Turkevich-Frens method in order to promote the convenient use of citrate-stabilized AuNPs. In particular, the mechanism of AuNPs formation using this synthetic route has been examined in details [34-38]. Kimling et al. indicated that a high concentration of citrate more rapidly stabilizes AuNPs of smaller sizes, whereas a 6 low concentration of citrate leads to large-size AuNPs and even to the aggregation of AuNPs [34].

Remarkable research on the mechanism of the Turkevich-Frens method in multiple-step process was published by Kumar's group [37]. The initial step of this multiple-step process, with reactions occurring in series and parallel, is the oxidation of citrate that yields dicarboxy acetone. Then, the auric salt is reduced to aurous salt and Au 0 , and the aurous salt is assembled on the Au 0 atoms to form the AuNP (Fig. 1). Thus, in theTurkevich-Frens method, the actual AuNP stabilizer is dicarboxy acetone resulting from the oxidation of citrate, rather than citrate itself.

In addition, the presence of a citrate salt modifies the pH of the system and influences the size and size distribution of the AuNPs [38]. On this basis, nearly monodispersed AuNPs with sizes ranging from 20 to 40 nm have been synthesized upon variation of the solution pH [39-42]. Other improvements of the Turkevich method involved the control of the reaction temperature [43], the introduction of fluorescent light irradiation [44], and the use of high-power ultrasound [45,46]. Citrate-stabilized AuNPs are always larger than 10 nm, due to the very modest reducing ability of trisodium citrate dihydrate. An intriguing result from Puntes's group [47] concerning citrate-stabilized AuNPs that appeared in 2010 is the use of D 2 O as the solvent instead of H 2 O during the synthesis of AuNPs. As shown in Fig. 2, the size of the AuNPs was tailored to 5 nm. It was concluded that D 2 O increased the reducing strength of citrate.

2.1.1.2. Citrate as a stabilizing agent only. In general, citrate plays a role as a stabilizing agent with preparations of AuNPs requiring relatively high temperatures due to its weak reducing strength. Slot discovered a new way to prepare AuNPs using a mixture of tannic acid/citrate solution whereby tannic acid plays the role of a reducing agent instead of citrate, and the AuNPs are obtained at 60 • C [48]. Then Natan's group introduced a method using citrate as a stabilized agent only and NaBH 4 as a reducing agent [49]. The AuNPs were obtained upon adding the NaBH 4 /citrate mixture into the HAuCl 4 solution at room temperature. With this method, the size of AuNPs is tailored to 6 nm, which compares with sizes beyond 20 nm using the traditional Turkevich method.

2.1.1.3. Reversed addition method. Very recently, another remarkable modification of the Turkevich method involved the reversed order of addition that was conducted by adding HAuCl 4 to the citrate solution, producing monodispersed AuNPs with relatively small size (less than 10 nm) [50,51].

In summary, the size of the citrate-stabilized AuNPs produced by in situ synthesis is between 5 nm and 150 nm. When the size is decreased, relatively monodispersed AuNPs are obtained, whereas when the size is increased (especially > 20 nm) polydispersed AuNPs are obtained. Citrate-stabilized AuNPs were also used as intermediates in further preparations or functionalizations such as ligand substitution reaction and seed-growth-mediated syntheses.

2.1.2. Brust-Schiffrin method 2.1.2.1. Synthetic procedure. Thiolate-stabilized AuNPs were first reported by Mulvaney and Giersig [52]. These authors showed the possibility of using alkylthiols of various chain lengths to stabilize AuNPs. The two-phase Brust-Schiffrin method, published in 1994, was the first method able to prepare the thiolate-stabilized AuNPs via in situ synthesis, and it has therefore met great success [53]. Its high impact is due to (i) facile synthesis in ambient condition; (ii) relative high thermal and air stability of the AuNPs prepared in this way; (iii) repeated isolation and re-dissolution without aggregation or decomposition; (iv) control of the small size (less than 5 nm) with narrow dispersity; (v) relatively easily functionalization and modification by ligand substitution. The AuNPs are stabilized by relatively strong Au-Sbonds, their diameters are in the 2-5 nm range, and their shapes are cuboctahedral and icosahedral. Due to the nucleation-growth-passivation kinetics model by which the sulfur-containing agents inhibit the growth process [54,55], larger S/Au mole ratios give smaller average core sizes. Fast NaBH 4 addition and cooled solutions also produce smaller, more monodispersed AuNPs. During the reaction of the thiol with growing Au 0 NPs, the H atom of the thiol is lost, presumably by oxidative addition of the S H bond onto two contiguous Au 0 atoms of the AuNP surface. The fluxional properties of the Au H bonds on the AuNP surface can provide the fast walking path of the H atoms on the surface until two Au H bonds become contiguous for H 2 reductive elimination. Very recently, the formation surface Au S bonds was shown using Raman spectroscopy by Li et al. who actually demonstrated the overall mechanism including all the steps of the Brust-Schiffrin AuNP synthesis. In particular, they disclosed that, interestingly, these Au S bonds are formed only after adding NaBH 4 as indicated in Fig. 3. This work brings about a key understanding of the mechanism of this synthesis [56], while a previous widely accepted assumption had been that the thiol reduced Au(III) to Au(I) and formed [Au(I)SR] n [57].

Given the above advantages, the Brust-Schiffrin method is now much in use for the preparation and application on thiolateliganded AuNPs [58-62]. Precise Au clusters have been synthesized using this method and purified (see Section 2.1.2.2). From the nomenclature point of view, it is best to call "clusters" such small "NPs" (smaller than 1 nm) that are precisely defined without polydispersity (single molecules) and to reserve the "NP" nomenclature to the cases for which there is some dispersity (mixture of several clusters), even if it is low [63].

The Brust-Schiffrin method was extended in 1995 to an improved procedure upon which the p-mercaptophenol-stabilized AuNPs were synthesized in methanol solution without the phasetransfer agent TAOB [64]. In this way, the introduction of TAOB impurities was avoided. Indeed, methanol is an excellent solvent for a single-phase system, because both HAuCl 4 and p-mercaptophenol are soluble. Any thiol that is soluble in the same solvent as HAuCl 4 such as methanol, ethanol or water allows the use of a singlephase system for AuNP synthesis. In the following years, a variety of publications concentrated on functional thiol ligand-stabilized AuNPs using the single-phase procedure [54,55]. Brust's group prepared biocompatible and water-soluble AuNPs capped by (1mercaptoundec-11-yl) tetraethylene glycol [65] with less than 10 nm. Due inter alia to the hydrophobic alkanethiol interior and tetra (ethylene glycol) (TEG) hydrophilic shell, AuNPs are of great biomedical interest [66-68], in particular in the form of AuNPs capped with thiolated polyethylene glycols (PEGs) [69,70].

Very recently, a series of thiolate-stabilized AuNPs were prepared by a modified single-phase method. Sardar and Shumaker-Parry introduced 9-borabicyclo [3.3.1] nonane (9-BBN) as a mild reducing agent for the synthesis of a series offunctionalized alkylthiolate-stabilized AuNPs [71]. Other thiolate ligands such as bifunctional alkanethiolate [72-74], arenethiolate [75-79] and other functional thiolate-stabilized have been used to synthesize functional AuNPs [80] from the corresponding thiols.

Quantum mechanical calculations have suggested that, if heavier chalcogens (i.e. Se or Te) were used as the anchors, they would increase the conductance between the metal and the anchored ligand [81]. Thus, during the last decade, the synthesis of AuNPs with Se [82] or Te [83] ligand has been conducted with modified Brust-Schiffrin procedures.

The strength of the reducing agent used in Brust-Schiffrin method is much larger than that of citrate used in the Turkevich method, and according to Marcus theory the reaction rate in AuNPs synthesis using NaBH 4 is much larger than that of the Turkevich AuNP synthesis using citrate reduction. A direct consequence is that the size of the AuNPs synthesized using the NaBH 4 reductant is much smaller than that of Turkevich method using the citrate reductant.

2.1.2.2. Purification of precise Au clusters from AuNPs. Various methods of purification and isolation of precise Au clusters or mono-dispersed AuNPs from crude AuNPs that are more or less polydispersed mixtures of such clusters have been experimented. AuNPs with precise number of Au atoms and ligands such as Au 140 (SC6) 53 (SC6 = hexanethiol) [84] and Au 144 (SCH 2 CH 2 Ph) 60 [85] have been synthesized using the Brust-Schiffrin method. Murray's group [63,86,87] ] was confirmed by 1 H NMR, UV-vis., and mass spectrometry, and the X-ray crystal structure is illustrated in Fig. 4. Further, the yield was improved up to 50% by tuning the temperature and duration of the various steps, and the formation of oxidized neutral Au 25 L 18 was avoided.

The purification and separation of water-soluble thiolated 3-nm AuNPs is possible according to Sweeney et al. [88]. In this procedure, the solution of AuNPs is removed using a peristatic pump through a diafiltration membrane. Small molecule impurities or small NPs are eluted in the permeate, while the large NPs are retained. The expanded view is that of a hollow-fiber-type diafiltration membrane depicting the elution of small impurities and NPs and the retention of larger particles.

Kornberg's group [89,90] introduced the precipitation method in order to obtain Au 102 (P-MBA) 44 (MBA: p-mercaptobenzoic acid). The prepared P-MBA-protected AuNPs were precipitated by addition of ammonium acetate or NaCl and methanol and then collected in a microfuge by several centrifugation steps to yield purified Au 102 (P-MBA) 44 . This final Au cluster was characterized by SEM, MALDI-TOF MS, TGA and XPS.

2.1.2.3. Determination of the number of thiolate ligands. For thiolate ligand-stabilized AuNPs, the surface ligand coverage is determined using theoretical and experimental methods. The most commonly employed theoretical method for spherical AuNPs is that of Leff and co-workers [91]. In their assumption, the thiolate ligands are closepacked on the AuNP spherical surface. It is also assumed that each gold atom has a constant volume, v g = 17 Å3 , and each thiol occupies an area of S sulfur = 21.4 Å2 on the surface, so that both the number of Au atoms (n Au = 4 (R -ı) 3 /3v g , R -ı ≈ D/2) and the number of thiolate ligands (n thiol = 4 (R -ı) 2 /S sulfur ) are obtained through a simple measurement of the AuNP diameter by TEM followed by statistical calculations. For example, the average core diameter of 2.7 nm AuNP results in 577 Au atoms and 103 thiol ligands at the periphery [92].

Murray's spherical AuNP-core model introduces a functional relationship between the thiolate ligand coverage (ratio of alkanethiolates to surface Au atoms, ) and the mass fraction of alkanethiolate ( organic ) in the cluster with a given Au core radius (Eq. ( 1)) [93]. The surface ligand coverage is calculated using the measurement of organic by elemental and TGA analyses, and R core by TEM or SAXS. It was pointed out that the calculated result of spherical core was not very different [93] from those predicted for the face-centered cubic cuboctahedral model that was determined by Brust et al. [53] through the HRTEM measurement. As an example, an AuNP with a core radius R core = 1.19 nm and coverage = 0.66 contains 409 Au atoms and 126 alkanethiolate ligands in each AuNP.

↓ organic = (4 (R ↓ core -R ↓ Au) ↑ )2( ↓ HCP)((MW ↓ )thiol) )/ (4 (R ↓ core -R ↓ Au) ↑ 2( ↓ HCP)/(MW ) ↓ thiol - + 4/(3) R ↓ core ↑ 3( ↓ Au)(AW ) (1)
In Eq. ( 1), organic is the mass fraction of alkanethiolate in the AuNP, R Au is the crystallographic radius of a gold atom (0.145 nm), HCP is the density of surface gold atoms (13.89 atoms/nm 2 , 8 Fig. 2. Optical (UV-vis. spectroscopy) and morphological (TEM) characterization of AuNPs synthesized using various solvents: 100% D2O (top), 50% D2O/H2O (middle), and 100% H2O (bottom). Inset graphs in TEM images show the size distribution measurements of AuNPs. Inset images in UV-vis. spectra show the colors of the AuNPs solutions. These figures are representative of three separated experiments.

Reprinted with permission from [47] (Puntes's group). Copyright 2010 American Chemical Society. assuming hexagonal close packing), MW thiol is the alkanethiolate molecular weight in mass/molecule, is the coverage (ratio of alkanethiolates to surface Au atoms), Au is the atom density of bulk gold (58.01 atoms/nm 3 ), and AW Au is the Au atomic weight in mass/atom.

In previous work of our group [94], the thiolate ligand number and total gold atom number could be experimentally determined through measurement of the AuNP core radius by TEM and the organic fraction determined by element analysis. The result was in relatively good agreement with the reports by Murray and Brust. From these various determination methods, it results that Leff's theoretical method leads to an underestimation of the actual number of thiolate ligands. If the thiolated AuNPs are well washed to remove excess free thiol (a key condition), i.e. if the Au/S ratio is correctly provided by elemental analysis or thermogravimetric analysis, the AuNP size determination by TEM leads to the correct number of AuNP ligands. The AuNP surface coverage by thiolate ligands depends on the geometry and size and varies between one thiolate ligand per two surface Au atoms to 2 thiolate ligands per 3 Au surface atoms.

2.1.2.4. Other sulfur ligands. Other sulfur ligands such as disulfides [95-100], xanthates [101], thioacetates [102], dithiocarbamates [103], trithiolates [104], benzenesulfonate (SDBS) [105], and thioctic acid [106] have also been used in the synthesis of AuNPs via the Brust-Schiffrin two-phase method or ligand-substitution method. Some of these ligands bind less readily than thiolates to the AuNP cores of AuNPs [107-109]. However, an opposite view recently indicated that alkanethioacetates stabilized-AuNPs were as stable as alkanethiol-stabilized AuNPs [102]. It is expected that the alkanethioacetate ligands stabilize AuNPs with loss of the S-acetyl moiety and covalent attachment of the sulfur atoms as thiolates to the Au surface. Due to the abundance of sulfur ligand in DNA or other proteins, the sulfur ligands stabilized AuNPs as the bioprobe have a broad used in DNA or protein detection and other bio-applications [95].

2.1.2.5. Methods of functionalization.

2.1.2.5.1. Reactions of AuNP-citrate with thiols. Due to the weakness of the Au-citrate bonds, the citrate-stabilized AuNPs are easily functionalized upon substitution of the citrate ligands by stronger thiolate ligands; this substitution is experimentally very simple and involves reaction of the precursor citrate-AuNPs with the corresponding functional thiols under ambient conditions. Compared with the Brust-Schiffrin method of direct synthesis, the AuNPs prepared from AuNP-citrate are larger, which allows potential applications of the latter in nanomedicine; therefore, the plasmon band [110], observed only for AuNPs larger than 2 nm, is key to the diagnostic. Mulvaney and Giersig initiated this method to synthesize thiolate-stabilized AuNPs by substituting the citrate ligands by thiolate ligands using the corresponding thiols [52]. This research area is already largely developed, and Mirkin's group has used this strategy to load AuNPs with thiolated DNA [111]. In addition, citrate-stabilized AuNPs have been functionalized with thiolated PEG, and the PEGylated AuNPs formed in this way have been used as contrast agent for in vivo X-ray computed tomography 16 The icosahedral Au13 core is surrounded by six Au2(SR)3 semi-rings that are slightly puckered in the reduced AuNPs as shown for the semi-ring with more pronounced yellow and pink colors (right). The icosahedral Au13 core (minus the center Au) is slightly distorted; the blue Au Au bonds lying directly below the center of each semi-ring are on average 0.12 Å shorter than the yellow Au Au bonds (average 2.96 Å). Overall Au-Au average 2.93 Å. Au13 core diameter 9.8 Å; overall AuNP diameter 23.9 Å.

Reprinted with permission from [63] (Murray's group). Copyright 2010American Chemical Society.

imaging [112]. Very recently, Daniel's group [113] synthesized robust AuNP-cored nitrile-and amine-terminated dendrimers by reaction between 19.5-nm AuNP-citrate and poly(propyleneimine) dendronic thiols as efficient platforms for drug delivery (Fig. 5).

2.1.2.5.2. Brust-Schiffrin method with functional thiols. Since the seminal report by Brust et al. [64], it is known that some functional thiols can be used in the Brust-Schiffrin AuNP synthesis (Section 2.1.2.1) if the functional group of the thiol is compatible with the reactions conditions, in particular the reductant. This strategy has thus been largely exploited. Recently, 16-mercapton-hexadecanoic acid-capped AuNPs were used for immobilization onto silica [114]. Thiol-functionalized polymers or dendrimers are also used to stabilize AuNPs via Brust-Schiffrin synthesis (see Sections 2.2.1 and 2.2.2). The direct synthesis of AuNPs with functional thiols sometimes avoids the aggregation that may result from the post-functionalization method (Section 2.1.2.5.4).

2.1.2.5.3. Thiolate-thiol ligand substitution reaction: functionalization. The ligand-substitution reaction (occasionally called "place exchange" reaction) was first initiated by Murray's group who substituted alkylthiolate ligands by functional alkyl thiolate ligands using the corresponding functional thiols for the synthesis of functional AuNPs [115]. Then these authors developed precise Au cluster ligand-exchange reactions using toluene-3,4dithiol CH 3 C 6 H 3 (SH) 2 , for the substitution of the HSC 2 Ph ligand in [Au 25 (SC 2 H 4 C 6 H 5 ) 18 ] - [116]. The reaction at the AuNP or Au cluster core surface involves transfer of the SH hydrogen atom of the incoming functional thiol to the AuNP surface-bonded S atom of the coordinated thiolate to form the leaving thiol (Eq. ( 2)).

Au-SR

+ R SH → Au-S(R)• • •H• • •SR → HSR + Au-SR (2)
The thiolate ligand substitution is conducted by introducing excess thiol ligands. This method has been widely utilized [117], because it avoids using the reducing conditions of the direct Brust-Schiffrin synthesis with functional thiols. Usually, the excess of functional thiol and the exchanged non-functional alkylthiol are separated by dissolution in methanol or ethanol in which the AuNPs are not soluble. Examples include amidoferrocenyl functionalized alkanethiol groups that were introduced into AuNPs for the redox recognition of oxoanions [118], azide-or bromo-functionalized thiols for further "click" functionalization of AuNPs [119-124], carboxylic acid-functionalized thiols for controlled preparation of carboxylated AuNPs [125], and small thiolate molecules for the identification of antibiotics [126].

Some more labile ligand-stabilized AuNPs were also submitted to ligand substitution by thiol ligands. For instance, the so-called "thiol-for-DMAP exchange" was conducted [127-129] using thiol ligands to substitute the DMAP-caps in AuNPs for the synthesis 10 of liquid crystal (LC)-capped AuNPs [127], and polymer-capped AuNPs [128].

However, the ligand-substitution reaction has some drawbacks that are (i) risk of irreversible aggregation of AuNPs, (ii) incomplete ligand substitution, (iii) in some cases experimental difficulty in the determination of the amount of exchanged ligands, (iv) possible incompatibilities between the initial medium of the AuNPs and new capping molecules, such as tolerance of the new potential ligands towards solvent [119]. Nevertheless, it remains useful.

2.1.2.5.4. Functionalization of pre-formed AuNPs. Various functionalizations of pre-formed AuNPs by direct synthesis or ligand substitution have been conducted such as halide nucleophilic substitution reactions [117], nucleophilic addition reactions [130], amidic coupling reactions using amino termini [131], polymerizations [132], and "click" functionalization [133,134]. For example, AuNPs stabilized by 4-aminothiophenol were functionalized with folic acid by condensation reaction, with potential application in cancer therapy [135,136]. In our group, "click" functionalization of AuNPs has been applied for usage in drug delivery systems (Fig. 6) [124,136]. This functionalization of pre-functionalized AuNPs has the inconvenient, however, that aggregation often occurs in the course of reactions, in particular if a catalyst is used. Nevertheless, it remains very useful, because the aggregated AuNPs are easily separated by precipitation.

In summary, due to the strong Au S bond (47 kcal/mol) [137], sulfur ligand-stabilized AuNPs have gained considerable interest for functionalization with thousands of publications since their discovery. These four functionalization methods are all currently used and complementary.

Schmid's Au 55 cluster and the phosphorus ligands

The phosphorus ligand-stabilized AuNPs have possible future applications in catalysis, imaging, sensing, and new therapeutic approaches [138-151]. They are also excellent precursors for ligand-substitution reactions towards the functionalization of AuNPs and for building bimetallic cores of AuNPs [152,153]. In the early 1980s, Schmid published the illustrious phosphine-stabilized Au 55 cluster that is well known as Schmid's cluster with narrow dispersity (1.4 ± 0.4 nm) [11]. Schmid Au cluster was defined as [Au 55 (PPh 3 ) 12 Cl 6 ], was well characterized by X-ray crystal structure and showed the properties of a quantum-dot particle for the first time [154]. Weare and Hutchison [153] improved the Schmid method in 2000 using HAuCl 4 and N(C 8 H 15 ) 4 Br in a water-toluene mixture to which PPh 3 and NaBH 4 (instead of diborane in Schmid's 12 method) were added. The cluster [Au 101 (PPh 3 ) 21 Cl 5 ] formed with [Au(PPh 3 )Cl] as an impurity. Later, Moores et al. [151] used the precursor complex [AuCl(SMe 2 )] instead of HAuCl 4 as the gold source and sodium naphthalenide (C 10 H 8 Na) instead of NaBH 4 as the reducing agent to synthesize phosphinine-stabilized AuNPs. Phosphinines are well adapted to the coordination to Au 0 and are suitable for AuNPs formation. Very recently, the mild reducing agent 9-borabicyclo-[3.3.1]nonane (9-BBN) was used in a new simple and versatile method for the synthesis of triphenylphosphine-stabilized AuNPs with diameters of 1.2-2.8 nm and narrow size distribution (Fig. 7) [138].

Other recent examples of the synthesis of AuNPs stabilized by phosphines or phosphine derivatives are (i) triphenylphosphinestabilized small Au clusters where triphenylphosphine is considered as a proactive etching agent [155], (ii) calixarene phosphine-stabilized AuNPs that were used for catalysis [156,157], (iii) 1,1 -binaphthyl-2,2 -dithiol-stabilized AuNPs for optics [158], and (iv) tetrakis-(hydroxymethyl)phosphonium chloride (THPC)stabilized AuNPs for further preparation of bimetallic nanoshells [159]. In addition, proteins and lipids contain many phosphorus atoms that also stabilize AuNPs with these bioligands (see Section 2.2.1).

Other ligands

Other possible AuNP ligands are oxygen-and nitrogen-based ligands containing electronegative groups such as amine (or amino), carboxyl, carbonyl and phenol groups. In particular, amines have been popular AuNP ligands for a long time due to their presence in biological and environmental systems [160-166]. A seminal and remarkable example is Crooks's octadecyl amine-capped AuNPs [161]. A series of aminoalcohol-stabilized AuNPs [162], aminestabilized AuNPs used as bioprobes [163,164,] (Fig. 8), and cyclic phenylazomethine (CPA)-stabilized AuNPs used as electrochemical probes for metal ion sensing are known [165].

Among them, 4-(N,N-dimethylamino) pyridine (DMAP) is a frequent AuNP ligand, and DMAP-stabilized AuNPs have found broad use in AuNP ligand-substitution reactions [167] and heterogeneous catalysis [168]. DMAP-liganded AuNPs were first published by Caruso's group [168,169] and recently gained increased attention [170-173]. These AuNPs are obtained by adding an aqueous DMAP solution into the TOAB-stabilized AuNPs via phase transfer. Two other key nitrogen ligands, TOAB and CTAB will be discussed in Section 2.3.1.

Among oxygen ligands, carboxyl derivatives and phenol are known as AuNP stabilizer. For instance, folic acid binds AuNPs via a carboxyl group, which is useful for nanomedicinal applications [174]. Other carbonyl stabilizers include N-vinylpyrrolidone [175] and N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid (HEPES); the latter stabilizes flower-like AuNPs with its carbonyl and phenolate groups [176].

Macromolecule-stabilized AuNPs

Macromolecules stabilize AuNPs by both steric embedding effect and ligand interactions of their heteroatoms (N, O, P and S donors) with the AuNP surface [177].

Polymers

As reviewed by Shan and Tenhu, the use of polymers stabilizers for the synthesis of AuNPs has various advantages: (i) enhancement of long-term AuNP stability, (ii) adjustment of their solubility, (iii) increased amphiphilicity, (iv) high and tunable surface density of shell/brush, (v) tailored properties of AuNPs, and (vi) compatibility and processibility [178]. Polymer-stabilized AuNPs date from Helcher's treatise in 1718 [179] that indicated starch-stabilized water-soluble Au particles. With the rapid development of nanotechnology, polymer-stabilized AuNPs are becoming actively and widely used in catalysis [180], optics [181] and biology [182]. The two major synthetic routes to polymer-stabilized AuNPs are the "grafting to" and "grafting from" techniques [183-190]. With the latter method, polymerization occurs at the Au surface in the presence of initiators; thus it can be viewed as a method of AuNP functionalization. Otherwise, the "grafting to" method involves direct AuNP synthesis by attachment of polymers onto the Au surface. There are two strategies for the "grafting to" method. The first one uses functionalized polymers with sulfur, nitrogen or other ligands at the end or in the middle of polymers to stabilize the AuNPs. This synthetic route is relevant to the Brust-Schiffrin method or the ligand substitution reaction. For the Brust-Schiffrin route that is often used with polymers, the HAuCl 4 solution is mixed with the functionalized polymers, and the reducing agent is added to form the AuNPs (both in one phase or two phases). For instance, the polymeric AuNP ligands used in this way include thiolate end-capped polystyrene (PS) [191-193], thiolate poly(ethylene glycol) (PEG) [194], five-arm PEG-b-PCL star block copolymers [195], thiolate poly(N-isopropylacrylamide) (PNIPAM) [196], thiolate poly(vinyl pyridine) (PVP) [197], polypeptide with disulfide termini [198], poly(acryloylaminophenylarsonic acid) (PAAPHA) with amine and arsenic acid group [199], poly(ethylenimine) (PEI) with amine groups [31], thioether-functionalized polymer ligands (DDT-PVAc and PTMP-PVAc) [200], ionic polymers [201], and Oethyl-S-(1-methoxycarbonyl)ethyl dithiocarbonate functionalized poly(N-vinyl caprolactam) (PVCL) (Fig. 9) [202].

The AuNP size is controlled by the polymer/Au ratio that is detected by the change of intensity of the plasmon absorption in the UV-vis spectra [203]. Some polymers such as PS and PAAPHA act as both reducing agents and stabilizers. Thus, they form AuNPs without additional reducing agents, but the AuNPs formed with such weak reducing agents are much larger than those formed using NaBH 4 .

The functionalized co-polymers are also used as templates in AuNP synthesis. For instance, Suzuki developed thermosensitive hybrid core-shell AuNPs via in situ synthesis. Fig. 10 shows the route used in this study. Thiol-and amino-functionalized poly(Nisopropylacrylamide) (NIPAM)-b-(glycidyl methacrylate) (GMA) (poly NIPAM-b-GMA) have been used as templates to stabilize AuNPs inside the polymer shells [204].

The ligand-substitution reaction for the synthesis of polymerstabilized AuNPs has a significant advantage in that the pre-prepared AuNPs therefore lead to relatively monodispersed AuNPs by the Turkevich or Brust method. Thus, after the ligandsubstitution reaction by polymers, the polymer-stabilized AuNPs are also relatively monodispersed, because the AuNP core does not undergo size change (Ostwald ripening) during the ligandsubstitution process. A typical ligand-substitution reaction was conducted by Azzam et al.: PEO-b-PS-b-P4VP was added into the TOAB-stabilized AuNP solution in toluene and stirred for 24 h, and the PEO-b-PS-b-P4VP-stabilized AuNPs were obtained [205]; some such recent examples are known [206-208]. The other "grafting-to" strategy uses the polymer as a template to stabilize the AuNPs as core-shell NPs. This method is also defined as polymer-surfactantor reverse micelle-stabilization of AuNPs, and is discussed in Section 2.2.4.

Dendrimers

Dendrimers are highly branched, cauliflower-shaped monodispersed, synthetic macromolecules with well defined composition, dimension and structures [209,210]. Dendrimer-or dendronstabilized AuNPs are classified in three parts: (i) AuNPs that are entrapped into the dendrimers or dendrons by the functional groups (containing S, N, P and O) and the steric embedding effects. These AuNPs are also called dendrimer-encapsulated AuNPs (Au DENs), and are sometimes used in electrocatalysis [211-219]; (ii) AuNPs that are surrounded by several small dendrimers at their periphery are called dendrimer-stabilized AuNPs (Au DSNs); (iii) AuNPs prepared as dendrimer cores and stabilized by coordinating ligands located at the focal points of dendrons. With functional groups located at the dendron periphery, the AuNP assembly provides AuNP-cored dendrimers containing peripheral functional groups such as redox groups that are broadly used as redox sensors [220-225].

Au DENs were first published by Crooks's and Tomalia's groups using the polyamidoamine (PAMAM) dendrimers as stabilizers [226-228], followed by a number of reports dealing with the preparation of dendrimer-stabilized AuNPs of various kinds. The size distribution of dendrimer-stabilized AuNPs depends on the initial dendrimer-to-Au ratio [229-231] and dendrimer generation [232,233]. Other sulfur groups, hydroxyl groups [245,246], benzyl ethers [247], and amine groups [248], allow the synthesis of dendrimer-stabilized AuNPs [234-244] (Fig. 11). In "click" PEG dendrimer-stabilized AuNPs from our group, stabilization is provided by 1,2,3-triazole coordination, and the AuNPs are encapsulated in the dendrimers when the latter are large enough or surrounded by several dendrimers when the latter are too small (zeroth generation) [249,250].

Phosphorylcholine-functionalized dendrimers provide another example of AuNP stabilizer. Their stability and controllable surface properties indicated potential use in biosensing (Fig. 12) [251]. AFM and TEM were used to demonstrate the morphology and size distribution of the AuNPs formed. Recently, Voelcker's use of the NICISS technique proved useful to understand the inner structure of dendrimer-stabilized AuNPs [233].

Surfactants and reverse micelles

Surfactant-stabilized AuNPs without reverse micelle formation

Surfactants stabilize the AuNPs by electrostatic bonding between the Au surface and the surfactant heads. The surfactant TOAB is normally used in the Brust-Schiffrin two-phase method as a phase-transfer agent, and it also acts as AuNP stabilizer. The TOABcapped AuNPs is obtained by the Brust-Schiffrin two-phase method without adding the additional stabilizer and utilized in further functionalization or self-assembly [252-255]. Another example is Nikoobakht et al.' s synthesis of cetyltrimethylammonium bromide (CTAB)-stabilized AuNPs [256-259]. The AuNPs are obtained by quickly adding an ice-cold NaBH 4 solution into the mixture of CTAB and HAuCl 4 solution, yielding AuNPs of average size up to 4 nm with a net positive interfacial charge. It is frequently used as a seed for the preparation of monodispersed gold nanorods (AuNRs) [256,257] and size-and shape-controlled AuNPs [260,261].

Surfactant-stabilized AuNPs with reverse micelle formation

Besides the electrostatic bonding, surfactants are also used to form reverse micelles for AuNPs synthesis. Reverse micelle solutions are transparent, isotropic, thermodynamically stable water-in-oil microemulsions that are dispersed in a continuous oil phase and stabilized by surfactant molecules at the water/oil interface [262-264]. Water is readily solubilized in the polar core of reverse micelles, forming so-called water pools that play the role of templates for AuNP formation and also prevent the aggregation of AuNPs [265]. The sizes of the AuNPs are defined by the micelle volume. Both low-molecular-weight surfactants and copolymer surfactants form reverse micelles for the synthesis of AuNPs.

Sodium bis(2-ethylhexyl) sulfosuccinate (AOT) is a widely used low-molecular-weight surfactant in AuNPs synthesis. The reverse micelle is thus prepared by adding water/AOT/n-heptane [266] or water/ATO/isooctane [267,268] into an appropriate amount of HAuCl 4 or a gold salt. Then, the reducing agent hydrazine is added into the mixture to form the AuNPs. Recently, catanionic surfactants (mixture of octanoic acid and octylamine) such as lecithin [269,270], and trichromophoric dye-reverse micelles were also shown to stabilize AuNPs as shown in Fig. 13 [271].

However, the use of water/oil microemulsions for the synthesis of AuNPs has a major drawback. Large amounts of surfactant are required to stabilize the AuNPs, which introduces impurities. In order to solve this problem, Hollamby et al. purified reverse micelle-stabilized AuNPs in a single step. Thus, the AuNPs are stabilized by the microemulsion that forms in water/octane with butanol as co-additive. After adding excess water, the AuNPs are concentrated into the upper octane-rich phase, and the impurities remain in the water-rich lower phase [272].

Reverse micelles

Compared with classical surfactant-formed micelles, the copolymer surfactant-formed micelles have several advantages. First, the critical micelle concentration (cmc) of copolymers is much smaller, and their kinetic stability is larger than that of low-molecular-weight surfactants. Second, the size and shape of copolymer micelles is easily tuned by varying the composition of the copolymer, the length of the constituent blocks, and the architecture of the copolymer. Third, the stability of the AuNPs is enhanced upon increasing the length of the coronal blocks [195,273].

A large number of studies are available in polymer micellestabilized AuNPs, therefore only a few representative or recent examples are mentioned here. Poly(styrene)-blockpoly(vinylpyridine) (PS-b-PVP) [274-279] and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) [280-287] are the most widely used polymer micelles for AuNPs synthesis, and these AuNPs are applied in catalysis [275-277] and biosensing [287]. PS-b-PVP micelle-stabilized AuNPs are typically synthesized as described by Möller's group [279]. A solution of the block copolymer in dry toluene is mixed with HAuCl 4 in appropriate amount. The mixture is stirred for at least 24 h in order to allow complete dissolution of the gold salt in the core of the block copolymer micelle. The reducing agent hydrazine is added under vigorous stirring to form the AuNPs (Fig. 14) [274,278].

Various reducing agents introduced into PS-b-PVP micelle systems may result in the formation of AuNPs of various sizes [279]. The morphology, size and size distribution of the AuNPs formed were determined by AFM, DLS, and TEM.

In addition, microcalorimetry was used to investigate AuNP formation in the copolymer micelles. For instance, PEO-PPO-PEO-stabilized AuNPs were prepared in an efficient one-pot aqueous-phase synthesis from the reduction of Au salts by using PEO-PPO-PEO both as reducing agent and stabilizer [283]. The formation of AuNPs from Au salts comprises three steps: (i) initial reduction of Au ions in crown ether-like domains formed by block copolymer in solution, (ii) absorption of block copolymer on AuNPs and further reduction of Au ions on the surface of these AuNPs, and (iii) growth of particles stabilized by block copolymers [284]. Increasing the PEO chain length favors the reduction of Au salts and formation of AuNPs [284]. In addition, the temperature [285], micelle environment [286], and shapes and sizes of micelles [286] are also considered to be the main contribution factors in the overall growth kinetics leading to a specific morphology of AuNPs [286]. For example, the smaller micelle size with few surface cavities produced small AuNPs (Fig. 15) [286].

Other polymer reverse micelle-stabilized AuNPs were also recently prepared using similar approaches. For example, poly(ethylene oxide)-b-poly(-caprolactone) (PEO-b-PCL)stabilized AuNPs with 5-7 nm core size have potential for the exploration of drug-delivery systems with biodegradable PCL units and biocompatible PEO units [288]. Polyethylenimine-ghydrocaffeic acid (PEI-C)-AuNPs are also the subject of biomedical applications [289].

Liposomes

Liposomes, supramolecular assemblies of amphiphilic lipids, can be used as reverse micelles to prepare AuNPs. Early syntheses of liposome-stabilized AuNPs have been conducted by adding NaBH 4 to mixtures of HAuCl 4 and liposomes [290]. This method easily provides size-controlled AuNPs, but NaBH 4 is toxic and can modify the biological function [291]. Recently, a "green" and efficient methodology yielding liposome-stabilized AuNPs involved a simple, rapid, and controlled reaction. The AuNPs were obtained by adding ascorbic acid as reducing agent into the mixture of liposome-based giant unilamellar vesicles (similar to micelles) and HAuCl 4 [291]. Similarly, O'Sullivan's group used the biocompatible reducing agent glycerol for the synthesis of stabilized AuNPs (Fig. 16) [292,293].

Biosynthesis and "green chemistry"

Biosyntheses and "green" syntheses of AuNPs are further remarkable areas in AuNP preparation via in situ synthesis. In these syntheses, the biomolecule directly acts both as a stabilizer and reducing agent to form the AuNPs. The sources for bio-and green preparations of AuNPs are natural source extracts, chitosan, and microbes.

Natural-source extracts

Due to the abundance of carboxyl, carbonyl, hydroxyl and phenol groups, the natural-source extracts reduce Au III and stabilize the AuNPs with these groups. Since Goia and Marijevic synthesized AuNPs stabilized by Arabic gum (originated from a natural plant) [294], many environmentally friendly AuNPs have been synthesized, in particular by Sastry's group [295-297]. Non-toxic chemicals from natural source extracts were used in the syntheses in order to avoid adverse effects in medical and biology applications (Table 1). These AuNP syntheses using natural-source extracts are simple, just mixing the extracts with aqueous HAuCl 4 until the color changes to red or purple [295]. Besides this green-chemistry AuNPs stabilized by natural-source extracts.

Natural source

Groups for stabilizing AuNPs Publication year

Arabic gum [294] Phenol 1999 Lemon grass [295] Hydroxyl and carbonyl 2004 Emblica officinalis [296] Carbonyl and phenol 2005 Alove vera plant [297] Carbonyl and phenol 2006 Cinnamomumcamphora [298] Carbonyl 2007 Gellan gum [299] Carboxy, carbonyl and phenol

2008

Castor oil [300] Carboxyl 2008 Volvariella volvacea [301] Phenol 2009 Hibiscus [302] Phenol 2010 Bayberry tannin (BT) [303] Phenol and carbonyl 2010 Tannic acid [304] Phenol and carboxyl 2010 Zingiber officinale [305] Carbonyl and phenol 2011 aspect, AuNPs stabilized by natural-source extracts are also used in other fields involving AuNPs assembly [306].

Chitosan

Chitosan, the second-most abundant natural polymer in the world, is known as the deacetylated chitin and has a good water solubility and biocompatibility [307,308]. Chitosan stabilizes AuNPs with the amine groups and the steric effect of its own structure. Chitosan-stabilization of AuNPs by adding NaBH 4 as a reducing agent was first published in 2003 [308], then Huang's group [309] used chitosan both as a stabilizing agent and reducing agent and obtained AuNPs upon heating a HAuCl 4 -chitosan mixture for two hours at 70 • C, a fully "green" synthesis. From then on, chitosanstabilized AuNPs have been widely investigated in catalysis [310], biomedicine [311], and sensing [162]. In addition, carboxymethyl chitosan has later also been used to synthesize AuNPs, due to its higher sorption of metal ions than that of chitosan [312].

Microbes

The stabilization by microbes provides a remarkable mode of AuNP biosynthesis that has been studied for more than three decades. Beveridge and Murray showed that Bacillus subtilis reduces Au III to AuNPs with a size range of 5-25 nm sizes inside the cell wall [313]. Up to now, four major microbes have been used for AuNPs synthesis: bacteria, fungi, actinomycete and yeast. The abundance of carboxyl groups in microbes is considered to play a major role in the reduction of Au III [313]. Moreover, the abundant electronegative groups in microbes (amine, carboxyl, thiol, disulfur, etc.) contribute to the microbe stabilization of AuNPs. The synthesis of AuNPs with microbes follows two procedures: extracellular production and intracellular production. For the extracellular production, Au III is reduced by the cell wall reducing enzymes or soluble secreted enzymes. For the intracellular production, reduction occurs inside the cell. The extracellular production of AuNPs has wider applications than intracellular accumulation in optoelectronics, electronics, bioimaging and sensor technology [314].

In Table 2, some identified data from [314] are recollected together with some newer data showing recent microbe-stabilized AuNPs.

Seed-growth method

Principle of the seed-growth method

The seed growth method is another popular technique for AuNP synthesis that has been used for more than a century. Compared with the in situ synthesis, the seed-growth method enlarges the particles step by step, and it is easier to control the sizes and shapes of formed AuNPs. Thus, this procedure is widely used in the most recent size-and shape-controlled AuNPs syntheses. Reprinted with permission from [293] (O'Sullivan's group). Copyright 2011 American Chemical Society. The seed growth usually involves two steps. In the first step, small-size AuNP seeds are prepared. In the second step, the seeds are added to a "growth" solution containing HAuCl 4 and the stabilizing and reducing agents, then the newly reduced Au 0 grows on the seed surface to form large-size AuNPs. The reducing agents used in the second step are always mild ones that reduce Au III to Au 0 only in the presence of Au seeds as catalysts, thus the newly reduced Au 0 can only assemble on the surface of the Au seeds, and no new particle nucleation occurs in solution. Moreover, due to the use of a mild reducing agent, the second step is much slower than the first one, and it can be repeated to continue the growth process.

In the course of the seed-growth synthesis of AuNPs, the formation of seeds takes a significant place correlated to the size, shape and surface properties that are controlled by the amount and nature of reducing agent and stabilizer, and their ratio to the Au precursor. The earliest gold nano seeds were proposed by Natan [325] using citrate reductant and capped spherical Au seeds with diameter 12 nm for the overgrowth of spherical AuNPs. Murphy's group [326,345] reported the synthesis of 3.5 nm citrate-capped gold seeds by dropping an ice-cold aqueous solution of NaBH 4 into a solution of a mixture of HAuCl 4 and citrate. These seeds were originally used for the formation of AuNRs. This procedure of Au seed formation was modified by El-Sayed [327] using hexadecyltrimethylammonium bromide (CTAB) as the stabilizer instead of citrate. These Au seeds with a diameter smaller than 4 nm were used to promote the narrow dispersity of AuNRs. Subsequently, this seed formation was regarded as the most primary nucleation process in the synthesis of AuNPs. Other monodispersed, largesized, spherical or quasi-spherical AuNPs, AuNRs, and other shaped AuNPs have been synthesized using the seed-growth method, as follows.

Spherical or quasi-spherical AuNPs

The traditional in situ synthesis provides spherical or quasispherical AuNPs. The disadvantage is, however, that when the size increases it becomes out of control, and the shape is not controlled either. Therefore, the seed-growth strategy has emerged as a very efficient method to synthesize monodispersed AuNPs with large sizes (up to 300 nm) precisely and with well-defined shapes [327-331].

Natan and co-workers [325,332,333] pioneered the seedgrowth method in a European patent [334]. In this procedure, AuNPs between 20 nm and 100 nm were prepared by adding citrate-capped, NaBH 4 -reduced seeds into a "growth" solution containing a mild reducing agent such as citrate [332,333] or hydroxylamine [329]. These results provided an improvement of physical properties compared with the Turkevich and Frens method. However, this synthetic route also generated a small population of rod-shaped AuNPs as impurities. Later, Murphy's group [335] improved the method by using ascorbic acid as a reducing agent as mild as citrate in growth solution and using CTAB as a stabilizer to synthesize monodispersed spherical AuNPs up to 40 nm (the citrate-stabilized AuNPs with a relative standard deviation that was lower than 10% were considered as monodisperse [335,336]). This method was later used by Han's group for the synthesis of icosahedral AuNPs having controlled size (from 10 nm to 90 nm) (Fig. 17) [337].

In 2006, Liz-Marzan's group first reported the synthesis of gold nanoparticles via the seeded growth method up to 200 nm, also showing optical properties with quadrupolar modes [329]. Recently, highly monodispersed, spherical, citrate-stabilized AuNPs were synthesized up to 300 nm by using hydroquinone [330] or ascorbic acid (Fig. 18) [338] as reducing agent in the seedgrowth process. Reaction conditions such as temperature [331], pH [331], Au precursors to seed particle concentration [331] and citrate concentration [336] were considered to affect the size distribution and shape of the AuNPs.

Gold nanorods (AuNRs)

The AuNRs show two plasmon bands: a strong longitudinal band in the near-infrared region and a weak transverse band, similar to that of gold nanospheres, in the visible region. The band in the nearinfrared region, where tissue absorption is minimal, is very useful for potential in vivo applications. Consequently, due to their specific structure and shape, the AuNRs exhibit wide potential use in nanomedicine [339-341]. During the last few decades, AuNRs had been prepared using electrochemical and photochemical reduction methods in aqueous surfactant media and nanoporous templates [342-344]. However, the seed-growth method developed in particular by the Murphy group [326] then by the El Sayed group [327] now clearly appears as the most favorable one for synthesizing AuNRs, because it can easily generate high yields of well-defined and monodispersed AuNRs.

The current seed-growth method has indeed been first published by Murphy's group [326]. Briefly, 3-4 nm citrate capped AuNPs are used as seeds, then the seeds are added into the growth solution containing appropriate HAuCl 4 , CTAB and freshly prepared ascorbic acid, without further stirring or agitation. After 5-10 min, AuNRs with 4.6 ± 1 aspect ratio are generated in the solution. AuNRs with a high aspect ratio (up to approximate 20) are obtained by three-step growth: the first-step forms AuNRs that are used as seeds for the second growth, and the second-step-formed AuNRs are used as seeds for the third growth (Fig. 19) [326,344,346].

The AuNRs together with other shapeswere also obtained and purified by centrifugation. Indeed, this method has the disadvantage that large amounts of sphericalAuNPs and AuNPs with other shapes are formed as by-products, which greatly reduces the AuNR yield. The Mulvaney and Li-Marzan group then showed that when the temperature and CTAB concentration were reduced, nanorods with small aspect ratio of 1-6 could be obtained with up to 50% yields [328]. The situation is much improved in the presence of silver nitrate, as initially reported in Murthy'seminal articles where up to 50% bipyramidal AuNRs were obtained [326]. Nikoobakht and EI-Sayed very efficiently overcame the drawbacks by using silver nitrate in the presence of CTAB. The single-crystalline CTAB-capped seeds were used instead of citrate-capped seeds, and silver nitrate was used in the seed-growth process to control the aspect ratio of AuNRs. This optimized protocol resulted in high yield of AuNRs (99%) with ratios from 1.5 up to 5 and generated only traces of spherical AuNPs (Fig. 20) [327].

The yield, monodispersity and size of AuNRs were affected by many parameters. The addition of nitric acid into the growth solution leads to a high yield of AuNRs [347,348]. The presence of iodide at ppm concentrations in CTAB prevents the formation of AuNRs in the growth media [349,350]. Moreover, the purification of AuNRs from other shapes of AuNPs is also important for AuNRs synthesis. An efficient and relatively simple method for isolation of single-size rods from a mixture of different-sizerods, spheres and plates was reported by Jana [351]. Briefly, in a concentrated dispersion of a mixture of various-size rods, spheres and platelets formed when a surfactant (CTAB) was added, nanorods and platelets precipitate from the mixture leaving the spheres in solution. Under similar experimental conditions, the long rods precipitate more easily than the short rods, followed by platelets. Recently, it was disclosed that the micelle nature of CTAB was a key factor in NR purification from other shapes due to depletion attraction forces of surfactant micelles [352], and the bromide anions of the CTAB surfactant played a greater role than the CTA + cations for the process of AuNRs formation [353]. In addition, it was figured out that bromide anions played a crucial role in 20 the formation of rod-like AuNPs in the sense that there was a critical [Br -]/[Au III ] ratio (around 200) to form AuNRs with a maximum aspect ratio; beyond this value, the aspect ratio of AuNRs decreased [354]. Details of the mechanism of AuNR formation have been carefully reviewed by the EI-Sayed's [339] and Murphy's groups [340].

The post functionalization of AuNRs has been exploited for the synthesis of various hybrid nanomaterials. For instance, silver coating was achieved upon silver nitrate reduction in the presence of various stabilizers, silicon passivation was conducted by mixing with 3-mercaptopropyl trimethoxysilane (MPTMS) or 3mercaptopropyl triethoxysilane (MPTES), and iron oxide coating Reprinted with permission from [327] (EI-Sayed's group). Copyright 2003 American Chemical Society.

22

was achieved by coprecipitation of iron saltsor adsorption of iron oxide NPs. AuNRs have been integrated with polymer beads for drug delivery, and microgel beads are widely used due to their unique thermal-responsive properties. Supramolecular assemblies of AuNRs have also been designed end-to-end or side-by side [339].

Other shapes of AuNPs

Besides spherical or quasi-spherical AuNPs, anisotropic AuNPs have also recently been synthesized and widely used in biomedicine [355] and nanotherapy [356]. Among the shapecontrolled strategies [357], seed growth is an efficient method for the synthesis of anisotropic AuNPs. It allows rational design of nanocrystal shape, size distribution and monodispersity through the adaption of nucleation and growth conditions. In particular, anisotropic AuNPs include AuNRs [57], Au nanocubes [358], Au nanohexapods [359], Au nanoribbons [360], Au hollow nanocages [361], Au nanobranches [362-364], and Au nanopolyhedra [365-367].

Herein we are focusing on the main examples of anisotropic AuNPs syntheses and their improvements. A seminal report of a solution-based "chemical" route to multiple shaped-controlled AuNPs in 2004 is that of Murphy [358] and co-workers. In a typical procedure, a HAuCl 4 solution is added to a CTAB solution followed by addition to the growth solution of an aqueous solution containing AgNO 3 , ascorbic acid, and HAuCl 4 . In given conditions, nanorods, and other particles with triangular and square outlines were formed and, by increasing the ascorbic acid concentration, hexagonal nanocrystal appeared. Then, upon further increase in ascorbic acid, cube-shaped particles were formed in high yield (90%). A variation of branched AuNPs were also synthesized by controlling the various combinations of [seed]/ [Au III ] ratio or the concentrations of CTAB and ascorbic acid.

Huang's [368] group organized a systematic shape evolution process from CTAC-capped truncated cubic Au seeds to trioctahedral and rhombic dodecahedral AuNPs via a two-step seed growth process. Systematic shape evolutions from truncated cube to cubic, trisoctahedral, and rhombic dodecahedral structures have been controlled upon addition of various amounts of ascorbic acid.

Xia [359] and coworkers designed a facile method for the formation of thermodynamically unfavorable Au nano-hexapods by using N, N-dimethylformamide (DMF) to reduce HAuCl 4 in the presence of single crystal of octahedral Au as the seed. The shapes of the hexapods were controlled by variation of the amount of HAuCl 4 in the growth solution or changing the reaction temperature. Polyhedron is another shape of AuNPs that has recently been obtained by adding the CTAC-capped Au seeds into the growth solution containing HAuCl 4 , CTAC, and various salts, followed by reduction using ascorbic acid. The shapes of the AuNPs formed are controlled by the nature of various salts in the growth solution. For example, NaBr is used for rhombic dodecahedra, and KI is used for octahedral [367]. Very recently, Mirkin's group [361] synthesized octahedral AuNPs with hollow features. In the growth process, Au concave nanocubes are prepared as seeds [363], and the reaction is initiated by the addition of the seeds to the growth solution containing the appropriate ratio of HCl, HAuCl 4 , AgNO 3 , ascorbic acid, and cetyltrimethylammonium chloride (CTAC). The yield is higher than 90%, and the structure of the Au hollow is well characterized (Fig. 21 shows some examples of anisotropic AuNPs).

It was found that the temperature, pH, Au precursors/surfactants ratio and salts used in the growth solution influenced the growth of Au seeds on each facet, and finally affected the size and shapes of the AuNPs [369-372]. Usually, anisotropic AuNPs are formed when the Au ionsand a reductant are added, together with some shape-templating surfactant, and the seeds are grown into larger particles of particular morphologies. However, the mechanism of anisotropic nanocrystal formation is presently not well understood, although a detailed discussion has been proposed [369].

Other AuNP synthetic methods

Other methods involve both "top down" and "bottom up" strategies and are discussed here. They also include supported AuNPs and bimetallic AuNPs.

Pulse radiolysis

Pulse-radiolysis is another "bottom-up" method that involves gamma-ray irradiation for the reduction of Au III instead of the traditional addition of a chemical reductant [373]. As a suggested mechanism, radiolysis generates radicals by ionization and excitation of the solvent (usually water). A radical scavenger is introduced in order to trap the primary radical formed (OH • ) to give a new radical that is unable to oxidize the gold ions but exhibits strong reducing power, such as the radicals of secondary alcohols (2propanol is the most-commonly used one). This is followed by the disproportionation of Au II species giving Au I and Au III species. Meanwhile, the mild reducing agent reduces Au III to Au II , and Au I to Au 0 . During this stepwise process, Au III is progressively reduced to Au 0 , and the formation of AuNPs proceeds in the presence of a stabilizer that is required in order to avoid the overgrowth and aggregation of the AuNPs [374]. The stabilizer must be unable to reduce the gold salt directly before the irradiation. For instance, poly(vinyl alcohol) (PVA), octadecylamine(OA), and olyvinyl pyrrolidone (PVP) were used as stabilizers.

In a recent example, Abidiet al. [375] published a one-pot radiolytic synthesis of AuNRs of well-controlled aspect ratios in a CTAB micelle. The synthetic procedure involved addition of HAuCl 4 and TOAB to a stirred aqueous CTAB solution; after further stirring for 1 h at 50 • C, acetone, cyclohexane, and AgNO 3 were successively added into the previous solution, and then flushed with nitrogen and irradiated by a 60 Co panoramic gamma source. A series of AuNRs with various aspect ratios were obtained by variation of the Ag I concentration.

The radiolysis strategy yielded spherical AuNPs with diameters 6 nm and 13 nm [376] bimetallic Au-Pd [377] and Au-AgNPs [378], but altogether rather few publications have appeared.

Top-down methods

The top-down strategies start with a bulk gold substrate (generally film or pellet), followed by a nanoscale patterning procedure during which the major part of the gold film is removed, yielding AuNPs with predetermined scale and shape [339]. One of the most commonly utilized top-down techniques is the electron-beam lithographic method that results in the formation of multiple-shaped nanostructures with dimensional control on tens of nanometer length scale [379]. Another most popular topdown technique is the laser-based ablation [380], first introduced by Cotton et al. [381]. As a recent example, Meunier's group developed a two-step femtosecond laser-assisted technique producing size-controlled, low dispersed (20%), and functionalized spherical Au NPs in aqueous solution in the size range of 2-80 nm. In the first step, seeds were obtained by ablating a gold pellet substrate immersed in aqueous dextran (as stabilizer ligand) solutions. The seed-growth proceeds under femtosecond laser irradiation for 1 h [382]. Reprinted with permission from [362] (Mirkin's group). Copyright 2011 American Chemical Society.

Supported AuNPs

As in the synthesis of ligand-stabilized AuNPs, the primary role of the support is to avoid the aggregation of AuNPs. Moreover, the support also plays a direct or indirect role at the AuNP-support interface in gold-catalyzed reactions. AuNPs supported by mesoporous oxides including TiO 2 , CeO 2 , SiO 2 , Fe 3 O 4 , Al 2 O 3 , ZrO 2 , the forms of carbon, and metal-organic framework (MOFs) have broad applications in homogeneous and heterogeneous catalysis [383].

Oxides

The most widely used procedure for the preparation of AuNPs supported on these insoluble oxides solids is the precipitation-deposition method. Starting from an aqueous solution of HAuCl 4 , addition of a base leads to precipitation of a mixture of Au(OH) 4 or Au(OH) x -Cl 4-x -. Related gold oxy/hydroxides that adsorb into the solid are then reduced to metallic Au by boiling the adsorbed species in methanol or any other alcohol. After adsorption on the solid surface, AuNP formation occurs by nucleation and growth. The pH of the precipitation and the other experimental conditions (nature of the alcohol, temperature and time of the reduction, calcination procedure, etc.) provides a certain control of the particle size of the resulting NPs [384]. The ideal solid oxide support should have a high density of hydroxyl groups and a large surface area according to the deposition-precipitation mechanism in order to achieve the formation of AuNPs with narrow size distribution [383,385-393]. Characterization techniques for the oxide-supported AuNPs include TEM, SEM, XRD, Raman and UV-vis spectroscopies. Since Haruta' seminal discovery [384] in the 1980s of CO oxidation by O 2 to CO 2 at low temperature using TiO 2 -supported AuNPs that are smaller than 5 nm, these oxide-stabilized AuNPs are widely studied in catalysis for aerobic oxidations of various substrates including alcohol oxidation, CO oxidation, and hydrolytic dehydrogenation and for hydrogenation reactions [385-393].

Carbon

In the family of activated carbons, carbon blacks, graphites and carbon nanotubes (CNTs) are known to support AuNPs for catalytic applications [394] and sensing [395]. The precipitation-deposition method that is very useful with oxide supports results instead in aggregation with carbon supports [383]. Thus, in order to deposit AuNPs on carbons, two steps are required. This first step is the synthesis of the AuNPs by either the Turkevich method or the Brust method using a stabilizer such as citrate, thiol, polymer. The second step is the immobilization of preformed AuNPs on the surface of active carbons (also carbon blacks and graphite) or into the matrixes of CNTs. Concerning the immobilization step, as shown by the group of Prati and Rossi [15,396], the carbon support is added into a sol of preformed AuNPs with vigorous stirring, and the AuNPs are immobilized on the support with a certain amount of AuNP loading. A high stabilizer/Au ratio maintains the original sizes of the AuNPs during the immobilization step [396]. Sol-carbon interactions are critical to control the resulting AuNP size [396,397] of catalysts leads to high surface-area systems, in which the shapeselective behavior of mesoporous materials can be combined with the catalytic action of metal particles. This shape selectivity cannot be achieved with amorphous oxide-supported metal catalysts [398,399]. Among all the mesoporous materials, the mesoporous silicas have gained great attention due to the properties of MCM-41 (MCM: Mobil Composition of Matters) and related materials, including highly ordered mesopores, controlled pore size, specific surface areas and pore volumes [383,400,401]. Mulvaney' group published an early synthesis of silica-coated AuNPs in 1996. The silane coupling agent (3-aminopropyl)-trimethoxysilane was used to render the gold surface vitreophilic. After the formation of a thin silica layer in aqueous solution, the particles were transferred into ethanol for further growth using the Stober method. Varying the silica shell thickness and the refractive index of the solvent allowed control over the optical properties of the dispersions [110].

The use of task-specific ligands with calcining as post-treatment provides AuNPs that are encapsulated into the mesoporous channels or loaded on the surface of silica spheres [383,402]. For example, Corma's group [403] showed that task-specific ligands containing a cetylammonium moiety act as structure-directing agent with AuNPs containing a trialkoxysilyl group. The latter cocondenses with tetraethoxysilane (TEOS) to form mesoporous silica (Fig. 22). In another recent example [403], NH 2 and -NH functionalized mesoporous carbon nitride (MCN) was shown to stabilize AuNPs. The synthesis involved the reduction of AuCl 4 by NaBH 4 in water suspension in the presence of MCN.

Besides task-specific ligands, the AuNPs are also coated with polymers and then encapsulated by mesoporous materials as core-shell AuNPs. For instance, according to Jan et al. [404], a preparation of AuNP/mesoporous silica tubular nanostructures. Poly(L-lysine) (PLL) and poly(L-glutamic acid) (PLGA) multilayercoated membranes could be immersed in a HAuCl 4 solution to form AuNP/PLL/PLGA multilayers. Subsequently the membranes were taken out from the solution and inserted in a freshly prepared orthosilicic acid solution for 6-12 h, to allow precipitation of silica in this multilayer. After thoroughly rinsing with water and drying at room temperature, the as-prepared AuNP/meso-SiO 2 /PLL/PLGAmultilayer was obtained and purified by calcination (Fig. 23).

6.3.3.2. MOFs. MOFs are another form of mesoporous materials that are thermally robust and in many cases highly porous. It is expected that the crystalline porous structures of MOFs limit the migration and aggregation of AuNPs. Thus, MOF-supported AuNPs have gained progressive attention since the first report by Fisher and co-workers in 2005 [405]. In Fisher's method, the precursor AuNPs are mixed with MOF-5 and loaded into MOF-5 both by thermal metal organic chemical vapor deposition (MOCVD) and photo MOCVD. In addition, the MOF-5-encapsulated AuNPs were also prepared by solid grinding without organic solvent (Fig. 24) [406].

Very recently, Xu' group [407,408] published the synthesis of ZIF-8-encapsulated AuNPs (Zn(MeIM) 2 MeIM = 2methylimidazole). The pretreated ZIF-8 is dispersed in a HAuCl 4 /MeOH solution, and the mixture is pumped for 1-2 h to be mushy, then suitable amounts of MeOH are added into the slurry, and NaBH 4 in MeOH is added under vigorous stirring for the complete reduction of AuCl 4

-. The solid is recovered by filtration and thoroughly washed with MeOH. Very weak diffractions were detected from powder XRD patterns, indicating the formation of small AuNPs that was confirmed by TEM and HADDF-STEM.

Bimetallic AuNPs

Au bimetallic NPs are classified in two types of mixing patterns: core@shell NPs and alloy bimetallic NPs. The core@shell NPs consist of a metal shell surrounding a gold core, or gold shell surrounding another metal core. These NPs could be considered as heterogeneous. The alloy bimetallic NPs consists of a homogeneous mixture of gold and another metal in the NP. Many Au bimetallic NPs are known with Ag, Pd, Pt, Zn, Cu, ZrO 2 , CdS, Fe 2 O 3 , Eu, Ni, and Rh [9].

Core@shell NPs

Core@shell heterobimetallic NPs have appeared in the 1970s [409,410], and Toshima [411,412] has synthesized PVP-stabilized Au@Pd and Pd@Au NP catalysts, characterized them by X-ray absorption fine structures and shown that Au@PdNPs were more active than PdNPs for catalytic hydrogenation reactions, due to synergistic electronic effects [413-415]. The Au-Fe 3 O 4 bimetallic NPs are attractive materials for biological and medical areas, due to their theranostic (therapy + diagnostic) properties involving magnetic resonance imaging (MRI) and hyperthermia [416,417].

The Au-core@other metal-shell NPs are generally synthesized by sequential reduction of appropriate precursors [418]. For example, Crooks's group indicated various ways of forming Au-Pd bimetallic NPs in dendrimers. The reduction of metallic precursors is initiated either by the polyol method or by addition of NaBH 4 . The co-complexation of dendritic PAMAM interior ligands followed by reduction results in a core@shell structure. For instance, the polyol reduction often results in the formation of Au-core@Pd-shell NPs, because of the difference in the reduction potentials of Au and Pd. The Au III precursor is easier to reduce than various transition metal cations including Pd II and provides a seed for Pd II to be reduced on for the synthesis of Au@Pd NPs. The sizes of the core and shell are controlled by the Au/Pd ratio used during the synthetic procedure. Alternatively, an elegant method used by Crooks is galvanic displacement that involves the redox reaction between a DEN and metal ions of another metal [418]. Another typical example of core@shell NPs from Xie's group is theAu@Pt@Au nano raspberries that are used for catalysis [419]. As shown in Fig. 25, the first step is the formation of an Ag shell around the Au core. The conversion from Au@Ag to Au@Pt particles is achieved via the galvanic displacement of Ag by Pt through the addition of hexa-chloroplatinic(IV) acid. Silver is deposited on the Pt surface in a second coating step, again using AgNO 3 and sodium citrate as reducing agent. In the last step of the synthesis, raspberry-like Au@Pt@Au NPs are formed via the concerted action of both reagent reduction and galvanic replacement (vide infra). As shown in the SEM image in Fig. 25b, this approach leads to the growth of the desired Au protuberances instead of the formation of a complete and smooth Au shell. The other metal-core@Au-shell NPs are usually synthesized by forming the Au shell on the other metal core, i.e. sequential loading and reduction of the metals leads to the choice of core and shell, because the metal that is first introduced forms the core [418]. A variation involves core surface modification with functional groups serving as templates for the nucleation of Au [409,420]. For example, small Fe 3 O 4 NPs (5-15 nm) were first synthesized by reduction of Fe(acac) 3 by 1,6-hexadecanediol in the presence of the capping agents oleic acid (OA) and oleylamine. Then, the Fe 3 O 4 NPs served as seeds for the coating of the Au shell. Oleylamine is a crucial surfactant, providing an amine functional group that coordinates to the Au atoms, which is followed by reduction of Au(CH 3 COO) 3 by 1,6-hexadecanediol in the presence of oleylamine. As a result, the HR-TEM confirmed the formation of monodispersed Fe x O y @Au NPs with a 5-15 nm core and 0.5-2 nm Au shell thickness [421]. Core@shell AuNPs are also known with AuNPs coated with silica shells (Section 4.3.3.1). Murphy et al. disclosed silica nanospheres with a nanoscale overcoat of gold ("nanoshells") that have tunable absorption in the visible and NIR regions, which leads to remarkable potential use of these AuNPs in cellular imaging [422].

Bimetallic alloy NPs

The bimetallic Au alloy NPs are generally synthesized by simultaneous reduction of appropriate precursors [423,424]. For instance, theAu-Cu nanocube could be formed by simultaneous reduction of copper(II) acetylacetonate and HAuCl 4 by 1,2-hexadecanediol in diphenyl ether in the presence of 1-adamantanecarboxylic acid, 1-hexadecylamine, and 1dodecanethiol. The TEM image showed that the as-prepared particles were perfectly cubic in shape and uniform in size, averaging 23 nm, and the EDX spectroscopy line scanning analysis showed a homogeneous distribution of copper and gold across the entire nanocube [425].

Recently, Negishi et al. synthesized the precise alloy clusters Au 25-n Ag n (SC 12 H 25 ) 18 by reducing various ratios of HAuCl 4 /AgNO 3 mixtures and purified them by precipitation in acetonitrile. The structures were confirmed by MALDI and LDI mass spectra [426]. Later, this method was developed in order to obtain the alloy 12 H 25 ) by etching of the pre-formed Au/Ag clusters with excess thiol [426]. The optical absorption spectra of the nanoalloy clusters in the UV--vis. region exhibit three distinct features: a "plasmonic" peak around 430 nm (2.9 eV) and two shoulders around 310 (4.0 eV) and 560 nm (2.2 eV). The higher is the n value, the clearer is the appearance of these features (Fig. 26) [427,428].

Conclusion and outlook

The preparation of AuNPs that has been known for a very long time has met a considerable amount of variations involving multiple materials from biology to functional molecules and finally the solid state. The engineering of AuNPs has become finely tuned with size and shape control towards targeted applications. The intrinsic lack of toxicity of the AuNP cores and their topological, optical, sensing, catalytic and biomedical properties will obviously lead to a huge expansion of their applications in nanotechnology [429-431].

In summary, AuNPs with size below 10 nm are easily prepared by the Brust-Schiffrin method, the mechanism having recently been disclosed [56], or by high-temperature reduction with oxide and other solid supports for catalytic applications with supported AuNPs smaller than 5 nm. In molecular chemistry, thiolate ligands provide robust AuNPs of 1-10 nm size, and direct syntheses, ligand substitution reactions with functional thiols and post functionalizations using for instance olefin metathesis, click reaction and amide bond formation provide rich means of multiple functionalizations for potential biomedical applications [432].

AuNPs larger than 2-3 nm present the plasmon band resulting from the interaction of light with the collective conducting surface electrons of the AuNPs that is not observed for AuNPs smaller than 2 nm due to the localization of electron in molecular Au cluster bonds. The plasmon band of relatively large AuNPs is thus most useful for various imaging and other optical techniques. Therefore the improved Turkevich method using citrate Au III reduction is still much in use for the synthesis of such AuNPs in the 10-50 nm-size range that are functionalized with biomolecules, drugs, receptors and imaging agents for diagnostic and therapy applications. Here again, recent mechanistic investigations have successfully delineated the two roles, reductant and stabilizer of citrate [37]. The seed-growth method with spectacular recent progress, in particular by both Murphy's and El sayed's groups, however, is now considered as more precise than the Turkevich method, because it provides narrower size distributions. For the synthesis of AuNPs larger than 100 nm, the seed-growth method is also specifically efficient. AuNPs with various shapes (rods, cubes, triangles, hexapods, ribbons, hollow cages, branches, polyhedra) are synthesized using this method by careful controlling of the synthesis conditions. Thus the formation of AuNPs of a specific shape is possible, but its rationalization is awaiting more progress because the mechanisms are not yet well understood.

The technology of solid state-supported AuNPs that started with glass decoration (e.g. the IVth-century Lycurgus cup) continues to fascinate scientists with the remarkable catalytic properties of inter alia oxide-supported AuNPs. The latter, pioneered by Haruta 25 years ago, is still expanding using a variety of solids including mesoporous materials, MOFs, nano-and supracrystals [432] and heterobimetallic NPs towards promising "green-chemistry" applications.

Introduction

Carboranes that have a structure of 12-vertex icosahedral C 2 B 10 H 12 possess a high boron content and stability to catabolism, which can increase the thermal and chemical stability of complexes. Carborane is a key material for boron neutron capture therapy (BNCT) for the treatment of cancer. AuNPs have shown extraordinary performances in the areas of photothermal therapy, biosensing, imaging and drug delivery. Incorporation of carboranes into AuNPs could be a viable approach for the delivery of boron to the tumor tissues a nd the treatment of cancer through BNCT.

Since [Cu I tren(CH 2 Ph) 6 ][Br] (Cu(I)-tren) has been synthesized and utilized as excellent catalyst of "click" reaction in our previous reports, carborane and PEG were grafted onto the periphery of AuNPs with a satisfying yield in this chapter. AuNPs were also prepared in onestep through capping of a pre-functionalized thiolate dendron. Carboranes containg an ethynyl group were provided by the group of Professor Narayan Osmane with who we collaborated in the publications. ABSTRACT: Carboranes that have a high boron content are key materials for boron neutron capture therapy (BNCT), while PEGylated gold nanoparticles (AuNPs) are also most useful in various aspects of nanomedicine including photothermotherapy, imaging and drug vectorization. Therefore, methods to assemble these key components have been investigated for the first time. Strategies and results are delineated in this article, and the nanomaterials have been characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-vis., mass and multinuclear NMR data. A series of well-defined water-soluble bifunctional AuNPs containing carborane and polyethylene glycol (PEG) were synthesized through either two-step Cu(I)-catalyzed azide-alkyne cycloaddition CuAAC ("click") reactions at the periphery of azido-terminated AuNPs in the presence of the efficient catalyst [Cu I tren(CH 2 Ph) 6 ][Br] or simply by direct stabilization of AuNPs using a tris-carborane thiol dendron or a hybrid dendron containing both PEG and carborane.

■ INTRODUCTION

Carborane chemistry has been developed into nanomaterials during the past few decades because of the robustness and physicochemical properties of the carborane frameworks. Because of the structure of 12-vertex icosahedral C 2 B 10 H 12 , this carborane possesses high boron content and stability to catabolism, can increase thermal and chemical stability of complexes, and provides the possibility of use as building block and template in material science, 1 nanomedicine, 2 and synthesis of highly stable dendrimers, polymers, or other supramolecules. 3 It has been demonstrated that the use of carborane can enhance hydrophobic interactions between pharmaceuticals and their receptors and increase the in vivo stability of pharmaceuticals. Gold nanoparticles (AuNPs) have been among the most extensively studied nanomaterials over a century because of their remarkable optical properties related to their plasmon absorption and are now heavily utilized in chemistry, biology, engineering, and medicine because of their unique optical, chemical, electrical, and catalytic properties. 4 AuNPs have attracted the interest of scientists especially in the areas of photothermal therapy, biosensing, imaging, and drug delivery. 5 Therefore, incorporation of carboranes into AuNPs could be a viable approach for the delivery of boron to the tumor tissues and cancer treatment through boron neutron capture therapy (BNCT). BNCT is a binary radiation therapy for the treatment of cancer that is based on the capture of thermal neutrons by 10 B nuclei which have a large capture cross section, and these 10 B nuclei can be selectively delivered to tumor cells. 6 Polyethylene glycol (PEG) has been extensively used as modifying agent in many biomedical products because of its nontoxicity, water-solubility, and the enhanced permeability and retention (EPR) effect. The PEGylated NPs can postpone or prevent the rapid clearance by the reticular-endothelium system (RES). Therefore, drugs carried by PEGylated AuNPs can have a prolonged circulation time in blood. 7 Thus, the PEGylated AuNPs have been widely utilized in materials science, 8 biological and pharmaceutical applications. 9 The AuNPs containing both carborane and PEG could be regarded as valuable nanomaterials with useful optical properties, increased watersolubility and biocompatibility, and further suitable for biomedical studies. The Huisgen-type Cu(I)-catalyzed azidealkyne cycloaddition (CuAAC) reaction ("click" reaction) is the most efficient strategy to assemble a 1,2,3-triazole ring linking two molecular fragments together because of its atom economy, regioselectivity, wide substrate scope, and mild reaction conditions. 10 Moreover, the triazole group is completely biocompatible. The "click" modification of AuNPs has been a challenging topic of research in the past few years because of serious aggregation of AuNPs that induced low yield. 11 Recently, an efficient "click" catalyst [Cu I (CH 2 Ph) 6 tren][Br] (abbreviated Cu I -tren), has been reported, and only 7% of this catalyst is needed in toluene solution for the fabrication of sterically demanding "click" dendrimers. 12 Subsequently, this Cu I -tren catalyst has also been successfully employed for the "click" functionalization of AuNPs. High yields were obtained using this functionalization method, and the aggregation was largely avoided because of the dendritic effect around the catalytically active metal center. 13 With these tools in hands, it was desirable to investigate the possibility of synthesis, stability, and physicochemical behavior of assemblies between PEGylated AuNPs and carboranes using thiolate Au-ligands and "click" linkages. In this work, we have investigated the synthesis of bifunctional AuNPs containing both PEG and ortho-carborane units through "click" reactions between azido-terminated AuNPs and alkyne ligands in the presence of the Cu I -tren catalyst. We have also examined the direct AuNP formation and stabilization using a tris-carborane thiol dendron or a hybrid thiol dendron containing one PEG branch and two carborane branches. The morphologies of the core size, hydrodynamic diameter, and polydispersity of the carborane-functionalized AuNPs have been determined by transmission electron microscopy (TEM) and dynamic light scattering (DLS), while the distribution of various functional groups on AuNPs have been verified by 1 H NMR, and the functional properties of AuNPs were further characterized by infrared (IR) spectroscopy.

■ RESULTS AND DISCUSSION

Synthesis of Azido-Terminated AuNPs. Dodecanethiolate-AuNPs (AuNPs-1) were synthesized through the typical Brust-Schiffrin "two-phases" method, 14 and the prefunctionalization of AuNPs-1 was carried out by a classical ligand-substitution reaction. Briefly, AuNPs-1 was dissolved in CH 2 Cl 2 solution containing certain amount of undecanethiol, stirred under N 2 at 25 °C for a few days, during which time the ligand-replacement occurred on the surface of the AuNPs (Scheme 1). Three azido-terminated AuNPs (AuNPs-2a, AuNPs-2b, AuNPs-2c) with different dodecanethiolate: azidoundecanethiolate ligand ratio were obtained under different ligand-substitution conditions (Table 1): AuNPs-2b that contains 33% azido ligands on the surface was obtained with 1:1 ratio of azidoundecanethiol to dodecanethiolate ligands within 2 days. When the reaction time was prolonged to 5 days, AuNPs-2a containing 50% azido ligands was obtained under the same ligand ratio. With the purpose of introducing a higher % of azido groups, the ligand-exchange reaction was repeated 3 times with a ligand ratio of 2: 1, and the reaction time was 3 days for each time. This procedure yielded AuNPs-2c with 80% azido ligands in 80% yield, which was a little bit lower than the yield of the single substitution to obtain AuNPs-2a (90%) and AuNP-2b (>95%), because of increase of aggregation in the process of extended multiple-step ligand-substitution reaction.

The presence of azido group in the AuNPs has been confirmed by 1 H NMR (Supporting Information, Figure S1) and IR (Supporting Information, Figure S3) spectra. The spectra also indicated the percentage of azido-functional group on AuNPs, obtained through ligand substitution, depending upon both the reaction time and the feed ratio of the two different ligands.

"Click" Functionalization of AuNPs with Carborane and (or) PEG. The carborane-functionalized AuNP-3a was first obtained through a "click" reaction in the presence of the homogeneous [Cu I (CH 2 Ph) 6 tren][Br] catalyst (10%) in toluene solution, while an excess carborane alkyne per azido group (2: 1) was employed. The reaction was monitored by IR spectra until the ν N3 band had disappeared. The excess carborane and catalyst were removed by washing the AuNPs with ethanol and diethyl ether successively. AuNP-3a containing 50% carborane groups was obtained in 40% yield after removal of a precipitate of Au black by filtration. Subsequent stepwise "click" reactions were carried out for the synthesis of bifunctional AuNPs containing both PEG and carborane. In this method, azido-terminated AuNPs were reacted with carboranealkyne and PEG (M w 350 or 2000)-alkyne successively in the presence of 10% Cu I -tren catalyst (Scheme 2). When AuNPs-2b, containing 33% azido ligands, were used as starting material, AuNP-3b was obtained with 17% carborane and 17% PEG350 ligands involving 4:1:1 molar ratio of dodecanethiolate ligands, carborane, and PEG350, respectively (see Supporting Information, Figure S7). The yield of AuNP-3b was increased up to 65% with the decrease in functional group, when compared to that of AuNP-3a. With the purpose of obtaining water-soluble carborane-AuNPs, PEG2000-alkyne and Scheme 1. Synthesis of AuNPs-1 by the Brust-Schiffrin Method, and the Ligand-Exchange Synthesis of Azido-Terminated AuNPs AuNPs-2c containing a large percentage of azido groups were employed. Ultimately, water-soluble AuNPs-3c was obtained with the surface capped by 17% carborane, 66% PEG ligands, and 17% dodecanethiolate ligands. The molar ratio of dodecanethiolate-ligands: carborane: PEG350 on the surface of AuNPs-3c was 1: 1: 4, as confirmed by 1 H NMR spectra (Supporting Information, Figure S10). In this case, the hydrophilic PEG ligand properties led to improved water solubility and biocompatibility of AuNPs-3c. Another advantage of introducing a large percentage of PEG2000 was that the "click" reaction did not decrease the yield significantly, despite PEG being a longer chain and present in high proportion. This attributed to the flexibility of PEG in solution. The coating of PEG2000 onto the Au surface isolated the AuNPs from each other, which inhibited the AuNPs aggregation.

The UV-vis spectrum of AuNPs-3c, in Figure 1, exhibits a plasmon band at 524 nm, while its TEM image indicates the average core diameter of 4.75 nm. The hydrodynamic diameter of AuNPs-3c in aqueous solution, provided by DLS measurement, is 10.2 nm. The complete disappearance of the ν N3 band at 2090.9 cm -1 in the IR spectrum was evident after the second set of "click" reactions, when compared to the strong ν N3 peak of AuNP-2c (Figure 2). IR spectrum of AuNPs-3c also shows clearly the σ B-H stretching vibration at 2580.84 cm -1 . The core size determined using TEM images for AuNPs-3a, AuNPs-3b, and AuNPs-3c are similar to those recorded before the "click" reaction (Supporting Information), which demonstrated that the "click" reactions with AuNPs have no adverse effects on the polydispersity, despite the reaction yields being decreased because of aggregation. The DLS measurement showed the hydrodynamic diameters of AuNPs-3a (15.6 nm) and AuNPs-3b (15.4 nm), but the hydrodynamic diameter of AuNP-3c was much smaller (10.2 nm), presumably because of the effect of suppressing long linear PEG2000 branches during assembly among individual AuNPs. The DLS size-distribution histograms of AuNPs-3a, AuNPs-3b, and AuNPs-3c displayed narrow polydispersity of the "clicked" AuNPs, as TEM indicated.

Direct Synthesis of Bifunctional AuNPs. The triscarborane thioacetate dendron 3, a precursor for dendritic AuNPs, was synthesized by "click" reaction between the trisalkyne dendron 1 and carborane-azide 2 in the presence of copper sulfate and sodium ascorbate (Scheme 3a). The hybrid thioacetate dendron 4, containing one carborane branch and two PEG branches, was synthesized by "click" reaction between Scheme 2. Schematic Illustration of the "Click" Functionalization of AuNPs the dendron 1 and only 1 equiv of compound 2 and then further "click" reaction with 2 equiv of PEG2000-azide (Scheme 3b). Interestingly, the direct synthesis does not undergo any aggregation, contrary to the multistep synthesis. With the direct-synthetic approach, the tris-carborane thiol dendron 5 was obtained first from dendron 3, in situ, and the AuNPs-4a stabilized by bifunctional PEG550-SH mixed-ligands were subsequently synthesized through a simple one-step reduction procedure (Scheme 4a).

The direct synthesis involved 1: 1: 1 molar ratio of triscarborane thiol dendron 5, PEG550 thiol ligand, and HAuCl 4 3H 2 O, respectively. After purification, AuNPs-4a was shown to contain 20% dendron 5 and 80% PEG550 thiolate ligands at the periphery and, consequently, the ratio of carborane group to PEG ligand was found to be 3: 4 (Supporting Information, Figure S26). An increase in the number of carborane moieties was accomplished by utilizing the tris-carborane dendron 5 (Scheme 4a). With the purpose of precisely controlling the ratio of two functional groups (herein, the PEG/carborane ratio), the hybrid tris-dendron 6, obtained from dendron 4 in situ, was used for direct AuNP synthesis. The water-soluble AuNPs-4b capped with dendron 6 was synthesized in methanol solution through a simple reduction of Au(III) by NaBH 4 (Scheme 4b). The ratio of carborane to PEG in AuNPs-4b was exactly the same as with dendron 4, revealing that the invaluable advantage of this strategy is the control of relative amounts of two functional groups. The AuNPs that were obtained by this reaction essentially preserve the properties of the dendron. The TEM images of AuNPs-4a and AuNPs-4b are presented in Figure 3 showing the average core diameter of 3.43 and 7.59 nm, respectively. The size-distribution histograms showed that AuNPs-4a and AuNPs-4b are quite monodisperse.

The average core diameter of AuNPs-4b is larger (7.59 nm, Figure 3b) than those of other AuNPs, described above, which presumably results from slower thiol-gold interactions due to dendronic bulk inhibition around the thiol group. The hydrodynamic diameters of AuNPs-4a and AuNPs-4b were also measured in aqueous solution by DLS analysis. The results indicate that the hydrodynamic diameters of AuNPs-4a and AuNPs-4b are 19 and 14.5 nm, respectively.

The FT-IR spectra of dendron 4 and AuNPs-4b are shown in According to the core size determined by TEM and the ligand ratio estimated by 1 H NMR, the amounts of carborane units and of PEG ligand per AuNP are calculated using Leff's method based on both theoretical and experimental data. 16 As shown in Table 2, each AuNP carries more than 50 carborane units, and particularly, AuNPs-4b with more than 220 carborane units at the periphery could potentially find applications in BNCT for cancer treatment.

■ CONCLUSION

The present study showed two feasible approaches to synthesize the AuNPs-carborane assemblies. The two-step "click" modification of azido-terminated AuNPs in the presence of the [Cu I (CH 2 Ph) 6 tren][Br] catalyst gives bifunctional AuNPs in good yield, and the core size remains unchanged during the "click" reaction. The ratio of carborane to PEG functional ligands can be controlled by the feed ratio. Water-soluble AuNPs were obtained when a large percentage of long chain PEG (M w 2000) was employed. With this method, bifunctional AuNPs can be obtained with a narrow polydispersity. In the direct synthesis method, the mono-or bi-functional thiol dendron was introduced onto the surface of AuNPs through a simple reduction reaction. This direct synthesis method would be of great interest for multifunctionalization of AuNPs, because the prefunctionalization of a dendron can be efficiently achieved to avoid multistep AuNP synthesis. Remarkably, the hybrid dendron-stabilized AuNPs have an invariable ratio of two functional groups, and this ratio is thus exactly the same as that of the dendron. Furthermore, no aggregation of AuNPs occurred in this method. We conclude that the direct synthesis method would facilitate the procedure of AuNP functionalization. Thus, a series of well-designed bifunctional AuNPs containing carborane and PEG ligands with core sizes in the range of 3.42 to 7.59 nm were synthesized and fully characterized. The bifunctional AuNPs, in particular the water-soluble AuNPs such as AuNPs-3c, AuNPs-4a, and AuNPs-4b could provide a biocompatible platform in therapeutical BNCT investigation or some other applications. ■ EXPERIMENAL SECTION General Information. All chemicals and solvents were used as received. 1 H NMR spectra were recorded at 25 °C with a Bruker AC200, 300, or 400 MHz spectrometer. The 13 C NMR spectra were obtained in the pulsed FT mode at 50, 75, or 100 MHz with a Bruker AC 200, 300, or 400 MHz spectrometer. All the chemical shifts are reported in parts per million (δ, ppm) with reference to Me 4 Si (TMS) for the 1 H and 13 C NMR spectra. The mass spectra were recorded using an Applied Biosystems Voyager-DE STR-MALDI-TOF spectrometer. The DLS measurements were made using a MalvernZetasizer 3000 HAS instrument at 25 °C at an angle of 90°. UV-visible absorption (UV-vis) spectra were measured with a Perkin-Elmer Lambda 19 UV-visible spectrometer. The infrared (IR) spectra were measured with an ATI Mattson Genesis series FT-IR spectrophotometer. The elemental analyses were performed by the Center of Microanalyses of the CNRS at Lyon Villeurbanne, France.

Synthesis of Dodecanethiolate-AuNPs (AuNP-1). Dodecanethiolate-AuNPs (AuNP-1) were synthesized through the typical Brust-Schiffrin method. 14 Briefly, an aqueous solution of hydrogen tetrachloroaurate (15 mL, 106 mg, 0.27 mmol) was mixed with the toluene solution of tetraoctylammoniumbromide (20 mL, 600 mg, 1.09 mmol). The two-phase mixture was vigorously stirred until the hydrogen tetrachloroaurate was totally transferred into the organic layer, followed by the addition of dodecanethiol (28 mg, 0.14 mmol), then the mixture was further stirred for 20 min. A freshly prepared solution of sodium borohydride (11 mL, 131 mg, 4.8 mmol) was quickly added with vigorous stirring, and the mixture immediately became dark. After further stirring for 2 h, the organic phase was separated and dried over sodium sulfate, then concentrated to 5 mL under vacuum. The crude product was precipitated in 150 mL of ethanol and then washed again with ethanol, yield: 90%. 1 Ligand-Substitution Synthesis of Azido-Terminated AuNPs-2a, AuNPs-2b, and AuNPs-2c. AuNPs-1 (80 mg) and azidoundecanethiol (50 mg) were mixed in CH 2 Cl 2 solution, and the solution was stirred for 2 days under N 2 at room temperature. After removal of solvent by rotary evaporator, the obtained AuNPs-2b were then washed with ethanol to remove the excess thiols (yield: 95%). 1 "Click" Synthesis of Carborane-AuNPs, AuNP-3a. Azidoterminated AuNPs-2a (50 mg, 0.022 mmol azido-ligand) and 1-Me-9-benzyl-o-carborane containing an ethynyl substituent of the benzene ring 3 (carborane alkyne) (12 mg, 0.044 mmol) were dissolved in 20 mL of toluene; then the solution was degassed and flushed with N 2 . The "click" catalyst [Cu(I)(hexabenzyl)tren)]Br (Cu I -tren) (0.1 equiv, 0.4 mg) was added, and the solution was allowed stir 2 days at 30 °C under N 2 . After removal of toluene under vacuum, AuNPs-3a were washed with ethanol and diethyl ether to remove the excess carborane alkyne and the catalyst, respectively (yield: 40%). 1 "Click" Synthesis of Both Carborane and PEG350 Functionalized AuNP-3b. AuNPs-2b (40 mg, 0.012 mmol azido-ligand) and the carborane alkyne (0.006 mmol, 0.5 equiv per N 3 branch) were dissolved in 15 mL of toluene, then the solution was flushed with N 2 . Cu I -tren (0.001 mmol, 0.1 equiv) was added, the solution was then stirred at 30 °C under N 2 . The reaction was finished in 1 day as monitored by FT-IR, which was indicated by the disappearance of the typical σ CC band at 2120 cm -1 . Then the propargylated PEG monomethyl ether (M w = 388 g/mol, 0.006 mmol, 0.5 equiv.) was injected and the second "click" reaction was carried out in situ. The second reaction was finished in 24 h. After removal of toluene under vacuum, AuNPs-3b was washed with methanol and diethyl ether to remove the excess carborane, PEG, and the catalyst (yield 65%). 1 ). UV-vis: SPB at 530 nm. IR: disappearance of both the σ CC band at 2121 cm -1 , and the ν N3 band at 2090 cm -1 . TEM: average core diameter = 5.18 nm. DLS: average diameter = 15.4 nm. "Click" Synthesis of Both Carborane and PEG2000 Functionalized AuNP-3c. AuNPs-2c (40 mg, 0.029 mmol azido-ligand) and the carborane alkyne (0.0058 mmol, 0.2 equiv. per N 3 branch) were dissolved in 15 mL of toluene, then the solution was degassed and flushed with N 2 . After addition of Cu I -tren (0.0029 mmol, 0.1 equiv.), the solution was stirred at 30 °C under N 2 . The reaction was finished in 1 day as the typical σ CC band at 2100 cm -1 disappeared. Then a toluene solution (2 mL) of propargylated PEG monomethyl ether (PEG2000) (M w 2038 g/mol, 0.023 mmol, 0.8 equiv.) was injected, and the second "click" reaction was carried out in situ. The second reaction was confirmed to be finished in 36 h, as monitored by IR. After removal of toluene in vacuum, AuNPs-3c were obtained after washing with methanol and diethyl ether to remove the excess carborane, PEG, and catalyst (yield 60%). 1 H NMR (CDCl 3 , 300 MHz):

δ ppm = 7.88 (1H, CH in trizaole), 7.86 (1H, CH in triazole), 7.55-7.46 (4H, CH Ar ), 4.67 (2H, -OCH 2 CH 2 -triazole), 3.64 (176H, -CH 2 CH 2 O-), 3.51 (2H, CH 2 -triazole), 3.41 (2H, CH 2 -carborane), 3.36 (3H, -OCH 3 ), 2.62 (2H, -CH 2 SH), 2.16 (3H, carborane-CH 3 ), 1.24 (22H, -CH 2 CH 2 CH 2 -), 0.88 (3H, -CH 2 CH 3 )
. UV-vis: SPB at 524 nm. IR: σ B-H stretching vibration at 2580 cm -1 ; disappearance of both the σ alkyne band at 2120 cm -1 , and the ν N3 band at 2090 cm -1 . TEM: average core diameter = 4.75 nm. DLS: average diameter = 10.2 nm.

Synthesis of Carborane-Azide 2. Carborane alkyne (0.8 mmol, 216.8 mg) and 1,4-bis-(azidomethyl)benzene (8 mmol, 1.5 g) were dissolved in 15 mL of tetrahydrofuran (THF). CuSO 4 5H 2 O (8 mmol, 200 mg) aqueous solution was then added, followed by the dropwise addition of a freshly prepared aqueous solution of sodium ascorbate (16 mmol, 316 mg) to obtain a 1: 1 THF/water ratio. The solution was stirred overnight at room temperature under N 2 atmosphere. After removal of THF under vacuum, CH 2 Cl 2 was added to dissolve the product, and the organic solution was washed 3 three times with water to remove the catalyst. After drying with anhydrous Na 2 SO 4 , the solvent was removed under vacuum. Purification by silica gel chromatography (petroleum ether/CH 2 Cl 2 1: 1) provided the pure product as white crystals (yield 92%). 1 Synthesis of the Tris-Alkyne Thioactetate Dendron 1. A dimethylformamide (DMF) solution of 5-(bromomethyl)-3,4,5-tris-(prop-2-yn-1-yloxy)benzene 17 (3 mmol, 996 mg) and potassium thiolacetate (4 mmol, 343 mg) was stirred at room temperature for 20 h under N 2 atmosphere. Then water was added to quench the reaction, and the product was extracted with CH 2 Cl 2 . The organic phase was washed 3 times with water, and then dried over anhydrous Na 2 SO 4 . The solvent was removed by rotary evaporation (yield 95%). 1 Synthesis of the Tris-Carborane Thioacetate Dendron 3. The dendron 1 (0.32 mmol, 106 mg) and carborane-azide 2 (0.96 mmol, 444 mg) were dissolved in 10 mL of THF. CuSO 4 5H 2 O (0.96 mmol, 240 mg) in aqueous solution was added, followed by the dropwise addition of a freshly prepared aqueous solution of sodium ascorbate (1.92 mmol, 380 mg) to obtain a 1: 1 THF/water ratio. The solution was stirred overnight at room temperature under N 2 . After removal of THF under vacuum, CH 2 Cl 2 and 5 mL of a concentrated (30%) aqueous ammonia solution were added. The mixture was stirred during 30 min to remove the Cu ions trapped inside the dendron as [Cu(NH 3 ) 2 (H 2 O) 2 ] 2+ , and the organic layer was washed with water. After drying with anhydrous Na 2 SO 4 , the solvent was removed under vacuum (yield 92%). 1 Synthesis of the Hybrid Dendron 4. The dendron 1 (0.65 mmol, 212 mg) and carborane-azide 2 (0.65 mmol, 300 mg) were dissolved in 30 mL of THF, and CuSO 4 5H 2 O (0.65 mmol, 162 mg) in 2 mL aqueous solution was added, followed by the dropwise addition of a freshly prepared aqueous solution of sodium ascorbate (1.3 mmol, 257 mg) to obtain a 1:1 THF/water ratio. This solution was stirred overnight under N 2 at room temperature. After removal of THF under vacuum, 20 mL of CH 2 Cl 2 and 5 mL of a concentrated (30%) aqueous ammonia solution were added. The mixture was stirred 30 min to remove the Cu ions trapped inside the dendron as [Cu(NH 3 ) 2 (H 2 O) 2 ] 2+ . The organic layer was isolated and washed with water. After drying with anhydrous Na 2 SO 4 , the solvent was removed under vacuum. The monocarborane dendron was obtained as a mixture of carborane in the m-branch (major) or in the p-branch. (yield 87%). 1 band at 1107 cm -1 , and σ CC band at 2121 cm -1 . Subsequently, the monocarborane dendron (0.24 mmol, 190 mg) and PEG-alkyne monomethylether (0.48 mmol, 975 mg) were dissolved in 10 mL of THF, an aqueous solution of CuSO 4 5H 2 O (0.48 mmol, 120 mg, 5 mL) was then added. After degassing and refilling with N 2 , the aqueous solution of sodium ascorbate (0.96 mmol, 190 mg, 5 mL) was dropwise added into the reaction solution. After stirring overnight under N 2 at room temporature, the solvent was evaporated with a rotary evaporator, and 30 mL of CH 2 Cl 2 and 10 mL of a concentrated (30%) aqueous ammonia solution were added. The mixture was stirred until the organic layer became colorless to remove the Cu ions trapped inside the dendron. The organic layer was then isolated and washed with water. After drying with anhydrous Na 2 SO 4 , the solvent was removed under vacuum. 1 The growing field of gold nanoparticles (AuNPs) now occupies a central place in nanoscience and nanotechnology because of their key applications in catalysis, 1 materials science, 2 optical biosensors, 3 and nanomedical diagnostics and therapeutics (theranostics). 4 A wide range of AuNP stabilizers including molecular and solid-state materials are known. Among them, citrate and cetyltrimethylammonium bromide (CTAB) offer remarkable key intermediate structures for further stabilization by thiolates. 5 Some undesired toxicity of ionic ligands sometimes appears, however, 3a,b and the search of additional useful ligands, in particular neutral ones, is called for. We wish to report here the first AuNP stabilization attempts using a simple ''clicked'' 1,2,3-triazole ligand, indicating that it can provide a good biocompatible platform towards various applications in water including sensing, catalysis and thiolate coverage. We have selected a PEG tail, because it brings the solubility in aqueous media and biocompatibility, but ''click'' chemistry 6 also further offers unlimited possibilities for terminal groups of such AuNP frameworks. We explore here the aspects of these AuNP assemblies including characterization by 1 The excess ligand and salts are removed from the solution of 1 (Au/L = 2) by dialysis. The plasmon band is slightly red-shifted from 535 to 538 nm (Fig. S4, ESI †), and the core size (TEM) largely increases from 6.0 to 17.5 nm upon increasing the Au/L ratio (Table S1, ESI † Fig. 1). The shape of the AuNP core in 1 shown by HRTEM in Fig. 1c is icosahedral. Dynamic light scattering provides a size of 12.6 AE 3 nm in solution for 1. The Energy-Dispersive X-ray Spectroscopy (EDS) of 1 (Fig. 1d) confirms the presence of Au and the elements (i.e. C, N and O) contained in the triazole-mPEG ligands. Determination of the number of Au atoms and capped ligands per AuNP using the TEM, 1 H NMR and element analysis data (from EDX) 7 shows that the N/Au ratio is 0.03 and that the 6 nm-cored AuNP 1 contains approximately 8850 Au atoms and 1140 triazole-mPEG ligands.

Thiols are transformed to thiolates upon coordination to the AuNP surface in classic thiolate-AuNPs as recently proven by experiment data, 8 but triazoles are less acidic than thiols. Raman spectroscopy shows here that the triazole is not transformed to triazolate upon coordination to the AuNPs 1. Compared to the free triazole-mPEG (Fig. 1e,bottom 9 These two enhancements reflect the SERS effect provoked by the coordination of the triazole N atoms onto the surface Au atoms of the AuNPs. However, the clear enhancement of the N-H wagging at 559 cm À1 shows that the H atom of the triazole ligand is not lost near the Au surface, which is opposite to what happens with thiolate AuNP ligands. This key difference explains why the stabilization of AuNPs is weaker with triazole ligands than with thiolate ligands, and this distinction is at the origin of the specific properties and applications of 1 in sensing, catalysis, ligand substitution and multiple functionalizations. 10 Reduction of 4-nitrophenol is an example to test the catalytic efficacy of the triazole-PEG-capped AuNPs (eqn (2)).

(2) 4-Aminophenol is very useful for a wealth of applications that include analgesic and antipyretic drugs, photographic developer, corrosion inhibitor, anticorrosion lubricant, etc. 11 The reduction of 4-nitrophenol to 4-aminophenol using aqueous NaBH 4 is thermodynamically favorable, but the presence of the kinetic barrier due to the large structural transformation along the redox reaction between donor and acceptor molecules inhibits this reaction. The AuNPs catalyze this reaction because of the favorable reactivity at the surface Au atoms of the AuNP cores that are only weakly engaged with the neutral triazole ligands. An aqueous solution (2.5 mL) containing 4-nitrophenol (0.37 mmol) and NaBH 4 (0.03 mmol) was mixed in a 3 mL standard quartz cuvette. This solution immediately changed from yellow to yellow-green (absorbance at 400 nm in Fig. 2a), which indicated the formation of 4-nitrophenolate ions. Then AuNPs containing 0.5% Au catalyst were added, and the reaction was monitored by UV-vis spectroscopy. The intensity of the absorbance peak at about 400 nm rapidly decreased after addition of the AuNPs, which indicated the consumption of 4-nitrophenol, while the appearance and increase of the absorbance peak at 300 nm showed the formation of 4-aminophenol.

The reaction was nearly complete within 280 s at 25 1C. The plasmon band at about 530 nm confirms the stability of the triazole-mPEG-capped AuNPs during this reaction. The kinetic constant of this reaction follows kt = Àln(c/c 0 ), and Fig. 2b shows the relation of Àln(c/c 0 ) vs. reaction time for the reduction of 4-nitrophenol over triazole-mPEG-capped AuNPs, providing k = 5.2 Â 10 À3 s À1 . Further, this reaction was carried out with various amounts of the AuNP catalyst 1, and the k values are gathered in Table S1 (ESI †). Compared with recent reports, 11 the triazole-mPEG-capped AuNPs 1 shows among the very best catalytic activities for this reaction.

The AuNPs are also an excellent optical sensor for the selective detection of Hg 2+ . The plasmon band shift of AuNPs in the presence of various metal ions including Mg 2+ , Ca 2+ , Ag 2+ , K + , Fe 2+ , Cu 2+ , Na + , and Hg 2+ was investigated and no shift was observed. Enhancement of the absorbance with Fe 3+ and Cu 2+ is due to their own colors. A clear plasmon-band red shift of 12 nm (Fig. S11, ESI †) was only observed by adding Hg 2+ (Fig. 3).

It is known that the triazole ring strongly and selectively coordinates to Hg 2+ , compared with other ions. 12 The weak Au-triazole linkage opens the possibility for this Hg 2+ ion to displace triazole from the AuNP surface and to form a Au-Hg 2+ bond or a triazole-bridging heterobimetallic interaction with both Au and Hg 2+ . The selective red shift of the AuNP plasmon band can thus be taken into account by this coordination change at the AuNP surface of 1. During the Hg 2+ titration, the AuNP solution remains homogeneous, i.e. no AuNP aggregation occurs.

Finally, one of the key properties of the AuNPs 1 is their function as a precursor for a variety of functionalizations, 13 as easily as with citrate ions that undergo, however, some transformation upon coordination. The weak AuNP-triazole bonds open the route to facile ligand-substitution reactions, whereas the well-known substitution of strongly bonded thiolate ligands is tedious. 6c For instance, complete triazole ligand substitution by functional thiols in methanol and water is fast and facile without any aggregation or core size change for Au : L ratio = 0.5 and 2. The triazole-mPEG ligands were substituted with a mixture of 11-mercaptoundecanoic acid and thiol-mPEG, with a predetermined ratio of 1 : 1 that remained the same on the Au surface (Scheme 1 Fig. S14,ESI †). Another example deals with the more difficult problem of the introduction of a polyfunctional dendron that possesses a single thiol group at the focal point, and a sterically bulky nonaferrocenyl thiol dendron with 1 -3 connectivity has been chosen. 14 The exchange reaction between 1 and 3 is conducted in the presence of dodecanethiol (Scheme 1), yielding the stable dendrimer-shape AuNPs 4 that contains 40% of dendronic thiolates, as determined by 1 H NMR (Fig. S19,ESI †), which corresponds to approximately 1000 ferrocenyl groups around the 6 nm AuNP core (size from DLS: 16.3 AE 3 nm).

The TEM data show that this core size remains almost unchanged (slight increase) upon ligand substitution in 2 and 4 vs. 1. The DLS data provide sizes of 16.3 AE 3 nm and 13.1 AE 3 nm for 2 and 4 respectively. The cyclic voltammogram of the AuNP-cored dendrimer 4 was recorded on a Pt electrode in CH 2 Cl 2 with decamethylferrocene (FeCp* 2 ) as the internal reference. 15,16 It shows a chemically and electrochemically reversible anodic oxidation to polyferricinium at E 1/2 = 0.610 V vs. [FeCp* 2 ] +/0 with strong cathodic adsorption due to the large size, also characterized by a Epa À Epc value of 40 mV, lower than the 59 mV expected at 25 1C in the absence of adsorption (Fig. S22, ESI †). 16 In conclusion, a simple ''1,2,3-triazole'' ligand resulting from the extensively used ''click'' chemistry stabilizes AuNPs with various core sizes, specific properties and possible applications. Given the broad scope of ''click'' chemistry, many variations should be open for multiple usages.

Financial support from the China Scholarship Council (CSC) (PhD grants to P. Zhao and N. Li), the Universities Bordeaux 1 and Toulouse III, and the CNRS is gratefully acknowledged.

Notes and references

Introduction

Gold nanoparticles (AuNPs) have emerged as a key field of nanoscience due to their quantum-related and supramolecular properties, [1] with promising applications in catalysis, [2] sensing, [3] and nanomedicine, in particular. [4] A variety of ligands have been shown to stabilize AuNPs since the citrate method of synthesis reported in 1952 by Turkevich et al. that allowed the formation of water-soluble AuNPs in the range 15-50 nm, which are still currently used in biomedical applications. [5] Sulfur ligands are popular stabilizers, in particular thiolate ligands in the Brust-Schiffrin method, which provide small monodisperse AuNPs (2-6 nm). [6] Cetyltrimethylammonium bromide (CTAB), [7] a remarkable Au-nanorod stabilizer, has restrictions in biological applications due to the biotoxicity of this free surfactant. [8] Besides, many other nitrogen donors, such as imidazoles, [9] pyridines, [10] and others, have also been introduced for the stabilization of AuNPs, with the advantage of a modest AuNP-ligand bond strength that allows the AuNP surface to be reached by ready ligand displacement. Such an introduction of substrates onto the AuNP surface upon weak ligand displacement leads to possibilities in optical, sensing, and catalytic applications. [11] For AuNP stabilizers, the use of a large class of biocompatible neutral and water-solubilizing ligands that are heterobifunctional is now considered to be a new, general, and environmentally friendly strategy that can produce a variety of side products and overcome the limitations indicated above.

The Cu I -catalyzed azide-alkyne cycloaddition (CuAAC) reaction ("click" reaction) is one of the most well-known methods of linking functionalities, [12] and this reaction forms 1,4-heterobifunctional 1,2,3-triazoles, which are very useful ligands. The 1,2,3-triazole ring is an amphoteric p-electron-rich aromatic, which is completely biocompatible and stable toward both oxidizing and reducing agents. So far, click chemistry has been one of the numerous methods to functionalize thiolate ligands of nanoparticles. [13] Herein, we show that the clicked 1,2,3-triazoles can be used as excellent neutral ligands to stabilize AuNPs with the following great advantages: 1) these ligands are neutral and mild and involve a weaker bond with the AuNP cores than the thiolate ligands, a property that will be shown to be crucial in sensing and catalysis; 2) the great potential of click synthesis allows the introduction of a large variety of functional groups; 3) the heterobifunctionality of the 1,2,3-triazole ligands synthesized by click reactions allows the introduction of two functional groups at a time, which makes them superior to, for instance, thiolate and amine ligands; 4) water-soluble AuNPs can be synthesized in this way by choosing the biocompatible polyethylene glycol (PEG) group as one of the triazole substituents, whereas the other substituent will lead to a specific property, function, and application in water. The 1,2,3-triazole ring can associate with AuNP surfaces through the lone pair of electrons on the sp 2 -hybridized nitro-gen atoms. [14] In addition, dendrimers that incorporate triazole rings have been shown to have an excellent ability to encapsulate and stabilize noble-metal NPs. [15] These new 1,2,3-triazole-AuNPs synthesized in this study with a biologically benign PEG chain (M w = 2000) and various useful functional groups are anticipated to potentially undergo various applications based upon their electrochemical, optical, fluorescent, supramolecular, encapsulating, and catalytic properties. For the purpose of utilizing the properties of triazole-AuNPs, including the properties of the ligand and AuNP core, each compound contains two groups on each triazole unit, that is, a solubilizing PEG chain and a functional fragment.

Results and Discussion

Design of bifunctional triazole-stabilized AuNPs

The functional fragments of 1-5 include polymeric and dendronic groups with various functionalities (Figure 1). A coumarin fragment, which was expected to have excellent photoluminescence properties, was introduced in 6.

Redox-robust organometallic compounds that contain a ferrocenyl group, tris-ferrocenyl dendron, or the "electron-reservoir" complex [CpFe(h 6 -C 6 Me 6 )][PF 6 ] (Cp = cyclopentadienyl) complex [16] were introduced to combine electrochemical and optical properties in triazole-AuNPs in 7-9, respectively (Figure 1). These iron sandwich complexes are redox catalysts that have been used as glucose biosensors in the case of ferrocene [16b,c] and as redox catalysts for the reduction of nitrate and nitrite moieties in water in the case of the mixed sandwich complex. The presence of a large number of these species in water-soluble NPs should further facilitate their use as redox catalysts and redox sensors. In addition, 10 has been assembled with a b-cyclodextrin (b-CD) unit, that is, a rigid cylindrical amphiphilic molecule, and has been extensively used to include hydrophobic molecules, such as anticancer drugs, to increase their solubility in water. Hence, AuNP-10 is a promising candidate for drug-delivery vehicles. The synthesis of the disubstituted triazole-stabilized AuNPs was conducted by using tetrachloroauric acid and a 1,2,3-triazole dissolved in deionized water in a standard molar ratio 1:2 of Au III /triazole (AuNP-6 = 1:0.5, AuNP-2 = 1:5) and followed by the addition of sodium borohydride as the reductant.

Characterization of the AuNPs

The triazole-AuNPs were characterized by using UV/Vis spectroscopy, TEM, and dynamic light scattering (DLS). Particular properties of AuNP-5, -6, -7, and -10 with special functional groups were displayed separately from the corresponding measurements. The TEM images depict the core size of the AuNPs to be in the range 3.0-11.2 nm.

Variations in the core size relies on the ratio between the Au III center and the triazole in the preparation, and secondary determinants of this variation are the rate of reduction, which result from the addition of the reductant, and the slight influence of the distinctive functional fragments in each triazole compound. For instance, AuNP-6 (d = 11.2 nm) is much larger than other AuNPs because it was prepared with a ratio of Au III / triazole ligand of 1:0.5, thus leading to an assembly of the Au atoms that proceeds much faster than the AuNP stabilization by the triazole ligands until a certain point. In addition, a trace of anisotropic nanocrystals was produced during this synthesis (Figure 2). With a diameter over 10 nm, AuNPs-6 should show prominent efficiency in fluorescence quenching assays. In contrast, AuNPs-2 was prepared with a low Au III /triazole ratio (i.e., 1:5) to obtain smaller AuNPs that could reveal excellent catalytic properties. The AuNPs that were prepared with the ratio of Au III /triazole of 1:2 displayed similar diameters (AuNPs-1 = 6.1, AuNPs-4 = 6.0, AuNPs-5 = 5.9, AuNPs-8 = 5.2, AuNPs-9 = 5.7, AuNPs-10 = 5.2 nm), except for AuNPs-3 and AuNPs-7 (4.0 and 3.8 nm, respectively). The location of the surface plasmon band (SPB) in the UV/Vis spectra (see the Supporting Information for details) of various AuNPs corresponds well to the coresize distribution of these AuNPs.

The behavior of AuNP-1-AuNP-10 in water were revealed through DLS analysis. The diameter obtained for each AuNP is subjected to deviations according to the nature of the functional groups (see the Supporting Information). AuNP-1 agglomerates in a large cluster, which includes many individual AuNPs due to extensive aggregating supramolecular interactions among the PEG tails of both triazole substituents (d = 396 nm) in aqueous solution. The PEG chains dominate the behavior of the AuNPs in solution because of their long flexible chain and interweave with one another among several adjacent AuNPs. On the other hand, AuNP-2-AuNP-6 that contain organic fragments on one triazole substituent only give mobility diameters in the range 140-250 nm because intermolecular PEG-chain interweaving proceeds only on one triazole side. Finally, AuNP-7-AuNP-9 that bear a ferrocenyl unit and related groups are well dispersed in water and show a mobility diameter of lower than 30 nm (see Figures S48,S50, and S51 in the Supporting Information). Consequently, it is concluded that the ferrocenyl group and its derivatives inhibit the gathering of individual AuNPs.

The electrochemical properties of AuNP-7 were recognized upon recording cyclic voltammograms (see Figure S50 in the Supporting Information). Both the electrochemical and optical properties of AuNP-7 have proven to be valuable for the design of sensory devices that are devoted to the selective recognition of extensively studied oxo anions (especially HSO 4 À , H 2 PO 4 À , and ATP 2À ions). [17] The surface-enhanced Raman scattering (SERS) effect of triazole-AuNPs was investigated by comparison between the Raman spectra of 5 and AuNP-5 (Figure 3). This comparison shows a distinct enhancement at l = 539 (NÀN wagging) and 1100 cm À1 (CÀN, C=C, and N=N stretching), both of these signals correspond to the triazole ring. [14b,c] In the Raman spectrum of AuNP-5, these two signals of the triazole ring were so This phenomenon shows that the triazole CÀN and N=N bonds are much closer to the AuNPs surface than any other part of the triazole group. Therefore, this finding indicates that the triazole neutral ligands stabilize the AuNPs by coordination of their nitrogen atoms to the AuNP surface.

These AuNPs are thermally stable at 100 8C in aqueous solution, which is indicated by the absence of plasmon-band variation after several hours at 100 8C (see Figure S46 in the Supporting Information), but these AuNPs agglomerate in the condensed phase, which inhibits thermogravimetric studies. Their stability to variations in pH values in aqueous solution was further studied by using UV/Vis spectroscopic analysis to record the SPB band of AuNP-5 at 25 8C (see Figure S46 After stabilization of the AuNPs with 6 (see the preparation details in the Experimental Section), the fluorescent emission is dramatically quenched. The fluorescence quenching phenomenon is due to the short distance between the coumarin unit and the AuNPs surface, thus indicating that AuNP-6 has potential applications in biosensing or metal-ion detection by using this fluorescence "turn-on" method upon the substitution of fluorescent ligands.

AuNP-6 was employed in the fluorescent sensing of 2-chloroethyl methyl sulfide (2-CEMS), which is a widely employed analogue of bis(2-chloroethyl) sulfide (2-CEES), a mortiferous chemical weapon that is commonly known as "mustard gas" and was infamous in World War II. For security, the detection of 2-CEES has always been used instead of its less toxic analogues. [9b, 18] The sensing capability of AuNP-6 was verified through a ligand-displacement process through progressive titration of a solution of 2-CEMS in methanol into a solution of AuNPs-6 in water/methanol 7:3 in a standard quartz cuvette (path length: 1 cm), which were mixed by inversion for 10 seconds (see the Experimental Section). The obtained solution was recorded by means of fluorescence emission spectroscopy immediately or afterwards (Figure 4 b). As expected, the fluo- rescence band of AuNP-6 raises with an increase of the 2-CEMS concentration. Indeed, a strong increase in band intensity was observed when 2-CEMS was added in a 10-fold concentration, and the ligand displacement reached equilibrium within 10 minutes of the titration.

As a comparison, the titration of 1-dodecanethiol was carried out by following the same procedure, and a similar performance was observed as monitored by emission spectroscopic analysis (see Figure S48 in the Supporting Information). Substitution of the triazole ligands by the sulfide or thiol analytes on the AuNP surface results in the release of fluorescence, which had been quenched by the AuNPs surface-energy-transfer effect. Facile bonding of the Au-triazole moiety to 2-CEMS means that the AuNPs can detect the chemical-warfare-agent sulfur mustard by using a fluorescence "turn-on" assay.

Catalytic performance of AuNPs-2 in the reduction of 4-nitrophenol

The reduction of 4-nitrophenol is widely used for the evaluation of the catalytic activity of various metal NPs owing to the high sensitivity of this reaction to the metal catalyst and the convenient determination of the reaction rate by UV/Vis spectroscopic analysis. [19] The triazole-stabilized AuNP-2 with a core size of d = 3.0 nm (that is, smaller than other triazole-AuNPs) were probed as catalysts for the reduction of nitrophenol insofar as the AuNP surface was expected to be readily available for the activation of adsorbed substrates subsequent to the substitution of weakly coordinated triazole ligands by the substrates. Briefly, a solution of 4-nitrophenol (0.09 mmol) and sodium borohydride (7.5 mmol) in water was prepared in a standard quartz cuvette (3 mL). This solution immediately turned yellow and showed a strong absorption at l = 400 nm in the UV/Vis spectrum. An aqueous solution of AuNP-2 (0.5 %) was injected into the quartz cuvette, and the reduction reaction was monitored by UV/Vis spectroscopic analysis (Figure 5).

The absorption of 4-nitrophenol at l = 400 nm was shown to rapidly decrease with the reaction time (Figure 5 a). The plot of the consumption rate of 4-nitrophenol Àln (C/C 0 ) versus the reaction times provided the rate constant k = 7.0 10 À3 s À1 (at 22 8C).

Comparatively, AuNP-2 is much more active in the catalytic reduction of 4-nitrophenol by sodium borohydride than AuNPs capped with monosubstituted triazole-PEG, which is attributed to the small size of AuNP-2 and possibly also to the ready displacement of disubstituted triazole ligands by the substrates. All in all, the triazole-stabilized AuNPs reveal a remarkable catalytic activity in the reduction of 4-nitrophenol, which is largely due to the flexibility of the mild AuNP-triazole bonding.

Encapsulation of the hydrophobic compound 1-adamantanol with AuNP-10

The encapsulation properties of AuNP-10 containing b-CD were investigated by titration of AuNP-10 into 1-adamantanol (AD) in solution (Figure 6). This result is in accord with reports of this well-documented host-guest inclusion phenomena. [20] Moreover, no SPB shift was observed in the UV/Vis spectrum before and after the association (see Figure S52 in the Supporting Information). This study suggests that AuNP-10 could potentially be used as a promising drug carrier in cancer therapy without the need to chemically modify a drug because most commercially available drugs are hydrophobic compounds. The delivery potential of such drugs by b-CD and other carriers is well known, and the combination of this species in a AuNP cargo has been highlighted for combined diagnosis and therapy. [21]

Conclusion

Nanogold chemistry has benefitted from the widespread use of "click" chemistry, which not only stabilizes AuNPs, but also provides a remarkably mild coverage that permits uses and applications that are more difficult to conduct with thiolate-type anionic ligands. The series of disubstituted 1,2,3-triazoles syn- thesized in this study by using straightforward CuAAC "click" reactions contain a PEG chain, which contributes to the water solubility and biocompatibility of the triazole-stabilized AuNPs, and a polymer, dendron, alcohol, carboxylic acid, tris-allyl dendron, fluorescent dye, ferrocene complex, or b-CD moiety. The range of AuNP sizes obtained (i.e., 3.0-11.2 nm) leads to a sizedependent selective choice of applications. The SERS properties of the triazole-AuNPs have been illustrated by comparing the Raman spectrum of AuNP-5 and 5, thus providing evidence that the triazole ring was very close to AuNPs surface, that is to say, coordinated. The stability of these triazole-AuNPs under the physiological pH range 4-9 suggests that they are qualified to be used in further studies in the cellular environment, in particular because of the PEG tail, which is not only biocompatible but also provides enhanced permeability and a retention (EPR) effect. The fluorescence-emission decrease of AuNP-6 relative to ligand 6 alone as a result of the quenching effect of AuNPs successfully designated the AuNPs-6 as a fluorescent sensor of the sulfur-mustard analogue 2-CEMS. In catalysis, the facile removal of the triazole ligands by the substrate from the small AuNP-2 (3.0 nm core) is responsible for faster catalysis in the reduction of 4-nitrophenol (k = 7.0 10 À3 s À1 , when 0.5 % AuNPs is employed) than with thiolate AuNPs from which thiolate-ligand removal is more difficult. Finally, the excellent encapsulation ability of the trapping of hydrophobic molecules of AuNP-10 has a potential application in biomedicine.

In summary, this new general strategy that involves the use of common click chemistry to stabilize PEG-tailed bifunctional AuNPs with clicked 1,2,3-triazole ligands opens promising applications toward sensing, supramolecular encapsulation, synthesis of highly functionalized thiolate-AuNPs, and catalysis of various types of reactions.

Experimental Section

CuAAC click synthesis of triazoles 1-10 After drying with anhydrous Na 2 SO 4 , the solvent was removed in vacuo. Compounds 1-9 were obtained as white powders in high yields (> 95 %) after reprecipitation. Compound 10 was synthesized by using the click reaction between the alkyne (1 g, 0.5 mmol) and azide (580 mg, 0.5 mmol) moeities in water/DMSO (1:1), with CuI (11.6 mg, 0.06 mmol) as the catalyst. The catalyst was subsequently removed by filtration.

Synthesis of 1,2,3-triazole-mPEG-capped AuNPs 1-10

General procedure for the synthesis of the AuNPs 1-6, 9, and 10: Tetrachloroaurate acid (8.5 mg, 0.025 mmol) and the triazole ligand (0.05 mmol; 0.125 and 0.0125 mmol for AuNPs-2 and AuNPs-6, respectively) were dissolved in deionized water (10 mL), and the obtained solution was stirred for 10 min. Freshly prepared sodium borohydride (0.1 mmol) aqueous solution (1 mL) was added dropwise to the reaction mixture with vigorous stirring for 5 min. AuNPs-7 and -8 were prepared with a reverse addition to avoid reduction of the Au III center by the ferrocenyl groups. An aqueous solution of tetrachloroaurate acid (5 mL, 8.5 mg, 0.025 mmol) was added dropwise to aqueous solutions of the triazole derivative (6 mL, 0.05 mmol) and sodium borohydride (6 mL, 0.1 mmol). After further stirring for 30 min, the AuNPs were purified by dialysis for 24 h to remove the excess ligands and salts. The triazole-AuNPs were kept in aqueous solution at 22 8C Catalytic reduction of 4-nitrophenol with AuNPs-2

An aqueous solution (2.5 mL) containing 4-nitrophenol (0.09 mmol) and NaBH 4 (7.2 mmol) was prepared in a standard quartz cuvette (3 mL, path length = 1 cm). The AuNP catalyst (0.5 %, 0.45 10 À3 mmol) was injected into this solution, and the reaction progress was detected by UV/Vis spectroscopic analysis every 40 s at 22 8C.

Encapsulation of 1-adamantanol in AuNPs-10

Solutions of AD and AuNP-10 were prepared separately in D 2 O/ [D 6 ]DMSO (7:3, 5 mm in each case), followed by the dropwise titration of the AuNP-10 solution into the AD solution. After mixing by inversion, solutions of AD and b-CD in various molar ratios (1:0, 1:0.5, 1:1, and 1:1.5) were studied by 1 H NMR spectroscopic analysis.

advantage of triazole-AuNPs is the easy substitution of the neutral triazole ligands by thiols and other ligands giving rise to applications. Indeed this ligand substitution of triazole at the AuNP surface yields a stable ferrocenyl-terminated nanogold-cored dendrimer upon reaction with a ferrocenyl-terminated thiol dendron, substitution of coumarin-linked triazole with cysteine, homocysteine and glutathione provides remarkably efficient biothiol sensing, and a ferricenium-linked triazole-AuNP catalyst is effective for NaBH 4 reduction of 4-nitrophenol to 4-aminophenol. In this catalytic exemple, the additional electrostatic AuNP stabilization modulates the reaction rate and induction time.

KEYWORDS: triazole, gold nanoparticle, ferrocene, ferricenium, coumarin, biological thiol, sensing, catalysis, nitrophenol.

INTRODUCTION

In the past few decades, a variety of ligands [1][2][3][4][5][6][7][8][9] have been synthesized and used to stabilize gold nanoparticles (AuNPs) 9 either in organic solvents or in water. The surface properties, including ligand type, binding force of Au with other atoms, as well as the ligand coverage of AuNPs control the solubility, stability and applications of AuNPs. For example, in thiolate-stabilized AuNPs, covalent bonding between the gold and sulfur atoms contributes to the passivation of the surface of thiolatestabilized AuNPs and makes AuNPs stable in solid state, 1 whereas citrate stabilized AuNPs were usually prepared in aqueous solution due to the multi-package of ionic species on the AuNPs surface. 2 In addition, the influence of the surface properties of AuNPs was also observed in several examples on the coordination-induced stabilization of AuNPs with nitrogen donors, particularly dendritic supramolecules. 9 Indeed PAMAM dendrimers show significant template effects in the formation of NPs in various solvents. 3 Copper-catalyzed azide alkyne (CuAAC) "click" chemistry has generated supermolecules 4 such as PEG-terminated dendrimers and polymers 5 also stabilizing AuNPs in aqueous solution. 6 Besides, other nitrogen ligands such as imidazoles, 7 pyridines 8 have also been utilized for the stabilization of AuNPs. 9 The dual property of triazole-AuNPs (trz-AuNPs) that on one hand are stable and on the other hand have modest AuNP-N bond strength allowing facile ligand substitution of trzs by other ligands for various applications makes this family of AuNPs particularly attractive. Indeed, upon trz ligand substitution, it is possible to synthesize various other liganded AuNPs, to use the ligand-exchange processes for sensing and to provide catalytically efficient AuNP surfaces. 10 Stabilization and uses of watersoluble PEGylated triazole-AuNPs have recently been shown. 11 Here we focus on the stabilization of trz-AuNPs linked to ferrocene, 12,13 ferricenium and coumarin termini using these monofunctional triazole-AuNPs that are not synthesized in water and are soluble in organic solvents.

For applications, ferrocenes have attracted attention due to the sensing and biomedical applications of the ferrocenyl (Fc) derivatives related to their reversible redox properties. 14 Catalytic properties will also be shown to result from fast triazole removal by substrates at the AuNP surface. Indeed, noble metal nanoparticles, 15 in particular AuNPs, are excellent catalysts for the reduction of 4-nitrophenol (4-NP) that is toxic and inhibitory in nature, in order to produce 4-aminophenol (4-AP) that has properties and applications as corrosion inhibitor, dying agent, and in particular intermediate for the synthesis of paracetamol. 16 Finally, coumarin (Cou) is a wellknown fluorescent dye 17 that we are linking to triazole-AuNPs fort he investigation of fluorescent sensing properties based on AuNP ligand substitution by biologicallyrelevant thiols. Indeed the thiols, cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) play key roles in the biological systems. 18 Many diseases are relevant to the abnormal contents of Cys or Hcy in the human body. For instance, an abnormal level of cysteine may cause skin lesions and liver damage. 19 Furthermore, Hcy is a risk factor for Alzheimer's and cardiovascular diseases. 20 Excess Cys has been associated with neurotoxicity and many other diseases. 21 Accordingly, the development of chemo-sensors for biological thiol derivatives (biothiols) is of great importance as recently indicated. 22 Sensing biological thiols using the fluorescence "turn-on" method on the surface of AuNPs either results from the formation of new fluorescent species or the release of dye adsorbed on the AuNPs as recently reported. 23

RESULTS AND DISCUSSION

In order to easily incorporate the redox complex and other functionalities into AuNPs in organic solution, two kinds of small mono-substituted trz molecules, Fc-trz and Cou-trz, were synthesized taking into account their electrochemical or photochemical properties. Each functional unit was linked to the trz ring by an ether group that increased the flexibility of the trz ligand to make sure that trzs smoothly arrange on the surface of AuNPs.

Ferrocene-and ferricenium-trz-stabilized AuNPs.

Late transition-metal sandwich complexes are known for their stability in several oxidation states and ability to effect efficient stoichiometric and catalytic electrontransfer processes 24 for multiple applications. 25,26 They are also rather easy to functionalize on the ligands, 25 and thus the introduction of alkynyl or azido groups has been reported in view of further CuAAC "click" functionalizing chemistry. 27 The ferrocenyl-linked 1,2,3-triazole (Fc-trz, see Figure 1) was synthesized using the CuAAC "click" reaction between trimethylsilylazide and propargyloxymethylferrocene in a DMF/MeOH solution at 100 o C with CuI as catalyst (Experimental Section). Fc-trz was characterized by 1 H, 13 C NMR, IR spectroscopies, ESI mass spectrometry, elemental analysis and cyclic voltammetry (Supplementary Information). Cou-trz was synthesized according to the literature, 28 but its photochemical property and applications were not reported. A one-step process to functionalize AuNPs with Fc-trz was carried out in organic solution. As compared to the above-mentioned ligand-substitution method, Fc-trz was used as stabilizer instead of ferrocene thiolate ligands. 12a The preparation of AuNPs stabilized by Fc-trz was performed using two pathways. In the first method, Fc-trz and NaBH 4 were dissolved in ethanol, a HAuCl 4 ethanol solution was slowly injected into this solution under nitrogen atomosphere with stirring. After being further stirred for 2 hours, AuNP-1 was purified by dialysis against a large volume of ethanol. In this process, Au(III) was reduced by NaBH 4 while Fc-trz played the role of stabilizer.

No oxidation of Fc was found according to UV-vis. spectroscopy analysis of AuNP-1, which is well taken into account by the fact that NaBH 4 is a much stronger and faster reducing agent than ferrocene. Indeed, as shown in Figure 2a, two bands at 321 nm and 430 nm belong to the ferrocene unit.

On the other hand, during the preparation of AuNP-2, no external reductant was involved. Then Fc group does not only work as the capping agent in ist oxidized form but also as the reductant. In this reaction, the Fc group was oxidized to ferricenium chloride, Au III being reduced to Au 0 atoms that form AuNPs. These AuNP-2 are stabilized by the ferricenium chloride-trz ligand, stabilization resulting from the positive synergy between the electrostatic factor and the trz ligand coordination to the AuNP surface (Figure 1). The oxidation of ferrocene to ferricenium was sucessfully recorded by UV-vis. spectroscopy, the band at 637 nm in Figure 2b corresponding to the ferricenium unit. In Figure 2a

4-Nitrophenol reduction

AuNP-2 was selected, because it was the only AuNP in this series that was water soluble, which facilitated the catalytic experiments. The lability of the AuNP-trz and AuNP-Cl -connections on the surface of AuNP-2 was employed to catalyze 4-NP reduction in water/ethanol (95/5) in the presence of NaBH 4 29 with catalyst amounts of 0.5% or 1%. This reaction was carried out in a standard quartz cuvette (path lengh: 1 cm) and was monitored by UV-vis. spectroscopy in every 40 s (Supporting Information, Figure S11). The plots of the decrease rate of 4-NP [-ln(C/C 0 )] vs. the reaction times (Figure 4) were collected (C and C 0 being the 4-NP concentrations at times t resp. t = 0). The rate constant k of the catalytic reduction of 4-NP was found to be 1.1×10 -3 s -1 with 0.5% of AuNP-2 as catalyst, and the k value increased to 1.6×10 -3 s -1 with the increase of catalyst amount to 1%. Moreover, an induction period (200 s) was required, due to the AuNP surface organization of the substrates in the mixed solvent. 30 As compared to the catalysis of 4-NP reduction with bulky watersoluble PEG-triazole ligands which provided k = 5.2 ×10 -3 s -1 (cat. 0.5%), 13a the catalytic efficiency of AuNP-2 that was stabilized by the present triazole ligands was relatively lower (1.1×10 -3 s -1 ) although the core of AuNP-2 is slightly smaller than that of AuNPs with PEG-triazole ligands. However, AuNPs-2 remained superior for

Synthesis of a nanogold-cored Fc-terminated dendrimer

Another way to utilize the lability of triazole in monosubstituted trz-stabilized AuNPs for the synthesis of Fc-containing AuNPs was achieved by facile reaction of the trz-AuNPs with Fc-terminated dendrons containing a thiol focal point as potential AuNP thiolate ligand. Thus the nona-branched Fc-terminated thiol dendron 6 was synthesized and utilized to substitute the trz ligand from the surface of AuNPs. This method allows the formation of the very stable thiolate AuNPs containing a bulky Fcloaded surface. 13a The nona-ferrocene dendron 6 with 1→3 directionalites 31 was synthesized via

CuAAC "click" reaction of azidomethyl-Fc with a nona-alkyne dendron. 13a This dendron was easily grafted onto the surface of AuNPs by fast and quantitative trz ligand substitution to give AuNP-5 (Scheme 2). In order to limit the Fc bulk at the dendrimer periphery the dendron 6 was introduced together with linear dodecanethiol that were found in 70% of total of 540 ± 80 32 yield. 1

Catalytic reduction of 4-nitrophenol

A typical procedure follow: An aqueous solution (2.5 mL) containing NaBH 4 (7.2 μmol) and 4-nitrophenol (0.09 μmol) was prepared in 3 mL a standard quartz cuvette (path lengh: 1 cm). AuNP-2 (0.5%, 0.45 × 10 -3 μmol; 1%, 0.9× 10 -3 μmol) in

EtOH was injected into the cuvette. The reaction progress was detected by UV-vis. spectroscopy every 40 s.

Sensing of homocysteine, cysteine and glutathione.

A gradually increasing amount of homocysteine (40 mM, 0.5, 1, 2, 5, 10 equivalents) in water was added to a 2.5 mL solution of AuNP-3 (0.05 mM, water/DMSO 3:7) in a standard quartz cuvette (path length: 1 cm). The solution was monitored using a fluorometer after 10 min after mixing. The titration of cysteine and glutathione were conducted according to the same process.

Ligand-substitution reaction of AuNP-4 by a mixture of thiolate-nonaferrocenyl dendron 6 and dodecanethiol to synthesize AuNPs-5.

3 mL of THF solution of the thiol-nonaferrocenyl dendron 13a (150 mg, 0.25 mmol)

and dodecanethiol (10 mg, 0.05 mmol, 12 μL) was added into 3 mL (1mM) thiazole-mPEG stabilized AuNPs (AuNP-4) 13a and stirred for 10 min. Then 10 mL dichloromethane was added into the mixed solution, the organic phase was then separated and dried over Na 2 SO 4. After evaporating the solvent, AuNPs was washed with acetone and then ethanol followed by precipitation in dichloromethane/methanol.

■ INTRODUCTION

Dendrimers that are classic synthetic macromolecules with welldefined topological flower-like structures have been demonstrated to have an extraordinary ability to hold hydrophobic guests by noncovalent bonding including physical encapsulations, van der Waals forces, hydrogen-bond interactions, and hydrophobic interactions. 1 The dendrimer-metal nanoparticle (NP) host-guest composites are synthesized through a template approach in which metal ions are entrapped in the interior of dendrimers due to the functional groups and the steric embedding effects and then reduced chemically. These dendrimer-NP composites have been modified to exhibit sufficient solubility and stability, resulting in their potential applications inter alia in catalysis, 2 photophysics, 3 diagnosis, 4 and sensing. 5 Catalysis by dendrimer-encapsulated metal NPs was pioneered by Crooks' group 6 using polyamidoamine (PAMAM) dendrimers. 7 Subsequently, many reports have appeared on the stabilization of and catalysis by precious metal NPs (Pd, Pt, and Au) in various dendrimers. 8 In particular, attention has been focused on the dendrimer-stabilized AuNPs that have applications in both drug delivery and surface plasmon-based photothermal diagnosis and therapy, 9 whereas the catalytic properties of dendrimer-stabilized AuNPs were often investigated in the form of bimetal alloys or nanocomposites. 10 "Click" dendrimers terminating with triethylene glycol (TEG) branched tris-dendrons 11 have been used for the stabilization of AuNPs subsequent to coordination of their trz ligands. It was supposed that AuNPs are either encapsulated in the dendrimers or surrounded by several dendrimers, which depends on both the size of the AuNPs and the morphology of the dendrimers. 11 It was also reported that the TEG dendronterminated "click" dendrimer-stabilized PdNPs displayed impressive activity in the Suzuki-Miyaura reaction in aqueous medium. 12 Significantly, numerous studies in catalysis under various "green" conditions have shown that the dendrimer plays the role of the nanoreactor and nanofilter. 2 One of our general goals is to design water-soluble and biocompatible nanomaterials in order to apply them to sensing, catalysis, and biomedicine. 13 For this purpose, PEG-terminated dendrimers fulfill these requirements and moreover provide the possibility to benefit from the enhanced permeability and retention (EPR) effect upon accumulating in tumoral tissues. 14 The association of AuNPs 15 with dendrimers is of particular interest given their multiple applications in catalysis, 16 photonics, 17 and biomedicine. 18 The activation of aromatics upon temporary π complexation by late transition metal groups, 19 in particular in the [Fe(η 5 -C 5 H 5 )(arene)] + complexes, 20 provides a powerful means of functionalization and, in particular, for the synthesis of dendritic cores, dendrons, and dendrimers with 1 → 3 connectivity. 21 Thus, we have applied the η 5 -C 5 H 5 Fe + -induced polyallylation 20,22 of polymethylbenzene, in particular the nona-allylation of mesitylene giving 1 (Scheme 1), 22 for the construction of nona-PEG-terminated dendrimers. 23 Therefore, the nona-allylated core 1 obtained as shown in Scheme 1 was hydrosilylated with dimethylchloromethylsilane, and the product 2 was submitted to nucleophilic substitution of chloride by azide via reaction with NaN 3 to yield 3 (Scheme 2). 23 The known nona-azide 3 is a very practical starting material for a variety of copper-catalyzed alkyne azide cycloaddition (CuAAC) "click" reactions and was applied here in the investigation of such reactions in order to introduce PEG tethers together with triazole (trz) rings onto the dendrimers using propargyl-PEG derivatives. Herein we report these syntheses and characterizations of the 1,2,3-triazole dendrimers obtained in this way, the complexation of Au(III) to the intradendritic trz ligands upon reaction with HAuCl 4 , and further introduction of AuNPs of catalytic interest by reduction of Au III using NaBH 4 . The variation of parameters leading to these AuNP syntheses has been examined for further catalysis of p-nitrophenol reduction by these AuNPs that are only mildly stabilized by the trz ligands, as well as the influence of the AuNP core size on the catalytic reduction rate.

■ RESULTS AND DISCUSSION Synthesis of Dendrimers. As a versatile substrate, the known arene-cored, nonabranched dendrimer with azido termini 3 was synthesized according to Schemes 1 and 2. 11 Synthesis of PEG-branched nonadendrimers was then con- veniently carried out by a selective "click" reaction (Scheme 3) through grafting PEG alkyne onto an azido-terminated nonadendrimer. The classic Sharpless catalyst was used, and the "click" reaction was launched in THF/H 2 O solution. These reactions were completed within 48 h, this long reaction time being necessary because of the steric bulk of the PEG tethers and the modest reactivity of Cu(I) under these conditions. By employing PEG alkyne with different chain lengths (PEG550 and PEG2000), two corresponding PEG-branched dendrimers (abbreviated as DEND550 and DEND2000) were obtained in satisfactory yields and characterized by 1 H, 13 C, and 29 Si NMR, IR spectroscopy, elemental analysis, size exclusion chromatograph, and mass spectrometry for DEND550 (see details in the Supporting Information).

Preparation and Characterization of the Dendrimer-Stabilized AuNPs. AuNPs stabilized by DEND550 and DEND2000 were prepared in aqueous solution with sodium borohydride as reductant as shown in Scheme 4. The dendrimer and HAuCl 4 were dissolved together in deionized water and stirred for 30 min in order to ensure access of AuCl 4 to the trz ring that is wrapped by the PEG chains. A freshly prepared NaBH 4 aqueous solution was then added dropwise into the as-prepared solution. The color of the solution gradually turned deep red or purple as AuNPs formed. For the purpose of synthesizing AuNPs with various sizes, the molar ratio of Au(III)/trz was varied as 1:1, 5:1, 10:1, and 20:1.

Accordingly, the obtained AuNPs with DEND550 are named Au-DEND550-1, Au-DEND550-5, Au-DEND550-10, and Au-DEND550-20, respectively, whereas with DEND2000 they are named Au-DEND2000-1, Au-DEND2000-5, Au-DEND2000-10, and Au-DEND2000-20, respectively.

Complexation of Au(III) with triazole molecules has been investigated by Bortoluzzi and co-workers. 24 In order to understand the complexation process between Au(III) and the trz dendrimers, a titration process was carried out by adding various amounts of HAuCl 4 into an as-prepared D 2 O solution of dendrimer. After stirring for 20 min, the reaction was monitored by 1 H NMR spectroscopy. The ratio of trz/Au(III) was progressively set to 1:0, 1:1, and 1:2. 1 H NMR spectroscopy demonstrated that with both DEND2000 and DEND550 the complexation of Au(III) was complete after addition of 1 equiv of Au(III) per dendritic branch. As depicted in the 1 H NMR spectrum of DEND2000 in Figure 1, the chemical shift of the trz proton (H trz ) is 7.9 ppm in pure D 2 O solution. After addition of 1 equiv of Au(III) per dendritic branch, the H trz signal is entirely shifted to 8.37 ppm, indicating completion of the complexation. No further shift was observed after addition of another equivalent of Au(III) precursor. The same result was obtained upon complexation of Au(III) with DEND550 (Supporting Information, Figure S11). For comparison, a monomeric trz derivative containing PEG2000 and an alkyl chain was synthesized, and with this monomer incomplete complexation resulting from an equilibrium was observed with the use of 1 equiv of Au(III) (Supporting Information, Figure S12). This comparison shows the benefit of the dendrimer structure over a linear analogue, owing to the additional driving force provided by encapsulation of the Cl 3 Au-trz moieties.

UV-vis spectroscopy also revealed that during the trz complexation the absorbance intensity of Au(III) at 302 nm decreased with time in both Au(III)/DEND2000 and Au(III)/ DEND550 solutions. No further decrease of the absorbance was observed after 20 min, suggesting that the complexation reached the equivalence point (Supporting Information, Figure S13). Therefore, it was necessary to leave enough time for the complexation, because the steric effect of both the dendritic structure and the package of PEG chain surrounding the trz ring slowed the coordination process.

In the case of the preparation of AuNPs involving lower Au(III)/trz ratios, the Au atoms were coordinated by trz, leading to the formation of AuNPs of small size with narrow dispersity. It seems reasonable to infer that, given their small size and formation from Cl 3 Au-trz moieties localized inside the dendrimers, these small AuNPs were encapsulated inside the dendrimers that were much larger than these AuNP cores (Figure 2, left). On the contrary, the AuNPs that were prepared with a high Au(III)/trz ratio mostly resulted from the formation of Au atoms outside the dendrimers because of the excess of Au(III) precursors over the number of trz rings. In this case the interdendritic AuNP stabilization does not prevent the formation of large AuNPs, and such AuNPs are much too large to be encapsulated inside the dendrimers. Thus, the stabilization of these latter AuNPs is due to several surrounding dendrimers (Figure 2, right).

The morphology and the core size of AuNPs were revealed by TEM images, in which the core diameter of the AuNPs was shown to regularly increase as the Au(III)/trz ratio was raised (Table 1 and Supporting Information, Figure S17-20). It was also found that with a certain Au(III)/trz ratio AuNPs stabilized by several dendrimers were diverse in core size, which was attributable to the difference in package density of dendrimers that influenced the mobility of Au(III), NaBH 4 , and AuNPs, as well as the leaching of Au atoms and small Au clusters. Upon stoichiometric addition of Au(III) to the dendrimeric trz bearing long PEG-2000 tethers followed by reduction with NaBH 4 , the formed AuNPs have a diameter of 1.8 nm, with about 180 Au atoms, which corresponds to one AuNP formed from Au atoms coming from 20 dendritic macromolecules on average. On the other hand, with short PEG-550 tethers AuNPs are interdendritically formed and stabilized with a 3.2 nm AuNP core, i.e., contain about 1000 Au atoms provided by more than 100 dendrimers (Figure 3 and Table 1). From these experiments, it is concluded that the Au atoms and very small primary Au clusters show a great mobility before definitive stabilization. The filtering effect of the PEG tethers is also clearly demonstrated upon comparing the influence of two PEG tether lengths on the limitation of the AuNP size.

The UV-vis spectra of the AuNPs displayed changes in the surface plasmon band (SPB) along with the variety of sizes. For instance, the SPB of Au-DEND550-1 was observed at a maximum of 516 nm, while the SPB of Au-DEND550-20 was found at 538 nm (Supporting Information, Figure S14). The difference in SPB was in agreement with the variation of core diameter as revealed by TEM. The plasmon band of Au-DEND2000-1 was weak and unresolved in the UV-vis spectrum, indicating that Au-DEND2000-1 has a small core size (1.8 nm). These AuNPs were also narrowly dispersed in aqueous solution, as revealed by the DLS measurements (Supporting Information, Figures S15 andS16). This result suggests promising applications of click-PEG-dendrimer-stabilized AuNPs in aqueous media.

Catalytic Reduction of p-Nitrophenol. Nitrophenols are toxic and hazardous micropollutants, and their degradation is challenging for environmental purposes. On the other hand, paminophenol is a potential industrial intermediate in manufacturing various analgesic and antipyretic drugs, anticorrosion lubricants, and hair-drying agents; thus efficient PdNP catalysis of p-nitrophenol reduction is of great value. p-Nitrophenol reduction also is a versatile reaction that is useful for the evaluation of the catalytic activity of various metal NPs, owing to its high sensitivity to metal catalyst and the convenient determination of the reaction rate through UVvis spectroscopy. 26 It has been previously reported that AuNPs possessed prominent catalytic activity in p-nitrophenol reduction. 27 In this context the loose coordination of trz ligands to the AuNP core surface provides a very favorable situation for efficient catalysis. The influence of the morphology of dendrimers, the size of AuNPs, and the catalyst amount on the kinetics of p-nitrophenol reduction was investigated in this study. Thus, the relatively smaller AuNPs, Au-DEND550-1 (d = 3.2 nm) and Au-DEND550-5 (7.3 nm), as well as Au-DEND2000-1 (1.8 nm) and Au-DEND2000-5 (5.7 nm), were provided as catalysts for the p-nitrophenol reduction. A typical catalytic reaction was processed as follows: a p-nitrophenol (0.09 μmol) aqueous solution was mixed with sodium borohydride (7.2 μmol) in a 3 mL standard quartz cuvette, to form a total volume of 2.5 mL. This solution immediately turned yellow and showed an intense absorption at 400 nm in the UV-vis spectrum. Various molar percentages of AuNPs (0.5%, 1%, 2%, and 5%, separately) in aqueous solution were then injected into the above-mentioned quartz cuvette, and the reduction reaction was monitored by UV-vis spectroscopy every 40 s throughout the reduction process (see Supporting Information, Figures S21-24).

The plot of the consuming rate [-ln(C/C 0 )] of pnitrophenol versus the reaction times is presented in Figure 4, and the k values of the reduction under various conditions are summarized in Figure 5. The catalysis results obtained with Au-DEND550-1 (3.2 nm) and Au-DEND550-5 (7.3 nm) show that the k value increases upon raising the amount of catalyst, and the k value decreases upon raising the AuNP size (Figure 5). Likewise, the large dendrimer (DEND2000)-stabilized AuNPs, Au-DEND2000-1 (1.8 nm) and Au-DEND2000-5 (5.7 nm), exhibit a similar trend.

Upon comparing the catalysis results of four AuNPs (Figure 5), it obviously appears that AuNPs stabilized by the small dendrimer (DEND550) exhibit higher catalytic activity than AuNPs stabilized by the larger dendrimer (DEND2000). This is taken into account by the less bulky periphery of the small dendrimer (DEND550) that allows easier access of the substrates to the AuNP core surface and makes their reaction easier on this surface. On the other hand, the filtering effect of the large PEG tethers in the case of PEG2000 causes kinetic limitations. In other reported examples of AuNP-catalyzed pnitrophenol reduction, the structural transformation at the AuNP surface was found to be rate limiting, 27 whereas here the diffusion of the substrate through the ligand shell can control the kinetics when the PEG tethers are long enough. In particular, Au-DEND550-1 (3.2 nm) displays a remarkable catalytic efficiency (k = 5.1 × 10 -3 s -1 , with the catalyst amount 0.5%) in such a low concentration of p-nitrophenol. This efficiency is among the best ever observed for this reaction, which can be attributed to the very mild interaction between the trz nitrogen ligand and the AuNP core surface, causing a very easy ligand exchange between the trz ligand and the substrate (p-nitrophenol and NaBH 4 ).

Concluding Remarks. The facile "click" synthesis of nona-PEG-branched macromolecules offers a valuable straightforward application of the η 5 -C 5 H 5 Fe + -induced nona-allylation of mesitylene. Then a positive and productive dendritic effect was disclosed for the quantitative intradendritic trz complexation to Au(III) upon reaction of the click dendrimers with HAuCl 4 in water according to AuCl 4 -+ trz → AuCl 3 (trz) + Cl -, whereas under analogous conditions complexation of a nondendritic triazole analogue leads only to an equilibrium.

Another original dendritic effect is that involving the influence of the PEG tether length on the formation of the AuNPs, in particular on their size. The NaBH 4 reduction of triazole-coordinated Au(III) was shown to lead to relatively small AuNPs, contrary to the reduction of free HAuCl 4 (when the Au(III)/triazole ratio was larger than unity), which led to large AuNPs. The influence of the parameters involved in the AuNP formation leads to the conclusion that interdendritic mobility of Au atoms (or native Au clusters) occurs more readily than in the case of PdNPs, which is of interest for late transition metal nanoparticle engineering for catalysis. Finally these trz dendrimer-stabilized AuNPs are excellent p-nitrophenol reduction catalysts.

In summary, the stabilization of metal NPs by easily synthesized "click" arene-cored trz dendritic macromolecules produces nanoreactors for nanoparticle catalysis with great efficiency thanks to the combination of the dendrimersupported nanoreactor and the flexible Au-trz bonding. The control of the fate of native Au atoms and small Au clusters by the structural and reaction parameters including "forced" leaching from the dendrimers has led to an understanding of how to optimize the catalytic efficiencies.

■ EXPERIMENTAL SECTION

Chemicals and Characterization Tools. All solvents and chemicals were used as purchased. Milli-Q water (18.2 MΩ) was used in the preparation of AuNPs. NMR spectra were recorded at 25 °C with a Bruker 300 (300 MHz) spectrometer. All the chemical shifts are reported in parts per million (δ, ppm) with reference to Me 4 Si for the 1 H and 13 C NMR spectra. The infrared (IR) spectra were recorded on an ATI Mattson Genesis series FT-IR spectrophotometer. UV-vis absorption spectra were measured with a PerkinElmer Lambda 19 UV-vis spectrometer. Elemental analyses were recorded on a PAR 273 potentiostat under a nitrogen atmosphere. The DLS measurements were made using a Malvern Zetasizer 3000 HSA instrument at 25 °C at an angle of 90°. Size exclusion chromatography of dendrimers was performed using a JASCO HPLC pump type 880-PU, TOSOHAAS TSK gel columns (G4000, G3000, and G2000 with pore sizes of 20, 75, and 200 Å, respectively, connected in series), and a Varian (series RI-3) refractive index detector, with THF as the mobile phase and calibrated with polystyrene standards Synthesis of DEND550 and DEND2000. A general procedure was employed and is described below: the azido-terminated nonabranch dendrimer 3 11 (0.08 mmol, 121 mg) and propargyl PEG (0.72 mmol) were dissolved in 5 mL of THF. CuSO 4 •5H 2 O (0.72 mmol, 180 mg) in aqueous solution was then added. The obtained solution was deaerated and refilled with N 2 , followed by dropwise addition of a freshly prepared aqueous solution of sodium ascorbate (1.44 mmol, 285 mg) to obtain a 1:1 THF/water ratio. The solution was stirred for 48 h at room temperature under a N 2 atmosphere. After removal of THF in vacuum, 5 mL of CH 2 Cl 2 and 5 mL of concentrated (30%) aqueous ammonia solution were added. The mixture was stirred for 30 min to release the Cu ions trapped inside the polymer as [Cu(NH 3 ) 2 (H 2 O) 2 ] 2+ . Then the organic layer was collected and washed with brine. After drying with anhydrous Na 2 SO 4 , the solvent was removed under vacuum. DEND2000 was recovered and purified by reprecipitation in CH 2 Cl 2 and diethyl ether. The PEGylated dendrimers were obtained in 91% (DEND550) and 88% (DEND2000) yields, respectively.

Titration of HAuCl 4 into the D 2 O Solution of Dendrimers (or Monomer). Taking Au(III)/DEND2000 as an example, HAuCl 4 (0.01 mmol, 2 mg, 1 equiv per branch) was added into a D 2 O solution of DEND2000 (0.0011 mmol, 21 mg, in 1 mL of D 2 O). After stirring for 20 min, the 1 H NMR spectrum (300 MHz) was recorded. Then, another equivalent of HAuCl 4 was added into the abovementioned solution, this solution was further stirred for 20 min, and the 1 H NMR spectrum was recorded again. The same operation was followed in the titration of HAuCl 4 with DEND550 (or the trz monomer) in D 2 O solution.

Preparation of Dendrimer-Stabilized AuNPs. AuNPs stabilized by dendrimers DEND550 (respectively DEND2000) were prepared under various Au(III)/trz ratios (1:1, 5:1, 10:1, and 20:1, respectively). Typically, HAuCl 4 •3H 2 O (0.009 mmol, 3.5 mg) and DEND550 (0.001 mmol, 6.5 mg) were dissolved in 5 mL of Milli-Q water. After being stirred for 10 min, 1 mL of freshly prepared NaBH 4 (0.045 mmol, 1.7 mg) water solution was added dropwise into the solution with vigorous stirring. The Au-DEND550-1 solution that was obtained was further stirred for 30 min and was dialyzed against a large volume of water (2 × 4 h). Subsequently, Au-DEND550-5, Au-DEND550-10, Au-DEND550-20, Au-DEND2000-1, Au-DEND2000-5, Au-DEND2000-10, and Au-DEND2000-20 were prepared following the same procedure. These dendrimer-stabilized AuNPs were kept in aqueous solution and were diluted before characterization and use in catalytic reactions.

Catalytic Reduction of p-Nitrophenol with Dendrimer-Stabilized AuNPs. An aqueous solution (2.5 mL) containing 0.09 μmol of p-nitrophenol and 7.2 μmol of NaBH 4 was prepared in a 3 mL standard quartz cuvette (path length: 1 cm). Then AuNP (0.5%, 0.45 × 10 -3 μmol) catalyst was injected into the as-prepared solution, and the reaction progress was detected by UV-vis spectroscopy every 40 s. The same processes were carried out with an increasing catalyst amount, successively 1%, 2%, and 5%. Gold nanoparticles (AuNPs) have recently received considerable interest for a variety of applications owing to their unique physical and chemical properties. 1 In particular, their extensive use in catalysis 2 has followed the seminal discovery of lowtemperature CO oxidation by small AuNPs by Haruta. 3 Among the transition metal-catalyzed redox reactions, the reduction of nitroaromatics is one of the most crucial ones. 4 Indeed, 4-nitrophenol (4-NP) is anthropogenic, toxic and inhibitory in nature. Its reduction product, 4-aminophenol (4-AP), finds applications as a photographic developer of black and white films, a corrosion inhibitor, a dying agent, a precursor for the manufacture of analgesic and antipyretic drugs, and in particular, as an intermediate for the synthesis of paracetamol. 5 Noble metal nanoparticle catalysts are widely employed for the reduction of 4-NP to 4-AP, [6][7][8] and this reaction, with an excess amount of NaBH 4 , has often been used as a model reaction to examine the catalytic performance of metal NPs, 6,7 as first shown by Pal et al. 8 AuNP catalysts that have been examined so far are solidsupported AuNPs 7 or various thiolate-AuNPs. The reaction mechanism is still unknown, although Ballauff's group provided strong evidence for a process fitting the Langmuir-Hinshelwood (LH) model. This mechanism involves adsorption of both reactants on the surface of the catalyst for AuNPs or PdNPs that are immobilized on the surface of spherical polyelectrolyte brushes with an induction time caused by dynamic restructuring of the nanoparticle surface. 6c,7b,d,9 For other AuNPs, Pal's group also showed that the catalytic reaction took place at the AuNP surface. 10 Ghosh's group showed that the rate constant increased with a decrease in the size of AuNPs and was proportional to the total surface area of AuNPs, 9 as reported by Ballauff's group; 11 and Liu et al. reported that surface functional groups influenced the catalytic behavior. 12 Katz suggested a completely different mechanism in which the active site was a leached gold species that was present in exceedingly small concentrations. 13 Zhang et al. suggested that the borohydride salt transferred a hydride to the AgNPs in the case of TiO 2 -supported AgNPs. 14 Scott's group showed that in the presence of excess borohydride salts, thiolate-AuNPs that catalyze 4-NP reduction grew to larger sizes. 15 Here we show the dramatic influence of the stereoelectronic effects of the ligand on the reaction rate, and we emphasize the electron reservoir behavior of the gold nanoparticle catalysts. Therefore, we compare, under identical conditions, the rates of the homogeneous 4-NP reduction by excess NaBH 4 catalyzed in water by water-soluble AuNPs stabilized by citrate, polyethylene glycol (PEG) thiolate of different lengths, and mono, bifunctional, polymeric and dendritic 1,2,3-triazoles terminated with PEG 400 or 2000 (Fig. 1). The catalytic 4-NP reduction is easily monitored via UV-vis spectroscopy by the decrease of the strong adsorption of the 4-nitrophenolate anion at 400 nm, directly leading to the rate constant. 8 Isosbestic points in the spectra of the reacting mixtures demonstrate that no side reaction occurs. 6c The various stable AuNPs that are studied have sizes around 3 nm, but larger AuNPs have also been examined for comparison (Table 1). The reduction rate has been observed, as in many preceeding cases, to be pseudo-first order in the presence of a large molar excess of NaBH 4 (here 81 equiv. NaBH 4 per mol 4-NP). All the apparent kinetic constants are summarized in Table 1. In order to obtain data that are independent of the surface, the rate constant (k 1 = k app /S) was also estimated normalized to the surface (S) with the assumption of the LH mechanistic model 7b,16 (see Table 1). The results clearly show that the best stabilizers, thiolates, provide the slowest AuNP catalysts, followed by the citrate. Citrate-AuNPs are large, but the comparison between thiolate-AuNPs and citrate-AuNPs of the same size (diameter: 13.5 nm) shows that the citrate-AuNPs are slightly less slow catalysts than the thiolate-AuNPs. The similarity of results with these two types of ligands, however, reveals a similarity of bonding to the AuNP, i.e. the citrate-AuNP bond should reflect the coordination of citrate to the AuNP surface, as the thiolate-AuNP bond, in spite of the difference in electronegativity and polarizability between these two chalcogen atoms.

All the 1,2,3-triazole (trz)-stabilized AuNPs 17 that are examined here are much more efficient catalysts than the AuNPs that are stabilized by the formally anionic thiolate and citrate ligands. This reveals the considerable advantage, in terms of catalytic reaction rates, of neutral ligands such as triazoles that form only weak coordination bonds with the AuNP surface given the impossibility of back bonding due to high-lying nitrogen p* orbitals. This weak bonding of the trz ligands, compared to the stronger bonding of thiolate and citrate ligands, is responsible for their easy displacement from the AuNP surface by substrates. It is also striking that the induction time (t 0 ), which is usually directly connected to the surface rearrangement on the AuNP surface, 7b,d is found only with the thiolate-AuNPs and citrate-AuNPs.

With these ligands, they are rather long, and by contrast, under these conditions, no induction times are found for all the trz-AuNPs, confirming the very facile trz displacement by the substrates. Among the trz-AuNPs, the dendrimer-stabilized trz-AuNPs are the less efficient catalysts. The polymer-stabilized trz-AuNPs are more efficient than the related dendrimer-stabilized trzAuNPs, but less so than the non-dendritic mono-and disubstituted trz ligands. Thus, it appears that this order of catalytic efficiency of the trz-AuNPs is related to their steric effects, the largest steric bulk being provided by the dendrimer framework that is bulkier than that of the polymer, whereas the less bulky non-macromolecular trz ligands provide by far the most efficient AuNP catalysts. These two AuNPs catalyze reactions that are so fast, under the same conditions as the other liganded AuNP catalysts, that they are too fast to observe a measurable rate at 25 1C. It is possible to compare these two AuNP catalysts, however, at lower temperatures. Then the monosubstituted trz-AuNPs appear to be more efficient than the bulkier disubstituted trz-AuNPs, as expected.

These results confirm that ligand displacement by substrates on the AuNP surfaces is the dominant feature of the mechanism that involves restructuration of the surface. This is in accord with the mechanism proposed by Ballauff and others following the LH kinetic model, which in particular also discards a leaching mechanism. With the series of trz-AuNPs, it also appears that diffusion of substrates towards the trz-AuNPs shows the filtration effect of the dendrimer and polymer frameworks, because the order of reaction rates follows the order of steric bulk of the trz-containing frameworks.

These data also confirm that the AuNPs play the role of an inner-sphere redox catalyst, 18 because BH 4

À transfers a hydride to the AuNP surface, resulting in the formation of a covalent Au -H bond. 7d This means that the negative charge is transferred to the AuNP, as already suggested, 7d,19 charge delocalization being largely facilitated by the low-lying conduction band of the AuNPs. Indeed, addition of NaBH 4 to the trz-AuNPs leads to a color change corresponding to a blue shift of the surface plasmon band (SPB) that indicates the accumulation of several negative charges at the AuNP surface. Such a shift that has been already observed in particular for thiolate-AuNPs is shown here for trz-AuNPs (Fig. 2). This effect is accompanied by another effect, AuNP aggregation, i.e. AuNP size increase (Ostwald ripening), 20 upon NaBH 4 addition, that is characterized by a red shift of the SPB. 1. The ligands of 7 and 8 are respectively HS-PEG400 and HS-PEG2000. After observation of the blue shift upon NaBH 4 addition, this red shift appears upon addition of more NaBH 4 . Gold precipitation then occurs when the amount of added NaBH 4 becomes too high (Fig. 2). The accumulation of negative charges at the AuNP surface proceeds along with the Au-H bond formation until the resulting electrostatic effect becomes too important. Along this line, electrochemical experiments conducted by Quinn's group by differential pulse voltammetry using well-defined thiolate-Au 147 NPs showed a series of 15 electrochemically and chemically reversible single electron transfer steps with Coulomb blockades (only limited by the electrochemical window) leading to stable multiply charged AuNPs. 21 In the presence of nitrophenolate anions on the AuNP surface, the electrochemical peak spacing that corresponds to the quantized capacitance charging was found to be decreased compared to that obtained in its absence, which corresponds to a small decrease of the AuNP capacitance. 22 This shows that the AuNPs behave as ''electron reservoirs''. 23 This role is efficiently fulfilled by the AuNP redox catalysts for 4-NP reduction in the transformation of inner-sphere single electron transfers from borohydride ions to the surface into a multi-electron transfer. A multi-electron transfer is necessary for each 4-NP reduction to 4-AP. The citrate anions, as hydrides, form coordination bonds with the AuNP surface involving partial charge transfer from the ligand to the AuNP surface. Such a coordination with a tripod of dihapto carboxylates that are coordinated to Au( 111) is known, 24 although the degree of AuNP-O covalency and charge transfer has not been addressed.

In conclusion, the role of the stabilizing ligands in the AuNP catalyzed 4-NP reduction has been shown here to be crucial. It is involved both in the restructuration at the AuNP surface following the LH kinetic model with considerable variation of efficiency from ''anionic'' thiolate or citrate ligands to neutral trz ligands and the steric or filtering effect 25 of the substrate through the bulk of the trz ligand framework. The difficulty in exchanging the thiolate ligands does not inhibit Suzuki-Miyaura cross carbon-carbon coupling reactions with analogous PdNPs, 26a because a leaching mechanism is involved. 26b On the other hand, it considerably slows down AuNP-catalyzed 4-NP reduction in contrast to the situation involving easily exchanged trz ligands. 17 The data and apparent accumulation of several negative charges in the AuNPs that occurs while the Au-H bonds form upon NaBH 4 reaction emphasize the role of ''electron reservoirs'' of these redox catalysts. Well-known precedents are found for instance in the role of PtNPs as redox catalysts in water photosplitting. 27 These findings should significantly contribute to shed light on the surface mechanism and optimize the design of effective catalysts for this intensively searched ''green'' aqueous reaction.

Financial support from Gobierno Vasco (R. C., post-dotoral scholarship), Universidad del Paı ´s Vasco, Chinese Scholarship Council (N. L., PhD grant), Ministe `re de la Recherche et de la Technologie (C. D., PhD grant), the CNRS (S. G. delegation), and the University of Bordeaux is gratefully acknowledged.

Notes and references

Introduction

The last chapter of this thesis is a review article that focuses on the synthesis, properties and applications of anisotropic AuNPs. Over 420 references are involved in this bibliographical work and more than 150 of them are in last 5 years. The investigations of anisotropic shapes and morphologies of AuNPs in particular have increased during the last years. Such syntheses of anisotropic AuNPs have attracted interest because the structural, optical, electronic, magnetic, and catalytic properties are different from, and most often superior to, those of spherical AuNPs and have potential applications. The recent development of preparation and applications of anisotropic AuNPs in catalysis, sensing, theranostics and drug delivery as well as the toxicity of AuNPs in biological system was reviewed therein with emphasis on historical aspects as well as recent literature reports. The preparation of this review article was essentially organized under the direction of Prof. Didier Astruc and Dr Pengxiang Zhao.

Introduction

Although nanogold represents one of the most remarkable areas of modern nanoscience and nanotechnology, spherical gold nanoparticles (AuNPs) have been known for millenia. Illustratory examples from history are the Lycurgus cup [1] and the Faraday publication. [2] Modern milestones from the second half of the 20th century are the Turkevitch [3] and Schiffrin-Brust synthetic methods, [4] Schmids magic number of gold atoms in stable colloids, [5] and Harutas discovery of the efficient oxidation of CO by O 2 at low temperatures by small (< 5 nm) AuNPs. [6] The outstanding properties of AuNPs are related to their easy stabilization and handling through thiolate ligands (Giersig and Mulvaney) [7] and the quantum size effect that is involved when the de Broglie wavelength of the valence electrons is on the same order as the size of the particle itself. [8] The free mobile electrons are trapped in a quantum box (the particle) and show the collective oscillation frequency of the plasman resonance, thus giving rise to the well-known plasmon absorption near 530 nm for 5-20 nm spherical AuNPs that causes the deep-red color and provides a variety of optical applications. [9][10][11] The resulting single-electron transitions result in the observed Coulomb blockade and, for example, the generation of up to 15 high-resolution quantized double-layer chargings in hexanethiol-Au 38 particles. [12] This Coulomb blockade can lead to applications as transistors, switches, electrometers, oscillators, and sensors. Finally, the large surface/volume ratio of AuNPs results in the AuNPs having an extraordinary reactivity that provides a large variety of catalytic applications. [13][14][15][16] Scientists already noticed the existence of anisotropic AuNPs at the beginning of the 20th century. In his book that was published in 1909, Zsimondy noted that gold particles "are not necessarily spherical when their size is 40 nm (mm) or below". He also discovered different colored anisotropic gold particles. Zsimondy invented the ultramicroscope, which allowed him to visualize the shapes of gold particles, and he received the 1925 Nobel Prize for "his demonstration of the heterogeneous nature of colloidal solutions and for the methods he used". He observed that gold often crystallized in a leaflike form with six sides. [17] In a well-known seminal article in 1908, Mie theoretically described the absorption and scattering to explain the colors (surface plasmon band) of gold particles. Although his theory applied only to spherical AuNPs, Mie attributed the deviations in some cases to nonspherical gold particles. [8] In 1912 Gans extended Mies theory and showed that aspherical particles absorb at a longer wavelength than spherical particles of comparable size. [18] Some years later, Svedberg, who discovered that particles could be separated with an ultracentrifuge, described experiments in which X-ray scattering was used to determine sizeand shape-dependent properties of gold particles, [19] and he was awarded the 1926 Nobel Prize for "his work on disperse systems". Following Einsteins description of Brownian motion as a diffusion process and development of a viscosity relationship in 1905 for dilute solutions of spherical particles, [20] in 1922 Jefferey extended the hydrodynamic calcu-Anisotropic gold nanoparticles (AuNPs) have attracted the interest of scientists for over a century, but research in this field has considerably accelerated since 2000 with the synthesis of numerous 1D, 2D, and 3D shapes as well as hollow AuNP structures. The anisotropy of these nonspherical, hollow, and nanoshell AuNP structures is the source of the plasmon absorption in the visible region as well as in the nearinfrared (NIR) region. This NIR absorption is especially sensitive to the AuNP shape and medium and can be shifted towards the part of the NIR region in which living tissue shows minimum absorption. This has led to crucial applications in medical diagnostics and therapy ("theranostics"), especially with Au nanoshells, nanorods, hollow nanospheres, and nanocubes. In addition, Au nanowires (AuNWs) can be synthesized with longitudinal dimensions of several tens of micrometers and can serve as plasmon waveguides for sophisticated optical devices. The application of anisotropic AuNPs has rapidly spread to optical, biomedical, and catalytic areas. In this Review, a brief historical survey is given, followed by a summary of the synthetic modes, variety of shapes, applications, and toxicity issues of this fastgrowing class of nanomaterials.

lations of the viscosity increase to solutions containing ellipsoidal particles. [21] The investigations of anisotropic shapes and morphologies of AuNPs in particular have increased during the last decade, most often relying on the development of seedmediated synthetic methods. In this respect, AuNPs are probably the class of nanoparticles that has provided the largest variety of shapes and has been studied the most. Such syntheses of anisotropic AuNPs have attracted interest because the structural, optical, electronic, magnetic, and catalytic properties are different from, and most often superior to, those of spherical AuNPs and have potential applications. In particular, compared to spherical (nonhollow) AuNPs, the main attractive feature of most anisotropic and hollow AuNPs is probably the appearance of a plasmon band in the near-infrared (NIR) region, where absorption by tissues is low. Therefore, the "water window" between 800 nm and 1300 nm can be used for medicinal diagnostics and photothermal therapy ("theranostics"). [22][23][24][25] Other noteworthy properties of nonspherical AuNPs are the enhancement of the SERS effect [26] and the catalytic properties of small AuNPs (< 5 nm) on textured surfaces. [12][13][14][15][16][17][18][19][20][21] In this Review, we highlight the synthesis, properties, and applications of the various shapes of AuNPs, with an emphasis on nonspherical and hollow AuNPs.

Pioneering Syntheses of Anisotropic AuNPs

AuNPs with hexagonal (icosahedral) and pentagonal (decahedral) profiles were synthesized by vapor deposition methods in 1979 [27] and 1981. [28] In 1989, Wiesner and Wokaun reported the first example of rod-shaped AuNPs by adding Au seeds to solutions of HAuCl 4 . [29] The Au seeds were formed by reduction of HAuCl 4 with phosphorus (as in Faradays synthesis), [2] and then gold nanorods (AuNRs) were grown by reduction of Au III with H 2 O 2 . Modern concepts of seedmediated synthesis were established by the Murphy research group at the beginning of the 2000s. They reported the synthesis of AuNRs by the addition of citrate-capped AuNPs to an Au I growth solution generated by the reduction of Au III with ascorbic acid in the presence of cetyltrimethylammonium bromide (CTAB) and Ag + (Figure 1). [30] The seedgrowth method has become established as the most efficient and popular one to synthesize specific shapes in high yields. [30,31] Bulk-solution methods have been reported to produce nanocrystals with multiple shapes, but with a low yield of a specific shape. [32] The first physical synthetic method used to make AuNRs was the photochemical reduction of auric acid reported in 1995 by Esumi et al. [33] In this method, Au III ions bound to rodlike cationic micelle surfactants to form ion pairs and when excited by UV light are reduced to Au 0 . A two-step process was proposed, involving the aggregation of metal nuclei to form primary particles followed by aggregation of these particles to form rod-shaped particles resulting from the stabilization of a specific crystal face by surfactant micelles. [34] Another early method to form AuNRs was the electrochemical reduction method, which was developed by the Wang research group in the late 1990s. [35,36] It was later shown that CTAB was crucial in this method for the high-yielding formation of AuNRs. In 2001 the El-Sayed research group already noted that CTAB induced the formation of a bilayer structure on the longitudinal surface of AuNRs, with the trimethylammonium head groups of the primary layer facing the AuNR surface. [37]

Methods and Techniques for the Synthesis of Anisotropic AuNPs

A large variety of bottom-up methods and techniques involving templates or capping agents have been used during the last decade to synthesize many types of anisotropic or hollow AuNPs. However, the versatile and more-complex seed-mediated method has become dominant, especially for the synthesis of AuNRs. Two or several of these methods are often combined (in particular, a template method with another method) for the synthesis of anisotropic AuNPs. Supramolecular chemistry also provides a means of assembling AuNPs. Top-down routes (mostly lithography) are much rarer, but are sometimes combined with a bottom-up technique.

Simple Chemical Reduction of Au III

Cetyltrimethylammonium bromide (CTAB) is a frequently used surfactant for the structuration of nonspherical AuNPs, and the role of its purity and mechanism of action have been debated. [22][23][24] Forms of silver, either as AgNO 3 or Ag nanoplates, [38,39] are also often used because of the key role silver plays in anisotropic growth. [40] For example, the addition of ascorbic acid to a mixture of CTAB, silver plates, and HAuCl 4 reduced orange Au III to almost colorless Au I . The rapid addition of NaOH then induced the formation of anisotropic AuNPs, whose color changed from pale blue to dark red within one day. The TEM images showed the formation of various shapes, including spheres (40 %), tadpole-like monopods (25 %), L-shaped, I-shaped, and Vshaped bipods (23 %), T-shaped, Y-shaped, and regular triangular tripods (9 %), and cross-like tetrapods (3 %; fcc structures, no incorporation of silver; Figure 2). NaOH plays a role in the branching of the NPs, as the use of NaBH 4 instead only yields spherical and rod-shaped AuNPs. The function of the silver plates is only to improve the yield, while the presence of Ag + is detrimental. [40] This example clearly shows the use of structuring agents for the formation of anisotropic AuNPs, but also the need for sophistication to improve the yields. [41]

The Seed-Mediated Method

Compared with the in situ synthesis, the seed-growth method enlarges the particles step by step, so that it becomes easier to control the sizes and shapes of the AuNPs. Thus, this procedure is widely used in the most recent size-and shapecontrolled AuNPs syntheses.

The seed growth usually takes place over two steps. In the first step, small AuNP seeds are prepared. In the second step, the seeds are added to a "growth" solution containing HAuCl 4 and the stabilizing (capping) and reducing agents, and the newly reduced Au 0 then grows on the seed surface to form large AuNPs. The reducing agents used in the second step are always mild ones that reduce Au III to Au 0 only in the presence of Au seeds as catalysts (in the absence of the Au 0 catalyst, ascorbic acid only reduces Au III to Au I in acidic medium). Thus, the newly reduced Au 0 can only assemble on the surface of the Au seeds, and no nucleation of new particles occurs in solution. Moreover, as a consequence of the use of a mild reducing agent, the second step is much slower than the first one, and it can be repeated to continue the growth process.

In the course of the seed-growth synthesis of AuNPs, the size, shape, and surface properties are controlled by the amount and nature of the reducing agent and stabilizer, as well as their ratio to the Au precursor.

A two-step method was devised by Zsigmondy, who combined his technique with Faradays method, which had Gold Nanoparticles remained ignored for 40 years before Zsimondy took notice. Zsimondys two-phase method was published in his book in 1909, [42] and it is this two-step method that was much later renamed as the seed-growth method.

In the seminal procedure by the Murphy research group for the formation of AuNRs, HAuCl 4 is reduced to HAuCl 2 by ascorbic acid in the presence of CTAB and AgNO 3 , then citrate-capped spherical AuNPs are added to this HAuCl 2 solution. [30] These spherical AuNPs catalyze the reduction of Au I to Au 0 by ascorbic acid. A three-step procedure in the absence of AgNO 3 allows longer AuNRs with aspect ratios (length/width) of up to 25 to be prepared. The AuNRs formed in the first stage serve as seeds for a second growth, and the latter are in turn used as seeds for the third growth (Figure 3). [41][42][43][44][45] The yield and monodispersity are improved by addition of nitric acid to the third seeding growth solution. [46] Other modifications from the El-Sayed research group, which improved the yield and polydispersity of the AuNRs, were the replacement of sodium citrate by the stronger CTAB stabilizer during the formation of the seeds and the use of AgNO 3 to control the aspect ratio of the AuNRs (Figure 4). [47] In this approach, the seeds are formed upon reduction of HAuCl 4 with NaBH 4 at 0 8C in the presence of CTAB followed by the addition of AgNO 3 ; this seed solution is then added to HAuCl 2 previously formed in the presence of CTAB. The AuNRs are obtained in 99 % yield with aspect ratios between 1.5 and 4.5 by using this method. Higher aspect ratios up to 10 or 20 are obtained upon addition of a cosurfactant (benzylhexadecylammonium chloride (BDAC) [47] or Pluronic F-127 [48] ) to the original growth solution and changing the AgNO 3 concentration. Even higher aspect ratios of up to 70 have been obtained by a fourth addition of growth solution. [42] Optimization studies have clearly shown that many parameters play a role in influencing the yield, shape, and dispersity. [41][42][43][44][45][46][47][48][49][50][51][52][53][54][55] In particular, impurities in the various commercial CTAB sources greatly affect the yield, dispersity, and aspect ratio. Traces of iodide, in particular, have a significant affect, because it binds selectively to the Au(111) facet, thus leading to the AuNRs. [54,55] Two AuNR growth mechanisms have been proposed [45,56,57] and discussed [22,[56][57][58][59] (Scheme 1). They involve a growth that is governed either by preferential binding of the head group of a cationic surfactant to the {100} face of the AuNP seed (rather than the less-favored rod end) [45,56] or by the electric field of the double layer between the positively charged seed and negatively charged AuCl 2 À on the CTAB micelle (Scheme 1 a,b). [57] Studies of the role of Ag + have shown that single-crystalline CTAB-capped seeds lead to single-crystalline AuNRs with {110} faces on the side and {100} faces on the end, whereas multiply twinned crystalline citrate-capped seeds grow into multiply twinned structures. (The role of Ag + and the effect of an under deposition potential (UPD) are discussed in Section 3.8.) Ag deposition on the {110} side of the rod is faster than on the {100} ends, and consequently seeds grow into rods. [50,60] Murphy and coworkers have also proposed a hybrid mechanism involving diffusion of AuCl 2 À on the CTAB micelles to CTAB-capped seed spheres resulting from electric-field interactions. Subsequently, silver ions preferentially deposit onto the {110} facets, on which CTAB is preferentially bound, thereby resulting in particle growth into AuNRs along the [110] direction (Scheme 1 c). [60] The critical role of the bromide counterion of CTAB or NaBr in the formation of AuNRs has been illustrated by the observation that there is a critical [Br À ]/ [Au 3+ ] ratio around 200 that leads to the maximum aspect ratio of the AuNRs, beyond which Br À is a poison. [61]

Photochemistry

Photochemistry is also a convenient method to generate anisotropic AuNPs. UV light can reduce HAuCl 4 to form AuNRs when a cationic micelle with a rod shape is bound to HAuCl 4 . The photoreduction to Au 0 atoms is followed in this case by controlled aggregation. [33,34] The micelle surfactant . stabilizes a specific crystal face, as in the seed-growth procedure, thereby leading to AuNRs. [34,[62][63][64] The presence of NaCl increases the AuNR aspect ratio and yield. [34] As in the seed-growth process, Ag + also improves the aspect ratio and yield. [63] UV light with a wavelength of 300 nm is optimal for these properties. [64] The method largely benefits from the improvement observed in the seed-growth method, namely the use of sodium borate, AgNO 3 , ascorbic acid, and CTAB; the advantage here is that the AuNRs are obtained in high yield in a single step, [65,66] unlike in the seed-growth method itself. The amount of sodium borate [65] and the temperature [66] can alter the aspect ratio. The photochemical route has also been performed in the presence of poly-(vinylpyrrolidone) (PVP) and ethylene glycol, [67] or TiO 2 colloids. [68][69][70] For example, the Zhang research group synthesized platelet-like AuNRs with an asymmetric five-twinned structure as well as, in combination with seed growth, six-star AuNPs. TiO 2 serves both as a photocatalyst and a stabilizer of the AuNPs (Scheme 2). [70] Graphene oxide was also used to assemble Au nanodots to patterned chains. [71] Although the photochemical method requires a long reaction time on the order of 30 h, [62,63] this period can be reduced to 30 min if HAuCl 4 is first reduced by ascorbic acid, and then exposed to UV irradiation in the presence of AgNO 3 . [72][73][74] The use of ketyl radicals with a short triplet lifespan (generated from 4-(2hydroxyethyloxy)phenyl(2-hydroxy-2-propyl)ketone, known as Irgacure-2959 (I-2959)) results in an especially efficient reaction, and causes the rapid reduction of Au III to Au 0 and formation of AuNRs upon irradiation at 300 nm. [75][76][77]

Electrochemistry

The electrochemical method to produce AuNPs was first reported by Svedberg in 1921. [78] In the modern electrochemical technique developed by Wang and co-workers, an Au plate anode and a Pt plate cathode were immersed in an electrolyte containing CTAB and tetradodecylammonium bromide (TOAB) as a cosurfactant. Electrolytic oxidation of the Au anode then formed Au III Br 4

À bound to the CTAB micelle, which then underwent migration to the cathode and cathodic reduction to Au 0 . AuNRs were formed with aspect ratios that were controlled by the presence of Ag + cations produced by the redox reaction between Au III and an Ag plate. The latter was gradually inserted into the solution. The AuNRs were separated from the cathode by ultrasonification. [35,36] This method produces AuNRs with aspect ratios of 3-7 that are single crystals without stacking faults, twins, or dislocations, as shown by HRTEM and diffraction studies (Figure 5). [79] Gold nanorods have also been electrochemically deposited inside porous membranes. [80][81][82] Although the electrochemical method is one of the pioneering methods for the synthesis of AuNPs, including anisotropic AuNPs, it remains actively investigated because of its simplicity, efficiency, and applicability. [83,84]

Sonochemistry

Ultrasound has become a useful tool for the synthesis of very small nanoparticles, because the effects derived from acoustic cavitation drives chemical reactions under extreme conditions. This method, however, yields nanoparticles with multiple shapes and a wide size distribution. [85] This problem is circumvented by the use of surfactants, as in other methods, and added alcohols also serve to control the particle shape and size. [86,87] For example, in the presence of a-d-glucose, the major reactions in the sonolysis process of aqueous HAuCl 4 follow Equations ( 1)-( 4). [88,89]

H 2 O ! HC þ OHC ð1Þ
sugar ! pyrolysis radicals ð2Þ
Au III þ reducing radicals ! Au 0 ð3Þ
n Au 0 ! AuNP ð4Þ
Han and co-workers have synthesized single-crystalline flexible Au nanobelts with a width of 30-50 nm and a length of several micrometers with this method (Figure 6). [90] 3.6. Templates

The synthesis of nonspherical AuNPs is often driven by the use of templates that have a complementary morphology to the AuNPs. [24] The templates are solid substrates, such as porous membranes, [91,92] mesoporous silica, [93] Si(100) wafers, [91] pyrolytic graphite, [94] polymers [92,95] (including block copolymers), [96] nanoparticles, [97,92] carbon nanotubes, [98] inorganic clusters such as LiMo 3 Se 3 , [99] surfactants organized in micelles, [35,42,43,100] or Langmuir-Blodgett films, [101] as well as biomolecules such as plant extracts, [102,103] microorganisms, [104] polypeptides, and DNA. [105] The essential role of the surfactant for the growth of anisotropic AuNPs (AuNRs) was already detailed in Section 3.2. Mulvaney proposed the electric-field model, according to which the rods grow because of a higher rate of mass . transfer of gold cations to the tips as a result of the asymmetric double layer around the rod. The CTAB bilayer confers a positive charge onto the Au rods, and the CTAB also binds Au I ions (produced by reduction of Au III by ascorbic acid), which retards its transfer to the Au rods. The surface potential decays more rapidly at the tips because of their curvature, thereby explaining why the rod tips grow faster than the lateral facets. [106] Other models have been proposed [57,106,107] and discussed, [58] in particular in terms of steric and chemical factors to direct preferential interactions between the cationic head group of CTAB and the growth sites. [107] Templates are frequently also used in combination with photochemistry, electrochemistry, and sonochemistry for the synthesis of anisotropic AuNPs. The template structure directs anisotropic growth by selective adsorption on specific crystallographic sites of the metal. This has been used in combination with surfactants, for example, with poly(vinylpyrolidone) (PVP). PVP also serves as a reductant, for example, for the synthesis of Au plates. [108,109] The reducing ability of PVP is due to its hydroxy termini and is dramatically enhanced in the presence of water bound to the PVP. Thus, kinetic control of the AuNP growth depends on the solvent and nature of the reductant and yields various morphologies that can sometimes be made selectively. [110] One of the most successful nanomaterials for the synthesis of AuNPs is silica nanospheres, which serve as templating cores around which Au layers can be coated. This powerful strategy was reported by Halas and co-workers, and allows the size of the Au nanoshell and the thickness of the nanogold layer to be controlled at will by changing the reaction time and the concentration of the plating solution (Figure 7). [111][112][113][114]

Galvanic Replacement

Galvanic replacement is a very simple approach in which gold cations in the plating bath are spontaneously (i.e. in a thermodynamically and kinetically favorable reaction) reduced by a metal without external current sources. [115][116][117][118][119][120][121][122][123][124][125][126][127][128][129][130] The driving force is the difference in the redox potential between the reducing metal and the Au 3+ /Au system. This method was proposed and exploited by Crooks and coworkers to prepare AuNPs encapsulated in dendrimers by metal exchange between dendrimer-encapsulated CuNPs and Au III from HAuCl 4 . [115,122,129] Au@Pt dendrimer-encapsulated nanoparticles have also recently been fabricated in this way. [129] The driving force for exchange is the lower redox potential of Cu II /Cu 0 (E8 = 0.340 V versus NHE) compared to AuCl 4 À /Au 0 (E8 = 0.99 V versus NHE). The reducing Ag metal (E8 Ag/Ag+ = 0.80 V versus NHE; E8 Ag/AgCl = 0.22 V versus NHE) in the form of an Ag nanostructure has been used extensively as a sacrificial template, in particular by the Xia research group [116,118,121,123,124,125] to fabricate hollow Au nanostructures. Gold nanocages (AuNCs) have been synthesized in this way starting from Ag nanocubes and an aqueous HAuCl 4 solution. These reactions proceed in two steps: 1) formation of seamless hollow structures with the walls made of Au-Ag alloys by galvanic replacement of Ag and Au III by Ag-Au alloying and 2) formation of hollow structures with porous walls by dealloying. [123] Aluminum foil is also often used as the metal source, because it is inexpensive and has a very low oxidation potential [E8(Al 3+ /Al=À1.67 V versus SHE] and can thus reduce many transition-metal cations through an exergonic reaction [Eq. ( 5)]:

Al þ Au III ! Al III þ Au 0 ð5Þ
This reaction is marred, however, by the thin layer of alumina Al 2 O 3 on the aluminum metal surface. The use of the metal chloride (AuCl 3 or HAuCl 4 ) in a high concentration [131] together with prior removal of the aluminum oxide with NaOH [132] can circumvent this difficulty. In the presence of chloride anions, oxidized aluminum cations form soluble tetrachloroaluminate salts that do not prevent the deposition of AuNPs. Fluoride forms stronger bonds than chloride, and NaF and NH 4 F have also been used to dissolve the alumina layer to allow the galvanic displacement through Al III dissolution [Eq. ( 6)].

Al 2 O 3 þ 6 H þ þ 12 F À ! 2 AlF 6 3À þ 3 H 2 O ½133 ð6Þ
This galvanic technique does not require the presence of template, surfactant, or stabilizer and is conducted at room temperature. The use of Al foil, NaF, and HAuCl 4 for the galvanic replacement of Al by Au III results in Au dendrites. [133] 3.8. The Effect of Ag + Salts and Underpotential Deposition A crucial finding by Murphy and co-workers was the favorable role of AgNO 3 in the controlled formation of long AuNRs. [41] By varying the parameters and using the seedgrowth procedure it was possible to direct the aspect ratio, optimize the yield (up to 50 %), and obtain long AuNRs (Figure 8). [60,134,135] Nikoobahkt and El-Sayed further improved the yield of the AuNRs up to 99 % by using CTAB [31] instead of the citrate-capped seeds used by Murphy and co-workers. The seed-mediated growth was much slower in the presence of CTAB. However, along with AuNR formation, the longitudinal surface plasmon band (SPB) is red-shifted in the absence of AgNO 3 (increase in the aspect ratio, that is, AuNR growth at the tips), whereas in the Gold Nanoparticles presence of AgNO 3 it is blue-shifted after 2 minutes. Thus, the length and aspect ratio quickly increases during this short time, then slowly decreases. [135] AgNO 3 immediately forms AgBr in the presence of CTAB. It was proposed that Ag + is not reduced at acidic pH values, (typically pH 3) because the ascorbic acid reductant is too weak, as confirmed by the lack of the AgNP SPB at 400 nm (AgNPs only form at a basic pH value, typically pH 8). Consequently, Ag + forms ion pairs [AgBr] that decrease the charge density on the Br À ion and the repulsion between head groups on the AuNR surface, thus inducing CTAB elongation. [31] It was alternatively argued that the adsorption of [AgBr] on the Au nanocrystal facets slows down the reduction of the gold ion and induces the growth of single-crystalline AuNR. [56] An underpotential deposition of Ag onto the AuNR has been invoked to explain the dramatic role of AgNO 3 on the growth of the AuNRs. [50] Underpotential deposition is the deposition of a metal adlayer onto a metal surface (Au) below the Nerst reduction potential (up to 0.5 to 0.9 V) of the metal ions (AgNO 3 /Ag), as a result of strong bonding interactions between the two metals in the adsorbed layer. [10,139] It appears that Ag + is essentially not reduced, although CIP analysis has shown the presence of Ag at up to 4.5 %. [60] However, it is the Ag + adsorption on AuNR (in the form of AgBr) that considerably slows down the growth of AuNRs. This rate decrease is selective and less efficient on the tips because of poorer coverage, which results in the formation of AuNRs.

The role of Ag + is not only crucial for the formation of AuNRs, but also for other shapes of AuNPs, as recently shown by several research groups. High-index concave cubic Au nanocrystals with {720} surfaces have indeed been synthesized by Mirkin and co-workers by the reduction of HAuCl 4 with ascorbic acid in the presence of a chloride-containing surfactant and small amounts of AgNO 3 . [137] Ag + has also been used for the synthesis of {730}-faceted bipyramids [50,138] and high-index AuNRs. [31,[139][140][141][142] 3.9. Supramolecular Arrangements Supramolecular chemistry [143,144] is another general method for assembling spherical [16] or nonspherical AuNPs (in particular AuNRs [59] ) into new nonspherical AuNPs in the form of liquid crystals, supracrystals, or supported ordered arrays. [145][146][147][148] Various examples will be described in the following. Large aligned structures of AuNRs were obtained upon drying a drop of AuNR solution on a copper TEM grid in air with the grid immersed halfway. [149] AuNRs pack together upon drying a rod-sphere mixture on a silicon wafer in a water vapor atmosphere. [150] AuNRs linearly align in a head-to-tail fashion after centrifugation, but a 2D parallel assembly is obtained after three rounds of centrifugation. [151] Liquid-crystalline structures of aligned AuNRs spontaneously form in a side-on arrangement with high aspect ratios in concentrated solutions. [152] Pileni has examined the ordering of transition-metal nanoparticles over long distances in 3D superlattices called supracrystals. [145,[153][154][155][156][157] In particular, it has been shown that this ordering of alkanethiolate-AuNPs into supracrystals depends on the solvent [153] and on the addition of water, [154] with their final morphology determined through either a layer-by-layer growth or a process of nucleation in solution. [153] Gold supracrystals can also grow from suspensions of gold nanocrystals, with the building process taking place in solution and at the air-liquid interface. These growth processes determine the crystallinity and mechanical properties of the supracrystals. [157] Anisotropic AuNP arrangements can also be formed by using bridging thiolate ligands. Feldheim and co-workers used phenylacetylene oligomers to synthesize 2D and 3D crystalline arrangements of AuNPs (e.g. dimers and trimers). The oligomers play the role of "molecular wires" between the AuNPs. In this way, well-defined, rigid arrays with a variety of geometries could be produced. [158] Examples of end-to-end AuNR assemblies include the use of biotin disulfide (in combination with streptavidin; Figure 9), [159] thioalkyl carboxylic acids (hydrogen bonding), [160,161] alkanedithiols (thiolate coordination), [162] thiolated DNA (hybridization), [163] thiolated alkynes and azides (click reaction), [164] and polymers such as polystyrene. [165,166] Mann and co-workers showed that DNA hybridization could also be used to assemble AuNRs side-by-side by first substituting CTAB with thiolated oligonucleotides. [167] Other research groups also described such arrangements (Figure 10). [168][169][170][171] Another way to achieve the side-by-side assembly of AuNRs is by appropriate choice of the thiolate ligands (e.g. 1,2-dipalmitoyl-sn-glycero-3-phophothioethanol) [172] or mercaptopropylsilane [173] ) followed by solvent evaporation.

Polymer engineering (see also Section 3.6) [108-114, 165, 166, 174-179] is a well-developed strategy to assemble AuNPs. Besides the previously discussed use of PVP, other polymers such as poly(N-isopropylacrylamide) and its acrylic acid derivative, [174,175] poly(viny alcohol), [176][177][178] and poly(styrene-b-methylacrylate) allow fabrication of anisotropic arrangements of AuNPs. [179] Figure 8. Increasing the amount of silver ions leads to gold nanorods of higher aspect ratios, which is consistent with the red-shifting LSPR. From Ref. [59]. Copyright 2009 Wiley-VCH.

Top-Down Syntheses: Lithography Methods

The most common lithography technique used to produce AuNPs is electron-beam lithography (EBL). [180,181] First, a substrate is coated with an electron-sensitive resist. The electron beam dissociates this coated substrate into fragments, which are removed with a developing agent. The nanoscale structure remaining is suitable for the deposition of AuNPs. Another lithography technique is focused ion beam (FIB) lithography. [180][181][182] A gallium ion beam sputters parts of a film to form the desired nanostructure shape. These lithography techniques have produced AuNRs [180][181][182][183] and Au nanodisks, [184] although a drawback is that AuNPs smaller than 10 nm as well as larger particles are usually not accessible. Ebbesen and co-workers have combined ionbeam and electron-beam techniques [185][186][187] to synthesize arrays of Au microholes, [185] grooves, and nanowire circuits. [186] Colloidal masks commonly used in lithography [169] have led to the production of, for example, hexagonal arrays of Au triangles [189] and Au nanorings. [190] A modern lithography method, on-wire lithography (OWL), has been reported by the Mirkin research group. [191,192] OWL is a template-based electrochemical process for forming gapped cyclindrical structures on a solid support. The size of the gaps and segment length can be controlled on a length scale of under 100 nm (Figure 11). This method allows the composition and distances between adjacent particles to be tailored down to 2 nm. [193][194][195] 1D arrays of Au nanostructures as small as 35 nm in diameter, which allow control of the segment length and spacing down to about 6 and 1 nm, respectively, were synthesized in this way. [196] Such nanostructures can be combined with organic and biological molecules to create systems with emergent and highly functional properties. [194] 4. The Morphology of AuNPs 4.1. Platonic AuNPs [123,178,179] AuNPs with the morphology of the five platonic solidsthe tetrahedron (four triangles), [197] hexahedron (cube, six squares), [137,[198][199][200] octahedron (eight triangles), [201] dodecahedron (twelve pentagons), [202] and icosahedron (twenty triangles) [203][204][205][206][207][208][209][210] -and their aqueous synthesis is often based on seed growth, as shown by the Murphy research group. [211] They are characterized by low-index facets ({111} for tetrahedron, octahedron, dodecahedron, and icosahedron and {100} for the cube). Yang and co-workers used a modified polyol process with PVP to form AuNPs in high yield with distinct highly uniform tetrahedra, cube, octahedra, and icosahedra (dubbed platonic nanocrystals) with sizes of 100-300 nm. Solutions of HAuCl 4 and PVP in ethylene glycol were injected simultaneously in boiling ethylene glycol, which also served as the reductant. The shape was controlled by the PVP/ HAuCl 4 ratio (4.3:1 to 8.6:1), with the AuNP shapes being highly sensitive to the concentration of the HAuCl 4 . [197] Han and co-workers carried out the synthesis of rhombic dodecahedral AuNPs from an aqueous solution of HAuCl 4 without the use of any seeds, surfactants, or foreign metal ions but only with DMF added as both the co-solvent and reductant at 90-95 8C for 15 h. Purification was carried out by centrifugation and washing with ethanol. It was suggested that reaction temperatures lower than the boiling point of DMF (156 8C) results in slower, kinetically controlled reductions that are responsible for the observed shapes formed. [201] Rhombic dodecahedral AuNPs (together with gold bipyramids) were also obtained by Mirkin and co-workers [210] by an Ag-assisted, seed-mediated growth process (Figure 12). [204] Dodecahedral AuNPs were transformed into other nonplatonic shapes such as rhombic cuboctahedron and then truncated octahedron in the presence of PVP at a low water content. At high water content, the dodecahedral AuNPs were transformed into rhombic cuboctahedra, then to truncated cubes, then cuboctahedra, and truncated octahedra. [211] Song and co-workers have used the popular polyol process, specifically 1,5-pentanediol at reflux, to successfully and quantitatively produce a variety of shaped AuNPs, including cubes, in the range of about 100 nm by incremental change in the AgNO 3 concentration. Smaller cubes and octahedral were also produced by using large amounts of PVP. [199] 4.2. One-Dimensional AuNPs: Nanorods, [212][213][214][215][216][217][218][219][220][221] Nanowires, [222][223][224][225][226][227][228][229][230][231][232][233] Nanotubes, [234][235][236][237][238][239][240][241][242][243] and Nanobelts [244][245][246][247] Nanorods are the most extensively investigated onedimensional anisotropic AuNPs to date. In Section 3, the electrochemical [35] and seed-growth methods leading to the formation of AuNRs were detailed, in particular the seminal studies by the Murphy [45] and El-Sayed [100] research groups, as well as several other synthetic techniques. For example, the seed-mediated growth leads to either single-crystalline or pentahedrally twinned AuNR structures.

Various other remarkable one-dimensional nanogold structures have also been synthesized, including nanowires, nanobelts, and nanotubes. When the aspect ratio of AuNRs increases, these AuNRs are called Au nanowires, thus there is a continuum between these two categories of AuNPs. AuNRs can also be assembled in line (Figures 13 and14) [176][177][178]212] or into other hybrid nanostructures such as nanotubes, [213] in particular by using polymer films as hosts. [214] Various aspects of AuNR materials, including their optical properties and . application properties, have been detailed in reviews by the research groups of Murphy, [45,22,215,216] El-Sayed, [59,101] and Liz-Marzan and Mulvaney. [217] The main interest in AuNRs arises because of their tremendous utility and multifunctionality for the diagnosis and treatment of cancer. They are indeed used as drugdelivery vehicles and as contrast agents for near-IR photothermal tumor ablation (see Section 6). [218][219][220][221] Gold nanowires (AuNWs) are synthesized by the tipselective growth of AuNRs. [222] AuNRs stabilized by CTAB and pentahedrally twinned are, for example, used for such processess. [223,224] This method produces crystalline AuNWs that are 12-15 mm long with a diameter of (90 AE 10) nm. AuNWs can also be prepared by UV irradiation (> 350 nm), photoreduction, and thermal reduction (at 70 8C) of HAuCl 4 in the bulk phase of the block copolymer made from PEG 20 -PPE 70 -PEG 20 (PPO = poly(propylene oxide) and Pluronic P123. [225] AuNWs have potential applications as nanoscale optical waveguides in the visible and near-IR regions. [226][227][228] AuNWs are indeed an ideal platform to produce surface plasmon waves by direct illumination of one end of the nanostructure. They can thus be used as tools for fundamental studies of subwavelength plasmon-based optics of wave propagation. This strategy was pioneered by Halas and coworkers, who used Ag-and AuNWs with longitudinal dimensions of more than 10 mm. The addition of an adjacent nanowire, substrate, or other symmetry-breaking defect enables direct coupling with the guided waves in a nanowire. Networks of plasmonic AuNWs can serve as the basis for optical devices such as interferometric logic gates, which can lead to nanorouters and multiplexes, light modulators, and a complete set of Boolean logic functions. [228] Bimetallic AuPtNWs are useful electrochemical sensors for glucose with increased selectivity, sensibility, and repeatability compared to monometallic nanowires. [229] CuAuNWs have been intensively studied because of their widespread applications. [230] Interestingly, the method of fabrication of AuCuNWs can also lead to the formation of AuCu nanotubes (AuCuNTs). Therefore, CuNWs were used as templates, and the AuCuNWs are formed as intermediates that ultimately lead to the AuCuNTs (Scheme 3). [231] AuNRs and AuNWs have also served as seeds for the fabrication of higher-order AuNRs and AuNWs containing periodic starfruit-like morphologies by Vigderman and Zubarev. [232] Nanowires are required for applications such as ultrasensitive and multiplex DNA detection through SERS and other biomedical SERS-based techniques. [233] Both AuNWs and Au nanotubes (AuNTs) can be prepared by electroless deposition onto the pore walls of porous polymer membranes, as shown by the seminal study by Wirtz and Martin. [234] In such membranes, the pores act as a template for the nanostructuration. A commercially available example of a support membrane is a polycarbonate filter with cyclindrical nanopores. Long Au deposition times leads to the preparation of AuNWs, while short deposition times give AuNTs. The AuNWs and AuNTs synthesized in this way can be used as nanoelectrodes, molecular filters, and chemical switches. [234] AuNTs are also sensitive refractive index reporters. [235] Finally, AuNTs have also been designed for specific catalytic applications (see Section 6.1). 4-(Dimethylamino)pyridine is a powerful auxiliary reagent for this electroless deposition method (Figure 15). [236] Indeed, AuNTs are currently synthesized by the galvanic replacement reaction, [235,237] for example, by using an anodized aluminum oxide template [238,239] and a polymer NT as a sacrificial core to produce hollow AuNTs that have exquisite sensitivity to the refractive index. [235] Thiol-functionalized nanoporous films can also act as a scaffold for the development of such highly ordered Au arrays. [240] Conical AuNT/pores can be advantageous to avoid unwanted plugging and are ideally suited to detect proteintype bioanalytes. [241] Inorganic nanostructures that serve as templates for the formation of AuNTs include goethite (FeOOH) rods. [242] Finally, density functional theory can be of tremendous help when designing discrete AuNTs such as Au 32 units with a zigzag structure. [243] Among the 1D AuNPs, Au nanobelts (AuNBs; or Au nanoribbons) have attracted interest, because they may represent a good system for examining dimensionally confined transport phenomena and for fabricating functional nanodevices. Han and co-workers synthesized single-crystalline AuNBs by using a combination of ultrasound irradiation and a-d-glucose as a directing agent under ambient conditions (Figure 16). The sugar is pyrolized to provide radicals that reduce Au III to Au 0 . [90] Gemini surfactants have also been used as a template in an aqueous phase by using dimethylene bis(tetradecylammonium bromide) (14-2-14) as a capping agent and template. A two-step seed-growth method (to avoid secondary nucleation) was used, whereby the first seedgrowth step yielded AuNRs of high aspect ratio; then AuNBs that were several micrometers long and 5 nm thick were grown in the second step. [244] Au nanoarrays of single-crystalline AuNBs with longrange identical crystallographic orientation have been synthesized by directional solid-state transformation of an Fe-Au eutectoid followed by a precise electrochemical treatment (Figure 17). [245] Nanostructured reactive precursors have been employed as effective sacrificial templates for the controlled synthesis of 1D inorganic nanostructures with desired compositions. For example, porous AuNBs were fabricated from metal-surfactant complexes of precursor NBs formed by HAuCl 4 and a bolaform surfactant containing two quaternary ammonium head groups. [246] Plasmonic effects were studied in nanostructures for the transport of optical information, and tunable plasmon resonance was indeed disclosed in AuNBs with cross-sectional dimensions smaller than 100 nm and narrow transverse plasmon modes. [247] 4.3. Two-Dimensional AuNPs: Gold Nanoplates [248][249][250][251][252][253][254][255][256][257][258][259][260][261][262][263][264][265][266][267] The synthesis of gold nanoplates with specific shapes (stars, pentagons, squares/rectangles, dimpled nanoplates, [248] hexagons, and truncated triangles) of well-defined particle sizes have been extensively exploited for their specific properties and features. One of the simplest and versatile methods to produce gold nanoplates is the polymer template method, whereby the polymers have an extraordinary effect as stabilizers, templates, and reductants. In 2004, Lee and coworkers used bulk polymeric phases of PEO 20 PPO 70 PEO 20 (PEO = poly(ethylene oxide), PPO = poly(propylene oxide)) as both the reductant and stabilizer, with the large (10 mm in width and 100 nm in thickness) gold nanosheets being obtained by reduction of HAuCl 4 in THF. [225] In another example, Radhakrishnan and co-workers generated polygonal gold nanoplates in situ in a poly(vinyl alcohol) matrix through thermal treatment. [249] Such a procedure occurs in high yield, does not involve the need for a reducing agent, and has been widely utilized in the synthesis of large singlecrystalline gold nanoplates. [250,251] In some cases the polymers acted only as stabilizers, such as in the formation of unprecedented starlike gold nanoplates through the reduction of HAuCl 4 by l-ascorbic acid in the presence of poly(N-vinyl-2-pyrrolidone) as reported by Nakamoto and co-workers. [252] On the other hand, Hojo and co-workers indicated that the morphology of polyhedral nanoplates was controlled with HAuCl 4 and polyvinylpyrrolidone (PVP) through a polyol process when using ethylene glycol as both the solvent and reducing agent. [253] In addition to the polymer process previously discussed, a wet-chemical route has also been proposed and exploited in the last decade. It was demonstrated that the morphology could be finely adjusted through control of the reactant ratio and the reaction conditions. In 2004, Wang and co-workers also reported a mild large-scale synthesis of micrometer-scale Au nanoplates. In this process, HAuCl 4 is reduced by ortho- phenylenediamine in aqueous medium to form hexagonal single-crystalline Au nanoplates with preferential growth along the Au (111) plane. This result suggests that the molar ratio of ortho-phenylenediamine to gold is key to producing Au nanoplates. [254] Huang and co-workers carried out the synthesis of trianglular and hexagonal gold nanoplates in aqueous solution by thermal reduction of HAuCl 4 with trisodium citrate in the presence of CTAB surfactant in just 5-40 min. The size of the gold nanoplates was varied from as small as 10 nm in width to several hundreds of nanometers. A [CTAB]/[HAuCl 4 ] ratio of 6:1 in the reaction solution was favorable for the formation of gold nanoplates (Figure 18). [255] A shape transformation from triangular to hexagonal nanoplates was evident on selective etching of Au triangular nanoplates, growing of Au nanodisks, then ripening of hexagonal nanoplates, which corresponded to the change of the medium upon alternate addition of HAuCl 4 and ascorbic acid. [256] Dong and co-workers introduced a mild and relatively "green" one-pot biomimetic method for the fabrication of gold nanoplates. The reactions were carried out at 25 8C in aqueous solution containing HAuCl 4 and aspartate as the reductant for 12 h. The hexagonal and truncated triangular nanoplates with a thickness of less than 30 nm were obtained after evaporation of the solvent. [257] This use of a biological reductant was investigated extensively over the last decade. For example, the use of brown seaweed Sargassum, [258] tannic acid, [259] or serum albumin protein led to the formation of single-crystalline gold nanoplates, [260] whereas glycine was used in the facile synthesis of concave gold nanoplates. [261] The advantage of this method is that it is environmentally friendly, and it has been applied to the synthesis of other shaped AuNPs.

Gold nanoplates exhibit a wide range of unique electrical and optical properties, for example, a significant surface enhanced Roman scattering (SERS), [262] tip-enhanced Raman scattering (TERS), [263] and a shape-and size-dependent surface plasmon absorbance in the visible to infrared region. These properties result in potential applications as sensors and probes. [263,264] A particular interesting morphology is triangular nanoprisms that exhibit three congruent edge lengths in the range of 40 nm to 1 mm and a defined thickness ranging from 5 to 50 nm. These triangular nanoprisms have plasmonic features in the visible and IR regions, can be prepared in high yield, and can be readily functionalized with a variety of sulfurcontaining adsorbates as well as other functional materials. [265][266][267] 4.4. 3-Dimensional AuNPs 4.4.1. Gold Nanotadpoles [268][269][270][271] Gold nanotadpoles are a class of anisotropic 3D gold nanostructures with tadpole-like appearance. These interesting nanostructures with special optical and electrical properties have unique potential applications in second-order nonlinear optics, nanoelectronics, and other fields. The successful synthesis of gold nanotadpoles was reported by the Tian research group in 2004. They used a simple chemical procedure in which the tadpole-shaped gold nanoparticles were synthesized in aqueous solution by the reduction of chloroauric acid with trisodium citrate in the presence of sodium dodecylsulfonate as a capping agent. These threedimensional and crystallized structures were characterized by TEM, AFM, and HRTEM methods (Figure 19). [268] Soon afterwards, tadpole-shaped AuNPs were synthesized in high yield by a temperature-reducing seeding approach, without any additional capping agent or surfactant. [269] An aggregation-based growth process was proposed as the formation mechanism. Very recently, Au nanotadpoles were synthesized through the normal seed-mediated process in the presence of Ag + ions and CTAB at room temperature. [270] A hybrid nanostructure consisting of Au heads and Pd tails and the mechanism of formation of this morphology were demonstrated by Xia and co-workers. [271] It was expected that these Pd-Au tadpoles would combine the properties of both the Pd nanorods and AuNPs. 4.4.2. Gold Nanodumbbells (AuNDs) [272][273][274][275][276][277] The seed-mediated growth of gold nanodumbbells (AuNDs) was described and particularly well investigated by the Liz-Marzµn research group. [272,273] It was reported that the presence of tiny amounts of iodide modified the growth of gold nanorods in such a way that tip growth was greatly enhanced, thereby resulting in the formation of well-defined dumbbell morphologies (Figure 20). Gold-containing hybrid nanodumbbells are of a great interest because of their ability to carry multiple functions that can be simultaneously utilized. The Au/Ag core/shell nanodumbbells have recently been synthesized either through the deposition of silver onto the surface of the AuNDs, [274] or by galvanic replacement and reagent reduction. [275] Another hybrid morphology is that of gold-tipped metal nanorods (nanodumbbells), such as CdSe-Au nanodumbbells, [276] The nucleation and growth mechanism of the formation of CoPt 3 / Au, FePt/Au, and Pt/Au nanodumbbells were systemically studied by Prakapenka and co-workers. The authors were able to propose a general strategy for the synthesis of dumbbells with precise control over the size distribution and yield. [277] 4.4.3. Branched AuNPs: Nanopods and Nanostars [278][279][280][281][282][283][284][285][286][287][288][289][290][291] Branched gold nanostructures, including monopods, bipods, tripods, tetrapods, hexapods, [278] and multipods such as nanoflowers, nanostars, and urchins [279] are highly desired because of their sharp edges and the correspondingly high localization of any surface plasmon modes. There are great expectations for such branched AuNPs as potential candidates in nanocircuits and nanodevices, and for in vivo application as a result of their high tissue penetration in that spectral range. So far, branched AuNPs have indeed been utilized in biosensing, [280] imaging, [281] targeting, [282] and photothermal therapy. [283] The synthesis of branched gold nanocrystals in aqueous solution was first reported by Carroll and co-workers in 2003. [40] They demonstrated the use of triangular Ag platelets as seeds for the synthesis of Au monopods, bipods, tripods, and tratrapods through the reduction of Au III with ascorbic acid in the presence of CTAB. Sau and Murphy introduced a seed-mediated synthesis whereby the dimension and number of branches were varied under various combinations of the [seed]/ [Au 3+ ] ratio. [211] Subsequently, Hao et al. reported the synthesis of new "branched" gold nanocrystals in high yield (over 90 %) by a wet-chemical route during which HAuCl 4 was reduced by sodium citrate in a solution of bis-(p-sulfonatophenyl)phenylphosphine dipotassium dihydrate (BSPP) and H 2 O 2 at room temperature. [284] Many other routes were subsequently used to synthesize branched AuNPs. [279,[285][286][287][288][289][290][291] The branched AuNPs are generally not as highly monodisperse as other shapes. It was expected that the branched nanoparticles would have complicated localized surface plasmon resonance (LSPR) spectral features that would be lost in ensemble measurements. To test this hypothesis, scattering spectra were measured on single Au nanostars, and the result showed that the spectra consisted of multiple sharp peaks in the visible and NIR region. [289] The LSPR spectra of highly branched AuNPs have been explored by simulating the simpler structural subunits. The finite-difference time-domain (FDTD) method was used to calculate the near-and far-field properties of a gold nanostar, whereby the nanostar was modeled as a solid core with protruding prolate tips. This study showed that the resulting LSPR energies can be thought of as a hybridization of the core and tip plasmons (Figure 21). [290] This hybridization greatly increases the overall excitation cross-section and field enhancement of the nanostar tips. This antenna effect of the nanostar core may be responsible for the relatively bright and narrow scattering spectra of nanostars in the single particle measurements. [291] 4.4.4. Gold Nanodendrites [292][293][294][295] The Au dendrites possess a hierarchical tree-type architecture with trunks, branches, and leaf components. Gold nanodendrites with hyperbranched architectures have attracted much attention because of their importance in understanding the fascinating fractal growth phenomena and their potential applications in functional devices, plasmonics, biosensing, and catalysis. [292][293][294][295] The most important synthetic method for the preparation of Au dendrites is electrochemical deposition because of its ease of control and simple operation, and because it generates highly pure and uniform deposits. Hung and co-workers, obtained hyperbranched Au dendrites . on a glassy carbon electrode by electrodeposition with a square-wave potential from a solution of HAuCl 4 containing cysteine as the blocking molecule. [292] Huang et al. reported the first shape-controlled synthesis of Au nanostructures in the presence of supramolecular complexes formed from dodecyltrimethylammonium bromide (DTAB) and b-cyclodextrin (b-CD). Well-defined planar Au nanodendrites were formed by the reduction of chloroauric acid in aqueous DTAB/b-CD solutions (Figure 22). [293] Another example of surfactant/reductant combination has been reported for which the three-dimensional dendritic gold nanostructures were prepared by an ultrafast one-step homogeneous solution method by reducing HAuCl 4 in the presence of decane-1,10-bis(methylpyrrolidinium bromide) ([mpy-C 10 -mpy]Br 2 ) as the capping agent and l-ascorbic acid (AA) as the reducing agent. [294] Dendritic AuNPs exhibited significant catalytic activities, and the good SERS sensitivity for the detection of biomolecules also indicated their potential applications in biosensing and nanodevices.

4.5.

Au Nanoshells [296][297][298][299][300][301][302][303][304][305][306][307][308] Core/shell nanomaterials containing a supporting core material and a thin Au nanoshell in the form of dielectric SiO 2 @Au particles have been designed by Radloff and Halas. Such Au nanoshells exhibit a strong plasmon resonance, and the SERS effect sensitively depends on the core radius and shell thickness. [297] The authors provided a seminal systematic approach for "nanoengineering" the optical resonance wavelength of metal NPs. This SPB wavelength could indeed be "tuned" across a large region of the visible and infrared spectrum in particular in the crucial near-infrared region between 700-1100 nm, for biomedical applications, by variation of the relative size of the inner and outer shell layer (see Section 6). Gold nanoshells are among the most important AuNPs and find considerable usage in both therapy and diagnostics (concept of theranostic). [298] Other core@shell nanomaterials with a metal or metal oxide core and Au nanoshells are also known. [296] The scattering spectra of single gold nanoshells were also measured by dark-field microscopy combined with high-resolution scanning electron and atomic force microscopy, [299] and it has been demonstrated that the gold nanoshells show the same fluorescence enhancements as dye molecules. [300][301][302] The fabrication of supported hemishell structures known as Au nanocups was also reported by the Halas research group. [303] Briefly, a 35 nm thick Au layer was deposited by electron-beam evaporation onto silica NPs (120 nm diameter), thereby forming an Au film on top of the silica NPs. The Au nanocap generates second-harmonic light, whose intensity increases as the angle between the incident fundamental beam and the nanocup symmetry axis is increased (Figure 23). These plasmon structures provide a promising approach for the design and fabrication of stable, synthetic second-order nonlinear optical materials.

SiO 2 @Au NPs were also applied to optical imaging, [304] biomedical detection, [305] and photothermal cancer therapeutic ability, [306] and may enable a new class of infrared materials, components, and devices to be developed.

Moreover, Fe 3 O 4 @Au core/shell NPs modified with antibodies and fluorescent dyes have been reported to act as contrast probes for the multimodal imaging of tumors. This finding illustrates the great potential of Fe 3 O 4 @Au NPs to improve the accuracy of tumor diagnosis. [307] Very recently, magnetic Co@Au core/shell NPs with a pure Co core, an intermediate Au shell, and a compact outer cobalt oxide shell were reported. [308] Figure 21. Principle of the plasmon hybridization of the LSPR of a gold nanostar. From Ref. [290]. Copyright 2007 American Chemical Society. 4.6. Hollow AuNPs 4.6.1. Hollow Gold Nanospheres (HAuNSs) [309][310][311][312][313][314][315][316][317][318][319] The investigation of HAuNSs was pioneered in 1997 by the Halas research group. [309] They described the properties of Au-coated dielectric nanoparticles, or gold nanoshells, in which the configuration of a dielectric core coated with a metal nanoshell occurs naturally during the growth of Au/ Au 2 S nanoparticles. It was found that gold nanoshells possess quite remarkable optical properties that differ dramatically from those of solid AuNPs. During the growth of the core/ shell cluster, the plasmon-related absorption peak undergoes very large shifts in its wavelength, from 650 to 900 nm. Such structures potentially exhibit tremendous importance for optical applications related to the absorbance of IR light. The plasmon resonant optical properties [310,311] and SERS effects [312] of nanoparticles consisting of core/shell structures were analyzed, and it was shown that they have a tunable plasmon resonance that depends on the ratio of the core radius to the total radius.

One of the most versatile approaches for the preparation of coated and hollow spheres is the layer-by-layer (LbL) assembly method. [111,313] Polyelectrolyte-modified polystyrene (PS) nanospheres were widely introduced as templates for the construction of HAuNS morphologies by the size-controllable LbL technique. The PS core was subsequently treated by either calcination, causing the removal of the core, or dissolution of the core into a specific solvent (such as, THF). [314,315] Wan and co-workers reported a facile and effective approach to fabricate HAuNSs with tunable cavity sizes. [316] These HAuNSs were fabricated by using Co nanoparticles (CoNPs) as sacrificial templates and varying the stoichiometric ratio between the HAuCl 4 starting material and the reductants. The formation of these hollow nanostructures is attributed to two subsequent reduction reactions: the initial reduction of HAuCl 4 by CoNPs, followed by reduction with NaBH 4 . This strategy allows the convenient one-pot synthesis of homogeneous HAuNSs.

Recently, biomacromolecules have been investigated as precursor materials for the synthesis of nanoparticles. Rosi and co-workers first reported that, in the presence of inorganic salts and reducing agents, properly designed peptide conjugates can orchestrate a one-pot reaction in which the synthesis and assembly of nanoparticles into complex superstructures occurs simultaneously. Hexanoic acid-AAAYSSGAPPMPPF (C 6 -AA-PEP Au ) conjugates direct the formation of well-defined HAuNS superstructures (diameter = 136.5 AE 2.6 nm) upon incubation in water for 24 h (Figure 24). [317] Remarkably, the C 12 -PEP Au conjugate, which has a slightly different composition, directs the dramatic formation of double-helical nanoparticle superstructures. [318] The impact of further modification of the peptide sequence on the resultant nanoparticle assembly was explored with the peptide sequence BP-A x -PEP Au (C 12 H 9 CO-A x -AYSS-GAPPMPPF; x = 0-3; BP = biphenyl). The studies showed . that the small modifications to the peptide sequence, in this case the addition of a single alanine residue, can have a significant impact on the diameter of the resulting spherical superstructure (29.4-270 nm). These investigations revealed the versatility of this bio-based method and the rich structural diversity that occurs when this method is employed. [319] 4.6.2. Gold Nanocages and Nanoframes [320][321][322][323][324][325][326][327][328] Au nanocages (AuNCs), a novel class of remarkable nanostructures possessing hollow interiors and porous walls, were pioneered by the Xia research group. [320] AuNCs are attractive for colorimetric sensing and biomedical applications. Galvanic replacement is a facile and general synthesis method for the preparation of hollow metal structures. The typical synthesis of gold nanocages can be described as follows: The controlled addition of HAuCl 4 solution to a boiling suspension of Ag nanocubes (as both template and reducing agent) leads to galvanic replacement because of the electrochemical potential difference between Au and Ag (the reduction potential of AuCl 4 À /Au (0.99 V versus the standard hydrogen electrode, SHE) is more positive than that of AgCl/ Ag (0.22 V versus SHE)). The redox reaction takes place according to Equation (7), with the consumption of Ag atoms and the deposition of Au 0 confined to the Ag nanocube surface. When more HAuCl 4 is added, the formation of the hollow, porous cagelike Au nanostructure is observed. The morphology was confirmed by Xia and co-workers by SEM and TEM. [123,320] 3 Ag ðsÞ þ HAuCl 4 ! Au ðsÞ þ 3 AgCl ðsÞ þ HCl ðaqÞ ð7Þ

The controlled selective removal (or dealloying) of Ag from the Au/Ag alloy nanoboxes was achieved with a wet aqueous etchant Fe(NO 3 ) 3 . Ag was selectively dissolved from Au/Ag-alloyed nanoboxes or nanocages by galvanic replacement [Eq. ( 8)], without the deposition of solid Fe.

Ag ðsÞ þ FeðNO 3 Þ 3 ! AgNO 3ðaqÞ þ FeðNO 3 Þ 2ðaqÞ ð8Þ
Nanoboxes derived from 50 nm Ag nanocubes were converted into nanocages and then cubic nanoframes by increasing the amount of the etchant Fe(NO 3 ) 3 in the dealloying process (Figure 25). The structural change was accompanied by the SPR peaks continuously shifting from the visible region to 1200 nm. [321] These nanoframes have sensitivity factors that are several times higher than those of gold nanospheres, gold nanocubes, and gold nanorods, as well as those of comparable-sized AuNCs. [322] AuNCs and Au nanoframes were investigated in catalysis [323] and biosensing in view of their controllable LSPR properties that depend on the size and, most importantly, the thickness of the walls. [324][325][326][327][328]

Optical Plasmonic Properties of Anisotropic

AuNPs: SPR and SERS

SPR Properties of Anisotropic AuNPs

The rates of absorption and scattering, as well as the position of the plasmon band (SPR peak), of AuNPs were found to depend on their shape, size, and structures. [322] Some anisotropic AuNPs have unique SPR properties that are different from those of spherical AuNPs. For example, the AuNRs (also the AuNDs and AuNTs) exhibit two plasmon bands: a strong longitudinal band in the near-infrared region and a weak transverse band, similar to that of spherical AuNPs, in the visible region. The band in the IR region, where tissue absorption is minimal, is very useful for potential in vivo applications in nanomedicine. [235,329] Moreover, the location and intensity of the SPR peaks of AuNRs are remarkably more sensitive to the local refractive index in the vicinity of the nanorods and can be altered by the binding of biomolecular targets to the surface of the AuNR, which forms the basis of their utility as attractive biosensors. Additionally, the nanorods have an inherently higher sensitivity to the local dielectric environment than similarly sized spherical nano- particles, thus making AuNRs excellent colorimetric probes. [330] The plasmon bands of other intricate-shape AuNPs have recently been explored, and the well-defined plasmon band in the ensemble spectra is observed in every case. [247,278,284,291,288,320] For example, gold nanostars typically show a plasmon band of the core and multiple plasmon bands corresponding to the tips and core-tip interactions. [284] The hollow AuNPs (AuNCs and other Au nanoframes) have the highest sensitivity factors, and their plasmon bands are tunable throughout the visible and into the near-infrared regions. The relative intensity of the scattering and absorption cross-sections of the hollow-structured AuNPs can be tuned by varying their size, and this feature makes them attractive for colorimetric sensing and biomedical applications. [320] Although silica@Au nanoshells are spherical and present a single broad plasmon absorption, the fine-tuning at will of the position of this SPB differentiates them from standard spherical AuNPs and allows their efficient use in vivo for cancer therapy and imaging (see Section 6). [22,114]

SERS Properties of Anisotropic AuNPs

The SERS effect originates from the dramatic amplification of electromagnetic fields in the NP ensembles. Considerable enhancement occurs at the AuNP surface, because the intensity of the Raman signals depends on the fourth power of the local electric field, which is very high at the AuNP surface because of the plasmon resonance. This enhancement also originates from electromagnetic coupling between adsorbed molecules and the AuNP surface as a result of charge transfer between the adsorbed molecules and the AuNP surface. Amplified electromagnetic fields at AuNP junctions contribute to SERS, because the selective enhancement of SERS is controlled by polarization-dependent resonance bands. In addition to the elastically scattered light by the AuNPs themselves, which can be imaged using a dark-field optical microscope, the AuNP surface provokes an inelastic SERS effect as a result of adsorbed molecules generating a Raman spectrum, which enables their identification. The two main strategies for SERS detection are direct identification of Raman-active adsorbed molecules and indirect detection of molecules that are incorporated into a biolabel. [330,331] The Raman scattering signal of molecules located on nonspherical AuNPs is distinctly enhanced by contributions from the strong absorptions in the near IR regime and the extremely high electric field intensities at their tips or hollow structure. [332][333][334][335] Thus, controlling the size, shape, and structure of anisotropic AuNPs is critical to enhance the sensitivity for effective molecular detection. [238,336] For example, El-Sayed and co-workers showed that oral cancer cells can align AuNRs that have been conjugated with anti-epidermal growth factor receptor antibodies on the cell surface, thereby leading to a SERS fingerprint specific to the cancer cell. [337] The Halas research group has discriminated between acidic cancer cells and healthy cells by monitoring changes in the Raman spectrum induced by pH changes of carboxy groups in a mercaptobenzoic acid layer on HAuNSs that were active in the NIR region. [338]

Fluorescence Enhancement

The interaction between AuNPs and a fluorophore results from a change in the electromagnetic field and the intensity of the photonic mode near the fluorophore. For AuNP-fluorophore distances less than 4 nm, the fluorescence is strongly quenched. At larger distances, the fluorescence intensity is increased, including by coupling with far-field scattering. For example, the quantum yield of indocyanine green (ICG), a near-infrared fluorophore, was increased by up to 80 % near the surface of an Au nanoshell. Such conjugates have been used for enhancing sensitivities in fluorescence imaging in vitro and in vivo. [114,301,302] 6. Applications of Anisotropic AuNPs

Catalytic Applications of Anisotropic AuNPs

The catalytic activity and selectivity of metal NPs (MNPs) is dependent primarily on the size and shape; therefore, nanoengineering is crucial in tailoring NP catalysts. As a consequence of the presence of sharp edges and corners, the number of active surface sites in anisotropic AuNPs is very high compared to spherical AuNPs. The shape of an NP is composed of a particular crystallographic plane, which ultimately determines the number of active surface sites present in that NP. For example, with 5 nm tetrahedral PtNPs, which have only {1,1,1} facets, 35 % of the surface atoms are located at corners or edges, whereas this proportion is only 6 % for cubes, with {1,0,0} facets. With such PtNPs, the average rate constants were found to increase exponentially as the percentage of surface atoms at the corners and edges increased when surface coordination was involved in the ratelimiting step of the reaction. [344] The {1,1,0} facets found in dodecahedral nanocrystals have the highest energy among the low-index facets. The synthesis of metal nanocrystals with high-energy facets is, thus, challenging in terms of catalytic applications, because these facets endow crystals with high activity.

Catalysis of carbon monoxide oxidation by dioxygen at low temperature by small (< 5 nm) AuNPs on titanium oxide was pioneered by Haruta et al. in the late 1980s. [339] This study was a breakthrough in catalysis science that opened up a wide area of AuNP-catalyzed oxidation reactions. [5,[13][14][15][339][340][341][342][343] For example, oxide-supported Au/carbon catalysts were found to have high catalytic activity in the selective oxidation of cyclohexene and cyclooctene. [340] It is widely accepted that CO molecules are preferentially adsorbed at edges and steps on the surface of nonspherical or hemispheical AuNPs, rather than on the facets. [341,342] Very recently, it was shown that the perimeter interfaces around AuNPs are the sites for CO oxidation (Figure 26). [343] Therefore, investigations in catalysis have recently focused on anisotropic AuNPs such as gold nanotubes, nanodendritic gold, gold nanopyramids, and polygonal gold. [344,345] Gold nanotubes were found to be very efficient catalysts for CO oxidation at room temperature. [346,347] For example, gold nanotubes deposited within the pores of polycarbonate membranes played the role of catalysts and nanoreactors, respectively, for CO oxidation. The catalytic efficiency of these nanotubes was found to be higher than simple supported AuNPs. [346] An et al. reported the oxidation of CO at room temperature by using single-walled helical gold nanotubes as catalysts. The high catalytic activity of the (5,3) nanotube for CO oxidation may be due to the presence of under-coordinated Au sites in the helical geometry. On the microscopic level, the high activity may be attributed to the electronic resonance between the d state of the Au atom at the reaction site and the antibonding 2p* state of CO and O 2 , and the concomitant partial charge transfer. [347] Huang et al. reported the synthesis of dendritic AuNPs with the cationic surfactant decane-1,10-bis(methylpyrrolidinium bromide) ([mpy-C 10 -mpy]Br 2 ). Widely exposed active surface provided active sites for selective adsorption to allow the efficient reduction of p-nitroaniline. [294] Very recently, Losic and co-workers highlighted the catalytic properties of gold nanotubes on porous anodic aluminum oxide. The system showed excellent catalytic activity for the reduction of 4-nitrophenol (4-AP) to 4-aminophenol (4-NP). [239] Some hybrid bimetallic anisotropic AuNPs also exhibit high activities in various catalytic reactions. [348,349,350] For example, Au-ZnO nanopyramids (hexagonal pyramid-like structure) demonstrated a higher photocatalytic efficiency than pure ZnO nanocrystals in the degradation of rhodamine B. [348] Bimetallic Au-Pt nanowires prepared by the electrodeposition method were used as electrocatalysts for the realtime nonenzymatic impedancimetric detection of glucose. [229] Cui et al. prepared Au/Ag-Mo NRs by deposition of AuNPs onto Ag-Mo NRs (Figure 27). This hybrid catalyst was a very efficient heterogeneous catalyst for the tandem reaction of alcohols and nitrobenzenes to generate N-alkyl amines and imines. [349] 6.2. Sensors and Molecular Recognition by Anisotropic AuNPs 6.

Detection of Toxic Ions

It is well known that heavy metal ions are widely distributed in biological systems and the environment, and play important roles in many biological and environmental processes. Thus, the analytical determination of toxic metals is an important issue in both environmental monitoring and clinical research. [330] AuNRs currently are the most widely used nonspherical AuNPs for detecting heavy metal ions. This is due to the extinction coefficient of AuNRs of around 10 10 m À1 cm À1 , which results in the variation of the longitudinal plasmon absorption (wavelength shift or intensity degradation) that is a highly sensitive probe for sensing applications. [351] For example, AuNRs with various functionalized ligands on the surface led to a characteristic change in the longitudinal plasmon absorption on coordination to different metal ions. Cysteine (Cys) modified AuNRs have been used as colorimetric probes in the titration of Cu 2+ ions. The strong coordination of Cu 2+ ions with cysteine results in a stable Cys-Cu-Cys complex and induces the aggregation of the AuNRs along with a rapid color change from blue-green to dark gray. [330] Dithiothreitol (DTT) modified AuNRs were used as a SPR sensor of Hg 2+ ions. In this case, the DTT was strongly adsorbed on the surface of the AuNRs through SH groups and induced the aggregation of AuNRs. The induced aggregation of AuNRs was inhibited in the presence of Hg 2+ ions, with the aggregation level dependent on the concentration of Hg 2+ ions. The degree of aggregation could be determined by the change in the intensity of the longitudinal plasmon absorption in the UV/Vis spectrum (Figure 28). [351] The detection of some other metals such as Cr VI and Pb II was recently reported by using a similar method. [352,353] Besides the alteration of the longitudinal plasmon absorption, fluorescence is also a very effective method for ion detection. For example, Chen et al. reported a fluorescence method for the detection of Hg 2+ ions in a homogeneous medium, with AuNRs used as a fluorescence quencher. Under optimum conditions, the method exhibits a dynamic response ranging from 10 pmol L À1 to 5 nmol L À1 , with a detection limit of 2.4 pmol L À1 . [354] In conclusion, the strong affinity between anisotropic AuNPs and heavy metal cations alters the position of the plasmon band of AuNRs or the fluorescence property of targeted ions. This method allows heavy metal cations to be detected in aqueous solutions with ultrahigh sensitivity and excellent selectivity without sample pretreatment.

Biosensors and Bioprobes

The SPR and SERS properties of nonspherical AuNPs have been widely used for biosensing. [355] Up to now, Au nanorods (including bimetallic Au@Ag nanorods), nanopyramids, nanotubes, nanocages, nanowires, and nanostars have been reported as biosensors and bioprobes. [356][357][358] The typical routes to biosensors are illustrated in Equation (9). The nonspherical AuNPs functionalized with specific molecules (small molecules, DNA, antibodies, and biotin) recognized particular nano-objects (protein, DNA, drugs, and streptavidin), which caused the change in the plasmon absorption or the SERS intensity of the AuNPs. AuNRs also gained the most interest of all the variousshaped nonspherical AuNPs for applications as biosensors and bioprobes. For example, AuNRs modified with double-stranded (DS-) DNA exhibited temperature-dependent assembly and disassembly. Modified AuNRs assembled at low temperature and disassembled at high temperature, with the reversible plasmon band observed during the process. [359] Bimetallic Au/Ag core/shell nanoparticles show a higher SERS activity than monometallic AuNRs, which results in 10 times lower detection limit. [356] Besides AuNRs, other shaped AuNPs also have advantages for use as biosensors. For example, Yoo et al. presented a SERS sensor based on a patterned Au nanowire (NW) on a film for multiplex detection of pathogen DNA through an exonuclease III assisted recycling reaction of target DNA. Multiple probe DNAs are added to the target DNA solution, and among them, only the complementary probe DNA is selectively digested by exonuclease III, thereby resulting in a decrease in its concentration. The digestion process is repeated by recycling of the target DNAs (Figure 29). The decrease in the complementary probe DNA concentration is detected by SERS, and the detection limit is only 100 fm. [233] The locally enhanced fields at the sharp ends and tips of Au nanostars were exploited to amplify SERS and allow molecular detection at the zeptomolar level. Dondapati et al. reported Au nanostars functionalized with biotinylated bovine serum albumin (BSA) as a label-free biosensor for streptavidin recognition, and the results showed that the concentrations of streptavidin as low as 100 pm were detected by a plasmon shift of 2.3 nm. [280] AuNRs and AuNCs were found to act as SPR-and SERSbased probes for the detection of thrombin at concentrations of 10 am and 1 fm, respectively. [357] Castellana et al. recently reported a lipid-capped AuNR biosensor for the capture and label-free detection of a membrane-active drug (amphotericin B) by mass spectrometry (MS). In this case, the signal for amphotericin B appeared in the mass spectrum after incubation. [358] This MS method should become a very important technique for biosensors and bioprobes in the future.

Fluorescent enhancement of Au nanoshell/fluorophore conjugates is a powerfull sensing technique that is used in biomedical fluorescence imaging. [144]

Molecular Recognition

Based on the same principles of biosensors described above and in Equation ( 9), anisotropic AuNPs are also used for molecular recognition. The introduction of a thiolate ligand is a key feature for application to molecular recognition. [361,362] For example, functionalized AuNRs and Au nanoplates have been used for toxin recognition and glucose recognition, respectively. [259,363] Mandal and co-workers investigated anisotropic AuNPs for the recognition of organic solvents. Cyclam-stabilized AuNPs (mixture of spherical and triangular) were "dip-coated" onto the wall of a quartz cuvette to form a thin film. The Au thin film showed characteristic changes in the plasmon band in various organic solvents, thus indicating the system to be an excellent refractive index sensor. [364] Figure 28. The absorption spectra of AuNRs (1), AuNRs-DTT (2), and the AuNRs-DTT-Hg 2+ (3-7) system. Hg 2+ concentrations: 1 (3), 5 (4), 10 (5), 20 (6), and 50 10 À9 mol L À1 (7). From Ref. [351]. Copyright 2012 Elsevier.

Nanoelectrodes

Nonspherical AuNPs have also been used in electrochemistry applications. Very recently, for example, Au nanotubes were investigated as nanoelectrodes. 3D Au nanoelectrodes were prepared by controlled chemical etching of long Au nanotubes. The cyclic voltammogram of 3D Au nanoelectrodes showed they had a much higher sensitivity (more than twice) than the embedded Au nanoelectrodes and could be used in many applications such as molecular detection. [365] 6.3. Biomedical Applications: Diagnostics and Therapy Spherical Au NPs, AuNRs, HAuNSs, AuNCs, and Au nanostars are the most remarkable AuNPs for biomedical applications because: 1) they have long body circulation times; 2) they selective accumulate at sites of interest through the enhanced permeability and retention (EPR) effect or by surface modification with specific coatings; 3) they have a large absorption in the near-infrared window for photothermal therapy; 4) their simple functionalization (e.g. with PEG) and structural features allows their use as nanocarriers for drugs, DNA, or RNA. [320,[366][367][368][369][370][371][372][373][374][375][376][377][378][379][380][381][382][383][384][385]386] The concept of "theranostic" AuNP nanocomposites has emerged for AuNPs that combine functionalities of both contrast agent and therapeutic actuators within a single nanoparticle. Au nanoshells were the first Au NPs to be used as efficient theranostic agents that combine imaging and phototherapy functions. [114]

Contrast Agents in Diagnostics

The development of new techniques to diagnose cancer early is contributing to an increase in cancer survival rates. For example, the resolution of conventional imaging tech-niques have been improved and new imaging modalities have been developed. [320] AuNPs are photoresistant and stable, thus offering long-time operation for optical imaging. Plasmonic nanoparticles, such as spherical Au nanoshells, [114] AuNPs, [387] AuNRs, Au nanocages, and Au nanostars are efficient contrast agents in optical imaging as a result of their unique interaction process with light particles (i.e., the excitation of the SPR by light). The most important in vivo diagnostic techniques include light scattering imaging, two-photon fluorescence imaging, and photothermal/photoacoustic imaging. [371] For example, the Au nanoshells developed by Halas and co-workers that scatter light in the NIR physiological "water window" have been used as contrast agents for dark-field scattering, [22,114,304] photoacoustic imaging, [114,325] and optical coherence tomography (OCT). [114,306] With a core radius of 60 nm and a shell thickness of 10-12 nm, Au nanoshells can both absorb and scatter light at 800 nm, and thus serve as both imaging and therapeutic (theranostic) agents. [304] Lee and coworkers compared three different AuNP shapes (cubic cages, rods, and quasispherical), each possessing at least one dimension in the 40-50 nm range. Each NP was covalently functionalized with an antibody (anti-thrombin) and used as part of a sandwich assay in conjunction with an Au SPR chip modified with a DNA-aptamer probe specific to thrombin.

AuNPs strongly scatter light at their plasmon wavelengths. The scattering cross-sections are 10 5 -10 6 times stronger than that of the emission from a fluorescent dye molecule. AuNRs that strongly scatter in the near-infrared region are capable of detecting cancer cells under excitation at spectral wavelengths where biological tissues absorb only slightly (Figure 30). [367] Moreover, nucleus targeting and imaging have also been achieved by conjugating the nanorods to the nuclear localization signal (NLS) peptides for potential in vivo imaging. [368] Oldenburg et al. demonstrated that AuNRs less than 50 nm in length are good contrast agents with little back scattering, thus rendering them appropriate for the highly scattering tissue phantom. [369] The sensitivity is comparable to the absorption-based in vivo transmission imaging. [370] Currently, dark-field imaging based on the light-scattering properties of nonspherical AuNPs (shells, spheres, rods, and cages) is widely used for cancer imaging through functionalized nanoparticle-receptor binding to cellsurface biomarkers. [371,372] Nonspherical AuNPs, especially AuNRs, exhibit enhanced two-photon luminescence (TPL), thereby making them detectable at single-particle levels under femtosecond NIR laser excitation. In comparison to confocal fluorescence microscopy, TPL has the advantages of higher spatial resolution and a reduced background signal. For example, the nanorod-enabled TPL intensity was three times stronger than that of two-photon autofluorescence, [374] and TPL imaging of AuNRs is 100 times stronger than the emission of a single fluorescein isothiocyanate molecule. [375] However, deleterious photothermal effects are induced by plasmon excitation upon continuous scanning with high-intensity pulsed laser light. Despite these problems, TPL has been used recently for cancer imaging in vivo. For example, He et al. reported the detection of circulating tumor cells in vivo by using the TPL technique. [376] In this experiment, CTCmimetic leukemia cells were injected into the blood stream of live mice, followed by injection of folate-conjugated AuNRs to preferentially label the circulating cancer cells in vivo. TPL imaging with an intravital flow cytometer detected single cancer cells in the vasculature of the mouse ear.

Photothermal (PT) and photoacoustic (PA) imaging are based on the laser-induced heating of materials, with the former relying on the direct detection of heat and the latter on the detection of acoustic waves generated by the thermal expansion of air surrounding the materials. Compared to fluorescence-based approaches, PT imaging has the advantages of a larger detection volume and a high stability of the photothermal signal. [371] The PT imaging technique has usually been employed in photothermal therapy, and thus this technique will also be discussed in the following section.

Furthermore, PA imaging is used much more often than PT imaging in biomedicine. This is because the PA technique combines the high contrast of optical imaging with the deep tissue penetration of ultrasound imaging. Numerous applications have been shown in recent years. One of these key areas is in tumor imaging. For example, Agarwal et al. used PT imaging to detect prostate cancer by using AuNRs conjugated with anti-HER2. [377] This technique was also used by Li et al. to perform multiplexed imaging of different cancer cell receptors by using AuNRs of varying aspect ratios and with various target molecules. [378] Another key area is in vivo imaging. For example, Xia and co-workers used PA to image the cerebral cortex of a rat before and after three successive administrations of PEGylated AuNCs. An enhancement of the brain vasculature by up to 81 % was observed (Figure 31 A,B). A difference image (Figure 31 C) confirms the enhancement achieved by administration of the Au nanocage. A photograph of the open skull (Figure 31 D) reveals that the anatomical features of the vasculature match well with those revealed by the PA technique. Moreover, when compared with Au nanoshells, the AuNCs appear to be more effective contrast enhancement agents for PA, which is likely related to their larger absorption cross-section and more compact size. [379]

Photothermal Cancer Therapy

Thermal therapy involves the destruction of cancer tissues by heating. Various energy sources have been applied, including radio frequencies, high-intensity focused ultra- sound, microwaves, and lasers. The heat energy can be delivered by external or internal means, through interstitial, intraluminal, or intracavitary approaches. However, because of absorption by normal tissues, the amount of energy delivered to the treatment volume is limited, which reduces the potency of the thermal effect. To improve the efficacy and tumor selectivity, light-absorbing materials (known as photothermal contrast agents) are introduced into tumor cells to mediate the photothermal effect. A temperature increase of 30-35 8C provokes cell death. Various Au nanostructures, including nanoshells, [388,389] nanorods, nanocages, and nanostars that absorb NIR light (wavelength 700-850 nm), have been shown to be effective in photothermal therapy. [366] The first use of anisotropic AuNPs in targeted photothermal therapy was conducted by the Halas research group by using silica@Au nanoshells functionalized with antibodies such as anti-HER2. The antibodies direct the AuNPs toward the cancer cells because they conjugate with surface cell markers that are overexpressed by cancer cells. These antibodies were linked to orthopyridyl disulfide-PEG-n-hydrosuccinimide (OPSS-PEG-NHS) that was bound to the Au surface through strong Au À S bonds. The advantage of the PEG linker is that it provides an enhanced permeability and retention (EPR) effect involving the new blood vessels formed at the tumor site. This photothermal therapy was first demonstrated in mice with subcutaneous tumors of 1 cm size. Analysis showed that phothermal treatment resulted in tissue damage over a similar sized area as that exposed to laser irradiation. Magnetic resonance thermal imaging (MRTI) revealed an aveage temperature increase of 37 8C after 5 min irradiation, which is sufficient to induce irreversible tissue damage. Nanoshell-free samples showed an average increase of 9 8C, which was considered to be safe for cell viability. [113] Further experiments determined animal survival times of over 90 days. [22] Theranostic experiments were conducted with the same antibody and Au nanoshells with a core radius of 60 nm and a shell 10 nm thick (plasmon absorption at 800 nm). The cells were imaged with a darkfield microcope, and irradiated with a NIR laser at 820 nm (0.008 W cm À2 , 7 mn). Only the cancer cells show a distinct enhancement in the dark-field scattering image and a dark circular area of cell death corresponding to the beam spot upon laser irradiation. [304] Au nanoshells have also been used to target tumor hypoxia, regions with reduced blood flow that are resitant to NP accumulation, by combining mild hyperthermia and radiation therapy. [22] Au nanoshells have also been examined in vivo for their ability to enhance optical coherence tomography (OCT) and to induce photothermal cell death. [306] Further use of theranostics by the Halas research group successfully involved multimodal imaging and therapy, that is, with two diagnostic capabilities, MRI and NIR fluorescence, in addition to photothermal therapy. The nanoensembles were prepared by encapsulating Au nanoshells in a silica epilayer doped with 10 nm Fe 3 O 4 nanoparticles and indocyanine green (ICG), functionalized with streptavidin, and conjugated with thiolated PEG. These ensembles showed very good results in vitro by targeting breast cancer cells and also in vivo in breast cancer xenografts. [390] El-Sayed and co-workers pioneered strategies for the application of AuNRs in photothermal therapy. Conjugation of AuNRs to anti-EGFR antibodies enabled selective photothermal therapy as a result of preferential AuNR binding to human oral cancer cells. [367] In more-recent studies, they linked macrolide to PEG-functionalized AuNRs, which preferentially delivered AuNRs into inflamed tumor tissues via tumor-associated macrophage cells (TAMs). [221] AuNCs with large absorption cross-sections also show a large photothermal effect. The absorbed photons are converted into phonons (lattice vibrations), which in turn produce a localized temperature increase. Xia and co-workers demonstrated the photothermal destruction of breast cancer cells in vitro through the use of immuno-Au nanocages. AuNCs with an edge length of 45 nm were selected because of their predicted large absorption cross-section. SK-BR-3 cells were treated with these immuno-AuNCs, then irradiated with a laser with a wavelength of 810 nm and a power density of 1.5 W cm À2 for 5 min. The treated cells were stained with calcein-AM and ethidium homodimer-1 so that live cells fluoresced green and dead cells fluoresced red. This analysis revealed a well-defined zone of cellular death consistent with the size of the laser spot. [380] Au nanostars were also investigated for phototherapy applications. They were successfully conjugated with anti-HER2 nanobodies and demonstrated specific interaction with HER2t and SKOV3 cells. The FACS data and the dark-field images clearly revealed that the Au nanostars conjugated with anti-HER2 specifically bound to the cells, while almost no binding was observed for the controls. Furthermore, the conjugate resulted in specific photothermal destruction of tumor cells in vitro. Exposing the cells to either only NIR light or nanoparticles did not affect cell viability. Nonspecific NPs conjugated with anti-PSA nanobodies did not result in any cell death upon exposure to laser irradiation, thus demonstrating the high specificity of these anti-HER2-conjugated Au nanostars. [283] Hybrid nanomaterials composed of two unique components not only retain the beneficial features of both, but also show synergistic properties. Hybrid AuNCs were also investigated in photothermal therapy. Single-wall carbon nanotubes (SWCNTs) were functionalized and attached to AuNCs through a thiol group (Figure 32). The as-prepared AuNCdecorated SWCNTs were then modified with RNA aptamer A9, which is specific to human prostate cancer cells. The photothermal response for the hybrid nanomaterial is much higher than for single nanomaterials. [243] In conclusion, the in vitro and in vivo studies described have proved remarkably successful, yet tumors that are treated are those that are easily accessible to NIR light and only a few centimeters under the skin surface. It has been suggested that techniques to deliver NIR light into deeper tissues should exploit fiber optic probes, and such a challenge remains crucial.

Drug and Gene Delivery

The search for nonviral drug or gene vectors is indispensible because of the risk of cytotoxicity of and immunologic Gold Nanoparticles responses to conventional virus-mediated drug or gene delivery. [382][383][384] Nonspherical AuNPs have recently been used as nanocarriers for efficient drug or gene delivery systems. [385,390] Lee et al. reported the use of cationic phospholipid functionalized AuNRs as plasmonic carriers that simultaneously exhibit carrier capabilities, demonstrate improved colloidal stability, maintain plasmonic properties, and show no cytotoxicity under physiological conditions. The in vivo studies demonstrated that these functionalized ANRs are stable under physiological conditions, thus retaining their unique plasmonic properties. Furthermore, the positively charged surface of AuNRs adsorbs cargos such as DNA oligonucleotides, RNA oligonucleotides, and siRNA. [387] Halas and co-workers described an SiO 2 @Au nanoshell that released single-stranded DNA from its surface when illuminated with plasmon-resonant light. This system allowed examination of DNA dehybridization induced by excitation of localized surface plasmons on the NPs (Figure 33). [391] In another study, the light-triggered release of the fluorescent molecule DAPI (4',6-diamidino-2-phenylindole) inside living cells was investigated from a host-guest complex with DNA bound to SiO 2 @Au nanoshells. [392] Diagnostic and therapeutic drug delivery based on SiO 2 @Au nanoshells was also investigated recently for the treatment of ovarian cancer [393] and the diagnosis of breast cancer. [394] With their hollow structures, AuNCs serve as "pockets" that are appropriate for drug release. For example, PEGcoated AuNCs have been used as nanocarriers for doxorubicin and triggered drug release under irradiation with NIR light. This drug delivery system was considered to be a dualmodality cancer therapy that combined both photothermal therapy and chemotherapy. An in vivo study of this delivery system indicated greater antitumor activity than either doxorubicin or AuNCs alone. [366] Moreover, the surface of AuNCs was functionalized with thermally responsive polymers to control the release through NIR laser irradiation or high-intensity focused ultrasound. Another reported controlled-release system used a phase-change material (PCM) loaded in the hollow interiors of AuNCs to achieve the controlled release. An increase in temperature uncaps the pores and releases the guest molecules from the AuNCs. The release is controlled by varying the power or duration of the ultrasound treatment. [395,396] Very recently, Wan and co-workers reported a bioresponsive controlled-release AuNC system. The AuNC was selected as a support and an ATP molecule was used as the target (Figure 34). AuNCs were functionalized with two kinds of thiol-modified singlestranded oligonucleotides (SH-DNAs) by means of Authiolate bonding on the surface of the AuNCs. The bases of the two immobilized SH-DNAs were partly complementary to that of the two ends of the ATP aptamer. Mixing the surface-modified AuNCs with the ATP aptamers resulted in hybridization of the ATP aptamers with the two immobilized SH-DNAs. This effected assembly of the ATP aptamers on the surface of the AuNCs, thereby capping the pores. The release of the guest molecule from the aptamer-AuNCs could . be triggered by the addition of ATP molecules, thereby opening the pores and releasing the cargo molecules. [397] 7. Toxicology A number of in vitro studies have shown that AuNP cores are, in general, nontoxic for cells. These findings are in contrast with other nanoparticles such as carbon nanotubes, asbestos, and metal oxides, which have been shown to cause drastic damage to cells. On the other hand, although it has been extensively demonstrated that anisotropic AuNPs have an enormous potential in theranostics, the in vivo toxicity of these AuNPs nedd to be established before drug administration can be allowed. Therefore, in vitro and in vivo toxicology studies have been undertaken to examine the risk of damage on cells, tissues, and organs of animals. Most of these studies have been conducted on AuNPs that were considered to be spherical [398] and only a few studies have concerned AuNRs that are the prototype of anisotropic AuNPs with biomedical applications. The toxicity of AuNPs has been the subject of excellent reviews. [371,399] Thus, we restrct the discussion to a few general aspects.

The first problem is the distinction between the toxicity of the AuNP core and that of the coating stabilizer, because it is not possible to study the AuNP core without a stabilizer. In vitro studies have shown that the toxicity of AuNRs is due to the CTAB ligands, not the core. Indeed, Murphy, Wyatt, and co-workers have shown that AuNPs that were coated with polyacrylic acid or poly(allylamine) hydrochloride resulted in almost no death of HT29 cells. [400] It is generally considered that AuNP cores larger than 5 nm behave as bulk gold in terms of reactivity, whereas AuNPs smaller than 2 nm are specifically reactive. AuNPs larger than 5 nm are indeed inactive in catalytic reactions, such as CO oxidation by O 2 , while oxide-supported AuNPs smaller than 2 nm are highly active. [12][13][14][15][16] This rule of thumb should be considered with caution, especially concerning anisotropic AuNPs, for which the smaller dimension is often much smaller than the largest dimension (AuNRs, AuNTs, HAuNSs, and AuNCs). The "large" AuNPs, that is, with sizes between 5 nm and 100 nm, show a plasmon band that reflects a quantum behavior that probably has an effect on their reactivity. Such particles are more reactive than bulk gold because of the large surface and, more specifically, because of the presence of edges, corners, and numerous structural defects (consider Au dendrites, for example). On the other hand, small AuNPs that are supposed to be highly reactive when they are naked can be well protected, for example, by biocompatible PEGylated thiolate ligands.

The second important problem is that of the oxidation state of gold. We know that AgNPs are very toxic, because of their relative ease of oxidation to Ag + salts, which have been generally recognized as intrinsically toxic. [401] Of course, Au 0 is much more difficult to oxidize than Ag 0 , but the oxidizability of Au 0 depends on the AuNP size, shape, and surrounding ligands. For example, with the most common citrate and thiolate ligands, the AuÀO and AuÀS bonds on the AuNP core surface are polarized (Au d+ ÀO dÀ and Au d+ ÀS dÀ , respectively). Moreover, cationic Au I and Au III atoms have been generally proposed to be responsible for the activity in the oxidation of substrates in aerobic AuNP-catalyzed oxidation reactions. [12][13][14][15][16]402] Redox reactions are intrinsic to all biological organisms and are facilitated by cytochromes; cytochrome P450, in particular, is a strong oxidation catalyst. Therefore, the potential oxidation of Au atoms to toxic Au I or Au III ions at the surface of AuNP core that could subsequently be leached should not be underestimated. Again, these potential problems and risks are more acute with anisotropic AuNPs than with isotropic ones because of their highly exposed AuNP surface areas and defects. Although from in vitro studies it appears so far that the AuNP cores of isotropic AuNPs larger than 5 nm are biologically inert, [399,371,402] more studies are needed with anisotropic AuNPs and in vivo studies.

The third problem is the dichotomy between the in vitro and in vivo studies in terms of results and number of studies. A large number of in vitro studies have been carried out using TEM, microscopy techniques, and ICP-MS, and they are interesting from a fundamental point of view and bring mechanistic information (for example, how AuNPs penerate cells as a function of size, coating type, and nature of the cell, as well as how some ionic capping agents such as CTAB or cationic ligands are toxic). [399,371] However, there are very few in vivo studies and there is no correlation with the in vitro results. In vitro studies by Chan and co-workers indicated that the optimum size for cell penetration is 40-50 nm because this is the maximized antibody-receptor interaction for receptormediated endocytosis. [403] In vitro studies involve a large variety of parameters that, as underligned by Murphy and coworkers, [399,371] do not provide a precise overall picture of toxicity. Xia and co-workers have distinguished AuNPs that entered cells from others by using a I 2 /KI mixture that selectively solvates surface-cell AuNPs that do not induce toxicity. [404] The aspect of AuNP aggregation has also been addressed. [405] Some in vivo studies have addressed the pharmacokinetics of AuNPs. Toxicity studies of citrate-capped AuNPs in mice by Chen et al. showed that small (3-5 nm) and large AuNPs (30 and 100 nm) were not toxic, whereas mediumsized AuNPs (8, 12, 17, and 37 nm) provoked severe sickness, loss of weight, change in fur color, and shorter life spans. The systemic toxicity was due to injury of the liver, spleen, and lungs. [406] On the other hand, 13 nm, citrate-caped AuNPs were shown by Lasagna-Reeves et al. not to cause any acute side effects. [407] Small 10 nm AuNPs were found by ICP-MS analysis in the liver, spleen, testis, lung, blood, and brain 24 h after intravenous injection, whereas larger AuNPs (up to 250 nm) were found only in the spleen and kidneys. [408] For clearance through the kidneys, the NPs must pass the crossfiltration glomeruli barrier (6-8 nm hydrodynamic diameter) and must, therefore, be smaller than 5.5 nm. Indeed, smaller AuNPs were shown to be excreted in the urine, whereas 18 nm AuNPs accumulated in the liver and spleen. [409,410] Agglomeration of small (2 nm) AuNPs to larger aggregates, however, can also prevent renal clearance. [411] As an example of the coating influence, it was shown that glutathione-coated small (2 nm) AuNPs underwent renal clearance more effi-Gold Nanoparticles ciently (because of low binding to serum proteins) than citrate-coated AuNPs, but larger gluthathione AuNPs (13 nm) did not pass the glomeruli barrier. [411] Studies have also been conducted on the environmental and ecological impact of CTAB-capped AuNRs in model estuarine systems (containing sediments, microbiol films, plants, clams, snails, and fish), and it was found that biofilms were the main route of entry into the food chain. [412]

Conclusion and Outlook

A gold rush at the end of the 20th century resulted from the recognition of the excellent catalytic properties of very small AuNPs (< 5 nm) and of the potential provided by the quantum dot and optical properties of larger AuNPs (> 3 nm). Emphasis was on the size rather than on the shape. Although anisotropic AuNPs have attracted the attention of scientists since the very beginning of the 20th century, increased focus on their investigation only occurred at the beginning of the 21st century with the discovery of myriads of shapes including platonic, 1D, 2D, 3D, and hollow structures. This multiplicity of crystallization modes has questioned the influence of many parameters on the growth mechanisms. Whereas the key role of some stabilizers such as CTAB, Ag + , and halide ions has been rationalized, the largescale reproducible production of AuNPs of specific shapes remains a crucial challenge.

The advantage of and great interest in anisotropic AuNPs compared to classic spherical AuNPs are not only their better definition as precise nanocrystals, but also their very important optical and catalytic properties. [344] Whereas the plasmon band is unique in the visible region for homogeneous spherical AuNPs, nonspherical AuNPs show a second plasmon absorption that can be shifted to the near-infrared region between 800 nm and 1300 nm. In this region the absorption by tissues is low, and thus allows phototherapy applications. Important factors for the wide application of anisotropic AuNPs are the scale of production, reproducibility, and toxicity. So far, only the toxic CTAB stabilizer can lead to the generation of such 1D structures. The discovery of alternative, nontoxic stabilizers is another challenging issue. It is remarkable that the preparation of Au nanoshells, AuNCs, and some other anisotropic AuNPs that absorb light in the NIR "water window" and are the subject of clinical anticancer "theranostic" applications do not suffer from the toxicity problem found with CTAB.

The catalytic performance of anisotropic AuNPs is related to the presence of edges, corners, steps, and other defects, which are considerably more numerous than in spherical AuNPs. [413,414] It can be predicted that this field will expand given the well-established role of AuNPs in catalysis. Precise studies at the interface between AuNPs and solid oxide supports should result in a better definition of the oxidation states of surface Au atoms, which are key intermediates in oxidation reactions. This issue is not only important for catalysis, but also for understanding the risk of the oxidation of surface Au atoms in vivo and possible subsequent toxicity. It is, thus, also essential for the future applications of nonspherical and hollow AuNPs as well as SiO 2 @Au nanoshells in nanomedicine. [415] Another aspect is the new bottom-up emergence of Au nanowires that reach longitudinal dimensions of several tens of micrometers for applications as plasmon waveguides and networks for optical devices, such as interferometric logic gates, which are promising for future information processing. [228,416] As an extension and comparitive study of linear PEG-triazole stabilized AuNPs, two arenecored nona-branched dendrimers that were terminated by various lengths of PEG chains were synthesized via "click" chemistry and were employed to stabilize AuNPs of various sizes. The dendritic structure significantly influences the formation of AuNPs, and the catalysis efficiency of 4-NP reduction catalyzed by these AuNPs. The dendrimer acts not only template but also as a nanoreactor owing to the combination of the dendrimer structure and the flexible Au-trz bonding.

Importantly, 4-NP reduction reactions effectively serves as a model reaction describing the size, species and the surface properties of metal NPs. AuNPs as electron reservoir redox catalysts show the strong stereoelectronic ligand influence in catalytic 4-NP reduction. In our investigation of AuNP catalyzed 4-NP reduction, it was found that the AuNPs surface behaves as an "electron reservoir", because the storage of electrons at the AuNP surface upon NaBH 4 reduction is crucial in the inner-sphere redox catalysis of substrate trtansformation at the AuNP surface (Chapter 4).

The systematic introduction of anisotropic AuNPs in the last chapter of this thesis broadens our view of synthesis, morphology and applications of AuNPs in many fields. The advantage of anisotropic AuNPs compared to classic gold nanospheres are not only their better definition as precise nanostructures, but also their excellent optical and catalytic properties.

These gold nanocrystals are promising materials in nanomedicine and nanotechnology in the close future.

In summary, this thesis has illustrated the state-of-the-art in AuNPs synthesis, properties, morphologies and various applications as well as the development of trends of Au nanoscience. With these basis, AuNPs with various functionalities and capabilities were prepared involving development of new strategies, with significant influence towards nanoscience applications.

Introduction

In the ANNEX of this thesis, a targeted anticancer drug delivery system was constructed involving biocompatible AuNPs. To prepare biocompatible AuNPs, a variety of water-soluble PEG-thiols were synthesized and used as ligands. The enhanced permeability and retention (EPR) effect of PEG species contributed to the biocompatibility and also vectorization of these PEGylated AuNPs. Folate that is a broadly used targeting agent was introduced to the periphery of the PEG-AuNPs. The bifunctional AuNPs were then loaded with docetaxel that is known as one of the most efficient anticancer drugs but of very poor water solubility. The cytotoxicity towards LnCaP prostate cancer cell was estimated in vitro to confirm the biocompatibility of multi-functional nanocarrier. It was found that the drug-loaded AuNPs reduced the cell viability in more than 50% in treatment.

Such in vitro studies in Bordeaux were initiated by Professor Jacques Robert from the Bergonie Anti-Cancer Institute of Bordeaux, and the first collaborative paper between the group of Professor Jacques Robert and our group was reported in 2011 (1). The present publication represent an on-going study report.

The present study published in the paper (2) of this ANNEX was conducted in collaboration with the group of Dr. Gillian Barratt from the Pharmacy department of Chatenay-Malabry (Institut Galien, Paris-Sud University). Rachel Oliveira from that group carried out experimental in vitro studies, and Dr. Pengxiaing Zhao (primarily) and myself in ISM constructed the nanovectors. All the chemical synthesis of ligands, and the preparations of various functionalized AuNPs containing PEG and folate at the periphery were completed in our laboratory of the Bordeaux University. (2) R. Oliveira, P. Zhao, N. Li, L. C de Santa Maria, J. Vergnaud-Gauduchon, J. Ruiz; D. Astruc, G. Barratt.Synthesis and in-vitro studies of gold nanoparticles loaded with docetaxel. Internat. J. Pharmaceutics, 2013, 454, 703-711.

Introduction

Prostate cancer is the second most important cancer in terms of incidence in both sexes and the sixth in terms of mortality for men throughout the world according to the International Agency for Research on Cancer (Ferlay et al., 2010). At the moment, this cancer is treated with hormone therapy, but this is usually only effective for 24-36 months before the patient develops resistance (Holzbeierlein et al., 2004). Docetaxel was the first cytotoxic therapy to show a survival benefit in castration-resistant prostate cancer. For this reason, it remains an important part of the treatment against metastatic prostate cancer despite its toxicity and other limitations (Hwang, 2012).

Like paclitaxel, docetaxel belongs to the family of taxanes, which are diterpenes produced by plants of the genus Taxus (Zhao and Astruc, 2012). Docetaxel is a semi-synthetic taxane that binds irreversibly to ␤-actin, thus altering microtubule polymerization dynamics, disrupting cell mitosis and interphase microtubule function and triggering apoptosis (Kraus et al., 2003). In phase-II studies, combinations of drugs including docetaxel have been shown to result in a significantly higher PSA decline ≥50% in hormone-refractory prostate cancer patients (Ferrero et al., 2006; Mezynski et al., 2012; Oudard et al., 2005). However, the toxicity of the drug remains a concern.

Much research is currently being directed toward developing targeted delivery systems for docetaxel, in order to increase the amount of drug reaching the cancer cells and spare normal cells. Lipid nanocapsules (Sánchez-Moreno et al., 2012), nanoparticles piloted by aptamers (Farokhzad et al., 2006) and block copolymer micelles (Gao et al., 2008; Gaucher et al., 2005; Mei et al., 2009; Ungaro et al., 2012) are some examples of nanocarriers containing docetaxel designed to target various types of cancer, such as lung, breast and prostate cancer. The encapsulation of this molecule within gold nanoparticles bearing folate on their surface and thus able to target the prostate cancer cells is an original therapeutic approach to improve its effectiveness.

Gold nanoparticles of various sizes and morphologies have attracted considerable interest for medical applications (Boisselier and Astruc, 2009; Zhao et al., 2013), including photothermal therapy (Lal et al., 2008 (Brown et al., 2010; Ghosh et al., 2008; Paciotti et al., 2006; Llevot and Astruc, 2012). The plasmon resonance properties of these nanoparticles allow their characterization and detection in biological systems (Jain et al., 2007).

There are several synthesis routes for this type of nanoparticles (Daniel and Astruc, 2003). Surface functionalization can impart the required properties to the nanoparticles; for example, specific recognition (Raschke et al., 2003; Wang et al., 2005) or biocompatibility (Mout et al., 2012). The effective use of these nanoparticles requires controlled interactions with biomacromolecules (Mout et al., 2012) and their toxicity must be carefully evaluated (Murphy et al., 2008).

Folic acid has high capacity for targeting cell membrane receptors on a range of cell types, allowing nanoparticle endocytosis (Mansouri et al., 2006). Moreover, folic acid has high affinity for prostate-specific membrane antigen (PSMA), overexpressed on prostate cancer cells (Ghosh and Heston, 2004).

This work concerns the development of a multifunctional drug delivery system (DDS) based on gold nanoparticles (AuNPs). The nanoparticle surface was modified with poly(ethylene glycol) to prevent opsonization, delay their capture by macrophages and thereby allow them to circulate longer in the body and reach the tumor by the EPR (enhanced permeation and retention) effect (Lyer et al., 2006). The targeting agent, folic acid, was introduced by the covalent linkage on the amino-terminus of PEG. Docetaxel was associated with the AuNPs by non covalent adsorption.

Materials and methods

Materials

All solvents and chemicals were used as received. 1 H NMR spectra were recorded at 25 • C with a Bruker 300 (300 MHz) spectrometer. All the chemical shifts are reported in parts per million (ı, ppm) with reference to Me 4 Si for the 1 H and 13 C NMR spectra. Absorption spectra were measured with Perkin-Elmer Lambda 19 UV-vis. spectrometer. The CellTiter 96 ® AQueous One Solution Reagent (MTS) reagent was purchased from Promega (Madison, USA). Cell culture reagents were from Lonza (Basel, Belgium). Docetaxel (DOC) and folate-binding protein (FBP) were purchased from Sigma-Aldrich (Illkirch, France) and DMSO was from Carlo Erba (Milan, Italy).

Synthesis of gold nanoparticles 2.2.1. Synthesis of gold nanoparticles modified with PEG: AuPEG550 and AuPEG2000

Gold nanoparticles containing PEG were synthesized by direct modified (single-phase) Brust type method using a mixture of HAuCl 4 methanol and of two thiol-PEG ligands: one type ligand is thiol-PEG550 or thiol-PEG2000, and the other type is functional thiol-PEG400-NH 2 •HCl. PEG-thiol ligands were described previously (Zhao et al., 2012). Only one PEG chain length was used for the functional thiol-PEG-ammonium: 400 Daltons (PEG 400).

2.2.2. Synthesis of gold nanoparticles modified with PEG and folate: AuPEGFol550 and AuPEGFol2000 2.2.2.1. Synthesis of HS-PEG-NH 2 •HCl. The thiolate-PEG-NH 2 •HCl (HS-PEG-NH 2 •HCl) was synthesized as described previously by anionic ring-opening polymerization of ethylene oxide using the allyl alcohol/potassium naphthalene initiator system (Yoshimoto et al., 2008). 1 H NMR (CDCl 3 , 300 MHz) 3.58 (40H, CH 2 CH 2 O ), 3.17 (2H, CH 2 NH 2 HCl), 2.70 (2H, HSCH 2 ).

Synthesis of HS-mPEG.

The synthesis route of thiolate-mPEG (HS-mPEG) was as described in Zheng et al. (2004). To summarize, the starting material for synthesizing HS-mPEG is mPEG-Br. After refluxing with thiourea in ethanol, the bromo group was converted to an isothiourionium bromide and then to a SNa group after a second refluxing with sodium hydroxide. Neutralization with dilute HCl generated mPEG-SH. Distillation of the extracted HS-mPEG produced the pure compound with 74% yield. 1 After evaporation of the solvent under vacuum, the crude product was dissolved in 30 mL distilled water followed by dialysis. Yield: 45 mg (61%). 1 acquisition (AMT) was used to evaluate the size and possible aggregation of nanoparticles containing PEG 550. A 120 kV electronic microscope 1400 equipped with wide-field CCD camera acquisition (Gatan) was used to observe the gold nanoparticles containing PEG 2000.

Surface plasmon resonance measurement

The interactions between FBP covalently immobilized on the surface of a CM5 sensor chip and the gold nanoparticles modified with folate were analyzed by surface plasmon resonance with a Biacore T100 instrument (GE Healthcare). Functionalization of the different channels of the chip was carried by amine coupling of FBP using EDC and NHS in the presence of ethanolamine.

Docetaxel loading

DOC was weighed accurately docetaxel (XP105 DeltaRange Analytical Balance -Mettler Toledo, Zurich, Switzerland) and a stock solution in DMSO at 29, 6 mg/mL was prepared.

Docetaxel was associated with the AuNPs by non covalent adsorption (Franc ¸ ois et al., 2011). 1 mL of a suspension of AuNPs at 10 mg/mL was added to 14.3 mL of an aqueous solution of DOC at 0.28 mg/mL. Both solutions were sterilized using a 0.2 m cellulose acetate sterile syringe filter and handled in a sterile environment in order to prevent microbial contamination during the association protocol. The mixture was stirred for 5 days at 30 • C. Previous work (Franc ¸ ois et al., 2011) using a spectroscopic technique showed that this protocol leads to association of all the added DOC. Therefore, the mixtures were used directly to evaluate cytotoxicity against human prostate cancer cells, without any separation step.

The addition of DOC to the different types of nanoparticles described above generated four batches of loaded nanoparticles: AuPEG550DOC -AuNPs containing DOC, PEG 400 and PEG 550 AuPEG550DOCFol -AuNPs containing DOC, PEG 400 with folate and PEG 550 AuPEG2000DOC -AuNPs containing DOC, PEG 400 and PEG 2000 AuPEG2000DOCFol -AuNPs containing DOC, PEG 400 with folate and PEG 2000.

Cytotoxicity evaluation

The human prostate cell line LnCap (ECACC Reference 89110211) was obtained from the Institut Bergonié, Bordeaux. Cells were routinely grown in RPMI-1640 medium supplemented 133 with l-glutamine, 10% of fetal bovine serum and 1% penicillin-streptomycin. Cells were incubated at 37 • C in an atmosphere containing 5% CO 2 and passaged once a week.

Cell viability was estimated by mitochondrial metabolic activity using the Promega CellTiter 96 ® Aqueous One Cell Proliferation (MTS) assay (Malich et al., 1997). The absorbance recorded in this colorimetric assay reflects the number of viable cells.

The cells were seeded in 96-well plates (5000 cells in 50 L per well). Twenty-four hours later, free DOC, unloaded AuNPs and AuNPs loaded with DOC were suspended in culture medium, serially diluted and added 96-well microtiter plates (50 L/well). The plates were incubated for a further 24, 48 or 144 h. Triplicate wells were used for each condition, and each experiment was performed in triplicate. Ninety minutes prior to the end of each exposure period, the MTS reagent (20 L/well) was added.

The absorbance of the formazan product was read with a 492 nm high-pass filter in a Multiskan MS microwell plate reader (Labsystem, Ramat-Gan, Israel). Background absorbance due to the non-specific reaction between the test compounds and the MTS reagent or to light scattering was measured in wells without cells and was subtracted from the values measured in the presence of cells (Hayes and Markovic, 2002).

The relative cell viability was calculated with respect to control wells containing cell culture medium without nanoparticles or DOC according to the formula

Statistical analysis

Cell culture data are reported as means of three experiments (each with triplicate wells) ±SD. Statistical analysis on the raw data was performed by one-way analysis of variance (ANOVA) followed by appropriate post hoc test (Tukey's multiple comparison) for comparison between groups. A significance level of 95% (p < 0.05) was accepted (Viviani et al., 2005).

Results and discussion

As shown in Scheme 1, AuNP were synthesized by modified Brust method (Brust et al., 1994). From the relative integrations of the peaks of the aryl group of the folate in the PEG-400-or PEG-2000-folate ligand and the PEG groups in the 1 H NMR spectrum of AuNP 1 (not shown), it can be seen that the AuNP surface was capped by 50% HS-mPEG and 50% HS-PEG-NH 2 •HCl. Indeed, the integration at 3.17 ppm belongs to the CH2NH2•HCl on HS-PEG-NH 2 •HCl and the integration at 3.3 ppm belongs to the OCH3 on mPEG, compared with the two integrations.

This indicated that 50% of capped ligand on AuNP modified with the two types of PEG (1 on Scheme 1) could be functionalized by NHS-folate through the coupling reaction, leading to AuNPs with bearing folate groups at their surface (2 on Scheme 1).

The folate functionalization at the termini of AuNP is confirmed by the 1H NMR spectrum (sSI). The plasmon band is observed at 530 nm which also confirms that the size of the AuNPs is more than 3 nm (not shown).

The AuNPs with or without folate were used to encapsulate DOC for the assessment of its in vitro activity. During the incubation of DOC with the nanoparticles numerous weak hydrogen bonds form between the OH and NH group of DOC (Scheme 1) and the numerous oxygen atoms of the PEG polymer. In this way, PEG acts as a solvent for the DOC molecules. The AuNPs prepared by the Schiffrin-Brust (Brust et al., 1994) method showed individualized particles with a gold core diameter of 7 ± 3 nm for the nanoparticles containing PEG 550 and 10 ± 5 nm for the nanoparticles containing PEG 2000 with a narrow polydispersity, as determined by TEM (Fig. 2). The freeze-drying process seems to provoke the aggregation of some nanoparticles because even after 30 min of treatment in an ultrasound bath, the TEM images showed aggregates with irregular shapes up to 400 nm (Fig. 1). This phenomenon has already been described in the literature (Franc ¸ a et al., 2010).

In vitro cytotoxicity toward LnCaP prostate cancer cells

The morphology of the cells was observed after 6 days of treatment using an optical microscope (Leitz Diaplan) at 100× magnification. Typical photomicrographs are shown in Fig. 3. There was no difference in the morphology of untreated cells and that of cells treated with unloaded AuNPs. However, both the nanoparticles loaded with docetaxel and the free drug at the same concentration clearly reduced the number of LnCaP cells remaining in the wells.

The toxicity toward LnCaP cells of the unloaded nanoparticles was estimated using the MTS after 24, 48 and 144 h of contact, as seen in Fig. 4. The concentrations of AuNPs were chosen to correspond to those of the loaded NPs and ranged between 35 pg/ml and 35 g/ml. The overall difference was not statistically significant for all concentrations and times (one-way ANOVA test). Fig. 5A shows LnCaP viability after a 24-h exposure to DOCloaded gold nanoparticles with PEG 550 chains, with or without folate, compared with free DOC. The presence of folate did not affect the cytotoxicity of the loaded nanoparticles at any concentration.

Free DOC produced a statistically significantly greater effect at the two lower concentrations than the loaded NPs. However, above 6.10 -7 M docetaxel, the difference decreased making the effects of all treatments statistically equivalent.

Fig. 5B shows the results of LnCaP viability after 24 h of exposure to DOC-loaded gold nanoparticles with longer PEG chains (PEG 2000). At the lowest concentration, the AuPEG2000DOCFol were less effective than AuPEG2000DOC; however this trend was not reproduced at other concentrations, where the two formulations yielded similar results. Unlike AuNPs with PEG 550, the effect of free DOC was statistically significantly greater than that of loaded AuNPs at all concentrations.

Fig. 5C shows the viability after 48 h of exposure to free docetaxel and AuNPs bearing PEG 550. The effect of AuPEG550DOC was concentration-dependent. AuDOCPEG550Fol showed a less evident effect of concentration. There was no statistically significant difference between the effects of free DOC and the loaded AuNPs at any concentration.

Fig. 5D shows cell viability after 48 h of exposure to free DOC and AuNPs (PEG 2000). Like the AuNPs with shorter PEG, AuPEG2000DOCFol demonstrated a statistically significant effect on cell viability for the two most concentrated suspensions compared with the control. The AuNPs without folate only had significant activity at 6.10 -5 M. At all concentrations, treatment with AuPEG2000DOCFol resulted in significantly lower viability than the same AuNPs without folate, except at 6.10 -9 M. 5E shows the cell viability after a longer exposure time (144 h) for the loaded AuNPs with the shorter PEG chains (PEG 550). The DOC-loaded nanoparticles produced a significant reduction in the cell viability at all concentrations. There was no difference between AuNPs with and without folate. Similar results were obtained with the AuNPS with longer PEG chains at 144 h (Fig. 4F). At this exposure time, the free drug showed a marked dose-response, with significant reductions in viability at all concentrations except the lowest concentration tested (6.10 -11 M).

In general, free DOC showed significant cytotoxicity toward LnCaP cells at all exposure times and concentrations except after 144 h at the lowest concentration, confirming the sensitivity of this cell line to DOC. The decrease in the efficacy of the lower concentrations of DOC at long incubation times could be due to precipitation of this very hydrophobic molecule in the culture medium.

Compared with free DOC, the loaded nanoparticles were less effective against LnCaP cells over the first 48 h. After 144 h of exposure the nanoparticles containing DOC were in general more effective than the free drug suggesting that there is a slower release of the incorporated drug and that its association with the AuNPs prevented its precipitation in the medium. An effect of the PEG chain length could only be observed after 48 h of exposure, when the AUNPS bearing shorter chains (PEG 550) had more effect on viability than those with longer chains (PEG 2000). This may indicate that the higher molecular weight PEG chains retained the DOC in the AuNPs longer and it was therefore less available to cells.

The DOC-loaded nanoparticles reduced the mitochondrial activity by 50% or more at concentrations of 6.10 -9 M and above after 144 h of treatment (Fig. 4E andF). However, no significant difference was observed between folate-bearing AuNPs and non modified ones. This might indicate that the folate residues are not orientated correctly for binding to PSMA, or that the non modified PEG chains cause steric hindrance, especially since these chains were longer than those carrying the folate. This hypothesis was confirmed by surface plasmon resonance measurements made with the Biacore instrument (data not shown). AuNPs bearing folate showed no specific interactions with folate-binding protein (FBP). A similar observation had been made concerning liposomes bearing PEG terminated with folate residues and was considered to indicate steric hindrance from the PEG chains (Botosoa et al., 2011).

The decrease in the number of cells after contact with DOCloaded nanoparticles was confirmed by observation with an optical microscope. Fig. 3 shows far fewer cells after 144 h of treatment with free or AuNP-associated DOC compared with control cultures or cultures treated with unloaded nanoparticles, where the cells can be seen to be growing as aggregates typical for this line. Cells incubated with AuPEG2000DOC and AuPEG2000FolDOC (data not shown) showed similar changes in the appearance of the cell monolayer to AuPEG550DOC (Fig. 3B) and AuPEG550FolDOC (Fig. 3D), respectively.

Conclusion

In this study, gold nanoparticles have been functionalized with PEG 550 and PEG 2000 by "click" chemistry in order to encapsulate docetaxel by non covalent interactions. The nanoparticles were also functionalized with folate. The unloaded nanoparticles were not cytotoxic to LnCaP cells at the concentrations used in this study (between 35 pg/ml and 35 g/ml). When DOC was adsorbed onto the AuNPs it produced a sustained cytotoxic effect on LNCaP cells in an in vitro test. These small particles could be useful for concentrating drug in solid tumors by the EPR effect, and the properties of the gold core could be exploited for tumor imaging and for thermal cytolysis of the tumor cells.

Nanoparticules d'or fonctionnelles pour les applications biomédicales et catalytiques

Résumé :

Le design et l'ingénierie de nanoparticules d'or (AuNPs) polyfonctionnelles suscitent un intérêt considérable en vue d'applications en nanomédecine, reconnaissance moléculaire, dans le domaine des capteurs et en catalyse dans un environnement aqueux. Cette thèse a été dédiée à une variété de fonctionnalisations, en particulier à l'aide de la méthode "click" impliquant la catalyse par le cuivre (I) de la cycloaddition des alcynes terminaux avec les azotures avec le catalyseur [Cu(hexabenzyltren)] Br pour l'introduction de polyéthylè ne glycol, carborane, ferrocène, coumarine, cyclodextrine, médicaments et molécules fluorescentes sur les AuNPs. Les ligands dits "click", c'est-à-dire des 1,2,3-triazoles fonctionnalisés en positions 1,4 et formés de cette façon ont été ici largement utilisés afin de stabiliser des AuNPs pour des applications biomédicales et catalytiques en collaboration.

Mots clés : Nanoparticule d'or, chimie "click", application biomédicale, catalyse, capteur fluorescent Title :Functionalization of gold nanoparticles for biomedical and catalytic applications Abstract : The design and molecular engineering of multi-functional gold nanoparticles (AuNPs) is of considerable interest towards applications in nanomedicine, molecular recognition, sensing and catalysis in aqueous environments. This thesis has been devoted to a variety of functionnalizations, in particular with the copper(I)-catalyzed Alkyne Azide cycloaddition (CuAAC) using the catalyst [Cu(I)(hexabenzyltren] Br for the introduction of polyethylene glycol, carborane, ferrocene, coumarin, cyclodextrin, drugs and fluorescent probes. The socalled "clicked" ligands, 1,4-bifunctional triazoles, that were formed in this way have been exensively used to stabilize AuNPs for biomedical and catalytic collaborative applications.

Fig. 1 .

 1 Fig. 1. AuNP synthesis using the Turkevich method.

Fig. 3 .

 3 Fig. 3. Mechanism for chalcogenate-protected metal NP synthesis by the Brust-Schiffrin method. Reprinted with permission from [56] (Tong's group). Copyright 2011 American Chemical Society.

Fig. 4 .

 4 Fig. 4. (left) X-ray crystal structure of [oct4N + ][Au25(SCH2CH2Ph)18 -].16 The icosahedral Au13 core is surrounded by six Au2(SR)3 semi-rings that are slightly puckered in the reduced AuNPs as shown for the semi-ring with more pronounced yellow and pink colors (right). The icosahedral Au13 core (minus the center Au) is slightly distorted; the blue Au Au bonds lying directly below the center of each semi-ring are on average 0.12 Å shorter than the yellow Au Au bonds (average 2.96 Å). Overall Au-Au average 2.93 Å. Au13 core diameter 9.8 Å; overall AuNP diameter 23.9 Å.

Fig. 5 .

 5 Fig. 5. Synthetic route for thiol-dendron-stabilized AuNPs. Reprinted with permission from [113] (Daniel's group). Copyright 2011 Royal Society of Chemistry.

Fig. 6 .

 6 Fig. 6. Encapsulation of docetaxel in PEGylated AuNPs considerably increases its solubility in water (Astruc's group). Reprinted with permission from [124]. Copyright 2011 Wiley-VCH.

Fig. 7 .

 7 Fig. 7. Synthesis pathway for TPP-stabilized AuNPs. Reprinted with permission from [138] (Shumaker-Parry's group). Copyright 2009 American Chemical Society.

Fig. 8 .

 8 Fig. 8. Synthesis and bioapplication of amine ligand stabilized AuNPs. Reprinted with permission from [164] (Suri's group). Copyright 2008 Elsevier.

Fig. 9 .

 9 Fig. 9. Synthesis of PVCL and coating of AuNPs. Reprinted with permission from [202] (Marty's group). Copyright 2011 Royal Chemical Society.

Fig. 10 .

 10 Fig. 10. Schematic representation of the synthesis of thermosensitive hybrid core-shell particles containing AuNPs. Reprinted with permission from [204] (Kawaguchi's group). Copyright 2005 American Chemical Society.

Fig. 11 .

 11 Fig. 11. Newkome-type dendron-stabilized AuNPs. Reprinted with permission from [244] (Hackley's group). Copyright 2011 American Chemical Society.

Fig. 12 .

 12 Fig. 12. Schematic representation of the synthesis of Au DSNs. Reprinted with permission from [251] (Ji's group). Copyright 2011 Springer.

Fig. 13 .

 13 Fig. 13. Formation of dye-functionalized AuNPs (GPN = AuNP). Reprinted with permission from [27] (Yoda's group). Copyright 2011 Elsevier.

Fig. 14 .

 14 Fig. 14. (a) Molecular structure of the PS-b-P4VP copolymer and schematic illustration of the micellation process with in situ synthesis of AuNPs in PS-b-P4VP. (b) Topography image of the surface (from AFM) of a spin-coated PS-b-P4VP film along with a schematic illustration of a self-assembled PS-b-P4VP (with AuNPs) micellar film on a substrate. (c) TEM image of PS-b-P4VP. The copolymer film is made up of a continuous bright PS matrix and dark spherical P4VP cores. (d) TEM image of PS-b-P4VP with AuNPs; PS-b-(P4VP/Au). The molar ratio of HAuCl4:P4VP is 0.1. Reprinted with permission from [278] (Leong's group). Copyright 2008 Wiley-VCH.

Fig. 15 .

 15 Fig. 15. A PEO-PPO-PEO micelle with the core occupied by PPO units and the corona constituted by PEO units. Reprinted with permission from [286] (Bakshi's group). Copyright 2011 American Chemical Society.

Fig. 16 .

 16 Fig. 16. (a) Designed liposomal nanoreactor. (b) Formation stages of AuNPs inside the glycerol-incorporated (15%, v/v) liposomes: liposomes before reaction (top left), liposomes during the reaction (middle) (scale bars = 10 nm), and purified AuNPs synthesized in the nanoreactor (bottom right, scale bar 20 nm). Photon correlation spectroscopy graph of the liposome size distribution before the reaction, PI = 0.232 (bottom left corner) and graph of the calculated AuNP size distribution using TEM (top right corner).

Fig. 17 .

 17 Fig. 17. TEM images of AuNPs in solution (a) 11.0 ± 0.8 nm, (b) 13.3 ± 2.0 nm, (c) 32.2 ± 1.8 nm, (d) 69.0 ± 3.7 nm and (e) 87.3 ± 12.1 nm. Reprinted with permission from [337] (Han's group). Copyright 2007 American Chemical Society.

Fig. 18 .

 18 Fig. 18. TEM images of AuNPs in solutions (a) 15 ± 2 nm, (b) 31 ± 3 nm, (c) 69 ± 3 nm, (d) 121 ± 10 nm, (e) 151 ± 8 nm and (e) 294 ± 17 nm. Scale bars are 200 nm for parts a-c and 500 nm for parts d-f.Reprinted with permission from[338] (Eychmüller's group). Copyright 2011 American Chemical Society.

Fig. 19 .

 19 Fig. 19. General methodology for the generation of AuNRs. Reprinted with permission from [346] (Murphy's group). Copyright 2004 American Chemical Society.

Fig. 20 .

 20 Fig. 20. TEM images of gold NRs synthesized in high yield with plasmon band energies at (a) 700, (b) 760, (c) 790, (d) 880, (e) 1130, and (f) 1250 nm. The scale bar is 50 nm.

Fig. 21 .

 21 Fig. 21. (a) Cubic AuNPs. Reprinted with permission from [358] (Murphy's group). Copyright 2004 American Chemical Society. (b) Trisoctahedra AuNPs. Reprinted with permission from [368] (Huang's group). Copyright 2010 American Chemical Society. (c) Start-like AuNPs. Reprinted with permission from [359] (Xia's group). Copyright 2011 Wiley-VCH. (d) SEM images of (A) the concave cube seeds and the products of the octahedra reaction with (B) 50, (C) 100, and (D) 150 L of a 10 mM HAuCl4 solution. Scale bars: 100 nm.

  .

6. 3 . 3 .

 33 Mesoporous materials and MOFs 6.3.3.1. Mesoporous materials. Mesoporous material-supported AuNPs are widely used as catalysts. The production of these types

Fig. 22 .

 22 Fig. 22. Synthesis of AuNPs encapsulated in mesoporous silica. Reprinted with permission from [403] (Corma's group). Copyright 2005 Royal Chemical Society.

Fig. 23 .

 23 Fig. 23. Procedure used for preparing mesoporous silica and AuNP/meso-silica tubes. Reprinted with permission from [404] (Jan's group). Copyright 2011 American Chemical Society.

Fig. 24 .

 24 Fig. 24. Synthetic procedure of MOF-5-encapsulated AuNPs. (PCP: porous coordination polymer.) Reprinted with permission from [406] (Haruta's group). Copyright 2008 Wiley-VCH.

Fig. 25 .

 25 Fig. 25. (a) Reaction scheme showing morphological and structuralchanges involved in the fabrication of Au/Pt/Au core/shell nanoraspberries and (b) SEM image of the product Au@Pt@Au.Reprinted with permission from[419] (Schlücker's group). Copyright 2011 American Chemical Society.

Fig. 26 .

 26 Fig. 26. UV-vis. spectra of Au92Ag52(SR)60 clusters for Au/Ag precursor ratio of 1:0.66 with phenylethane thiol (dotted), hexane thiol(dashed), and dodedane thiol (solid). Reprinted with permission from [427] (Dass's group). Copyright 2011 Royal Chemical Society.

"

  Click" Star-Shaped and Dendritic PEGylated Gold Nanoparticle-Carborane Assemblies Na Li, † Pengxiang Zhao, †, ‡ Lionel Salmon, § Jaime Ruiz, † Mark Zabawa, ∥ Narayan S. Hosmane,* ,∥ and Didier Astruc* , † † ISM, Univ. Bordeaux, 351 Cours de la Libeŕation, 33405 Talence Cedex, France ‡ Science and Technology on Surface Physics and Chemistry Laboratory, P.O. Box 718-35, Mianyang 621907, Sichuan, China § LCC, CNRS, 205 Route de Narbonne, 31077 Toulouse Cedex, France ∥ Department of Chemistry and Biochemistry, Northern Illinois University, DeKalb, Illinois 60115-2862, United States * S Supporting Information

Figure 1 .

 1 Figure 1. (a) UV-visible spectrum, (b) DLS size-distribution histogram (measured in water solution), (c) TEM image, and (d) the core sizedistribution histogram of AuNPs-3c.

Figure 2 .

 2 Figure 2. IR spectrum of AuNPs-3c (top, blue curve) compared to that of AuNP-2c (bottom, red curve). It shows the complete disappearance of the ν N3 band at 2090.9 cm -1 after "click" reactions, and the appearance of the σ B-H stretching vibration at 2580.8 cm -1 .

Figure 4 .

 4 The characteristic band due to the strong stretching mode of vibration of the C-O-C bond (σ C-O-C ) in the PEG chain at 1105 ± 10 cm -1 is the dominating feature in both spectra. The secondary strong and broad absorption around 2580 ± 10 cm -1 is due to σ B-H stretching vibration,15 that indicates the existence of the carborane cluster structure. The comparison of the FT-IR spectra of dendron 4 and AuNP-4b indicates a subsequent bonding of the carborane dendron to the AuNPs with a weak σ B-H vibration as a result of shielding of the carborane absorptions by nearby AuNPs. A similar characteristic σ B-H signal was also observed in the FT-IR spectra of other carborane-containing molecules (Supporting Information).

Scheme 3 .a

 3 Scheme 3. "Click" Syntheses of Carborane-Containing Dendrons a

Figure 3 .

 3 Figure 3. TEM images and diameter histogram distribution of AuNPs-4a and AuNPs-4b, respectively.

Scheme 4 .

 4 Scheme 4. Schematic Illustration of Direct Synthesis of (a) AuNPs-4a and (b) AuNPs-4b

  H NMR (CDCl 3 , 200 MHz): δ = 1.55 (2H, HS-CH 2 ), 1.26 (20H, -CH 2 CH 2 -), 0.87 (3H, -CH 3 ).

  H NMR (CDCl 3 , 200 MHz): δ ppm = 3.24 (2H, CH 2 -N 3 ), 1.58 (2H, HS-CH 2 ) 1.26 (20H, -CH 2 CH 2 -), 0.87 (3H, -CH 3 ). Upon continuing the reaction during 5 more days, after the same treatment, AuNPs-2a were obtained with yield 90%. 1 H NMR (CDCl 3 , 200 MHz): δ ppm = 3.24 (2H, CH 2 -N 3 ), 1.55 (2H, HS-CH 2 ) 1.27 (20H, -CH 2 CH 2 -), 0.88 (3H, -CH 3 ). Through a repeated ligand-substitution process (3 times) with prolonged reaction time (3 days) in each period, AuNPs-2c were obtained in 80% yield. 1 HNMR (CDCl 3 , 200 MHz): δ ppm = 3.24 (2H, CH 2 -N 3 ), 1.58 (2H, HS-CH 2 ), 1.28 (20H, -CH 2 CH 2 -), 0.88 (3H, -CH 3 ). UV-vis: SPB at 518 nm. TEM: average diameter: 4.43 nm. IR: ν N3 2090 cm -1 .

  H NMR (CDCl 3 , 200 MHz): δ ppm = 7.46 (3H, CH in Ar, and CH in triazole), 7.15 (2H, CH in Ar), 3.45 (4H, CH 2 -carborane and CH 2 -triazole), 1.59-1.25 (20H, -CH 2 CH 2 -), 0.89 (3H, -CH 2 CH 3 ). UV-vis: SPB at 522 nm. IR: disappearance of ν N3 band at 2090 cm -1 . TEM: average core-diameter: 3.42 nm. DLS analysis: average diameter = 15.6 nm.

Figure 4 .

 4 Figure 4. FT-IR spectra of dendron 4 (upper, in blue curve) and AuNP-4b (bottom, red curve).

  Part A. A wide range of AuNP stabilizers including molecular and solid-state materials are known since last century. We report here the first AuNP stabilization attempts using a simple ""clicked"" 1,2,3-triazole ligand with a PEG tail, indicating that it can provide a good biocompatible platform towards various applications in water including catalysis and thiolate coverage. This AuNP shows excellent performance in catalyzed nitrophenol reduction, selective detection of Hg 2+ and ligand substitution of thiolate ligands. This part was accomplished in collaboration with Dr. Pengxing Zhao who actually initiated the project.Part B. The one-step decoration of AuNPs with functional ligands is a priviledged topic towards applications. Since "click" chemistry is one of the most well-known methods of linking functionalities, a family of 1,2,3-triazole derivatives that contain both a PEG chain and another functional fragment (polymer, dendron, alcohol, carboxylic acid, allyl, coumarin, redox-robust metal complex or cyclodextrin) were synthesized and coordinated to AuNPs surface in aqueous solution in a size range 3-11.2 nm. These AuNPs have potential applications according to different functional groups.Part C. Monofunctional triazoles linked to ferrocene, ferricenium or coumarin, easily synthesized by "click" reactions between the corresponding functional azides and trimethylsilylacetylene followed by silyl group deprotection, provide a variety of convenient neutral ligands for the stabilization of functional gold nanoparticles (AuNPs) in polar organic solvents. These triazole-AuNPs are very useful towards a variety of applications to synthesis, sensing and catalysis. 3218 Chem. Commun., 2013, 49, 3218--3220 This journal is c The Royal Society of Chemistry 2013 Cite this: Chem. Commun., 2013, 49, 3218 How a simple ''clicked'' PEGylated 1,2,3-triazole ligand stabilizes gold nanoparticles for multiple usage † Pengxiang Zhao, ab Na Li, a Lionel Salmon, c Na Liu, d Jaime Ruiz a and Didier Astruc* a ''Click'' chemistry now offers access to a great variety of triazoles, and the first example of a strategy to stabilize gold nanoparticles (AuNPs) with a new 1,2,3-triazole-mPEG ligand is developed here together with preliminary examples of possible applications.

  H NMR, TEM, HRTEM, EDS, Raman spectroscopy, UV-vis spectroscopy, and dynamic light scattering (DLS), and evaluate catalysis of 4-nitrophenol reduction to 4-aminophenol, selective sensing of Hg 2+ , and usage as convenient starting materials for triazole/thiolate ligand substitution and heterobifunctionalization. The synthesis and post-treatment of triazole-mPEG capped AuNPs are carried out in water (eqn (1)).

( 1 )

 1 HAu III Cl 4 is reduced to Au 0 by dropwise addition of NaBH 4 in the presence of triazole-mPEG ligands at r.t. (ESI †). The three Au:ligand (L) stoichiometries (Au/L = 0.5, 2 and 10) yield stable AuNPs for which the purple color and plasmon band remain unchanged even after refluxing the aqueous AuNPs for one hour.

  ), 1 (upper) undergoes a remarkable enhancement of the Raman absorbance at 559 cm À1 and 1096 cm À1 . The former corresponds to N-H wagging, and the This journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 3218--3220 3219 latter shows the N-N, NQN and C-H vibration in the triazole ring.

Fig. 1

 1 Fig. 1 TEM of triazole-mPEG capped AuNPs 1 (a); size distribution of 1 (b); HR-TEM of 1 (c) and its EDS spectrum (EDS peaks of Cu and Si belong to the grid template used for the analysis) (d). Raman spectra of triazole-mPEG (bottom) and triazole-mPEG capped AuNPs 1 (upper) (e).

Fig. 2

 2 Fig. 2 (a) UV-vis absorption spectra during the reduction of 4-nitrophenol catalyzed by 1 (quartz cuvette, path length: 1 cm). (b) Plot of Àln(c/c 0 ) vs. reaction time during the reduction of 4-nitrophenol catalyzed by 1 at 25 1C.

Fig. 3

 3 Fig. 3 Plasmon band shift (vs. 535 nm) of 1 (0.4 mM) with MgCl 2 , CaCl 2 , KCl, Fe(C 6 H 5 O 7 ), Ag 2 CO 3 , CuCl 2 , Hg(O 2 CCH 3 ) 2 or NaCl (all 0.05 mM).

Figure 1 .

 1 Figure 1. Structures of the disubstituted 1,2,3-triazoles 1-10, and the stabilization of AuNPs with these disubstituted 1,2,3-triazole molecules. mPEG = methoxypolyethylene glycol.

Figure 2 .

 2 Figure 2. TEM images of AuNP-2 and AuNP-6 (core diameter = 3.0 and 11.2 nm, respectively).

Figure 3 .

 3 Figure 3. Raman spectra of 5 (upper curve) and AuNP-5 (lower curve).

  in the Supporting Information), whereby various pH values were adjusted by the addition of NaOH or HCl solution. As a result, no obvious plasmon-band shift was observed in the range pH 4-9, that is, the range of physiological pH values in the cellular environment, thus indicating that the triazole-AuNPs are stable in a physiological pH environment. Fluorescence sensing of 2-CEMS Triazole-AuNPs were also used as excellent fluorescence quenchers for fluorescence-based assays because of their extraordinary high molar extinction coefficients and broad energy bandwidth. The fluorescent dye coumarin-containing triazole 6 exhibits fluorescence under UV light (Scheme 1). After combination with AuNPs, the fluorescence of the donor ligand is decreased or even totally quenched (see Figure S47 in the Supporting Information, which shows the photographs of the fluorescent 6 and AuNP-6 under illuminance of UV light at l = 365 nm). Compound 6 displays an intense band with a wavelength of maximum emission l max = 472 nm, in the emission spectrum with excitation at l ex = 405 nm (Figure 4 a ).

Scheme 1 .

 1 Scheme 1. A sketch that illustrates the fluorescence quenching of 6 by conjugation with AuNPs and the fluorescence "turn-on" detection of the sulfurmustard analogue 2-CEMS through ligand displacement from the AuNP surface.

Figure 4 .

 4 Figure 4. a) Emission spectra of 6 (0.4 mm, upper curve) and AuNP-6 (0.4 mm, lower curve) in aqueous solution. b) Emission spectra of AuNP-6 without titration of 2-CEMS, with 5 equivalents of 2-CEMS, with 10 equivalents of 2-CEMS, and 5, 10, and 15 min after the addition of 10 equivalents of 2-CEMS, respectively (l ex = 405 nm).

1 H

 1 First, AD and AuNPs-10 were separately prepared in D 2 O/ [D 6 ]DMSO (7:3; 5 mm in each case), followed by the stepwise titration of AuNP-10 into the solution of AD. After being mixed by inversion, the obtained solution with various molar ratios of AD/b-CD (1:0.5, 1:1, or 1:1.5) was monitored by NMR spectroscopic analysis (see the full-scale spectra in Figure S54 in the Supporting Information).Three characteristic peaks of AD at d = 2.25, 1.82, and 1.75 ppm for H g , H b , and H d , respectively, showed chemicalshift changes due to the chemical environment of the protons in AD as a result of the formation of the b-CD/AD host-guest inclusion complex formed by trapping AD inside the b-CD cavity (Figure6). The largest shifts for these three peaks (d = 2.36, 1.87, and 1.78 ppm for H g , H b , and H d ) were observed in the AD/b-CD ratio of 1:1.5, and no further shift emerged when more AuNP-10 was added.

Figure 5 .

 5 Figure 5. a) UV/Vis spectra in which the reaction is monitored every 40 seconds (at 22 8C). b) Consumption rate of 4-nitrophenol Àln(C/C 0 ) versus reaction time.

  PEG (M w = 2000) azide or PEGalkyne (1 g, 0.5 mmol) and a given amount of a specific alkyne or azide were dissolved in THF (10 mL). CuSO 4 •5 H 2 O (0.25 mmol, 62 mg) in aqueous solution was added to the reaction mixture, followed by the dropwise addition of a freshly prepared aqueous solution of sodium ascorbate (0.5 mmol, 99 mg) to obtain a THF/ water in a ratio of 1:1. The solution was stirred overnight at room temperature under nitrogen. After removal of THF in vacuo, CH 2 Cl 2 (5 mL) and concentrated (30 %) aqueous ammonia solution (5 mL) were added to the reaction mixture, which was stirred for 30 min to remove the Cu ions trapped inside the polymer as [Cu(NH 3 )2-(H 2 O) 2 ] 2+ . The organic layer was collected and washed with water.

Figure 6 .

 6 Figure 6. Illustration of AD encapsulation with PEG-triazole-cyclodextrin stabilized AuNP-10. 1 H NMR spectra of AD (in D 2 O/[D 6 ]DMSO (7:3), 25 8C) and AD mixed with various amounts of AuNP-10 after titration (molar ratios of AD/CD = 1:0.5, 1:1, and 1:1.5).

Figure 1 .

 1 Figure 1. AuNPs stabilized by Fc-trz (AuNP-1) or oxidized Fc + -trz Cl -(AuNP-2) in organic solution.

3 .

 3 , a shoulder band around 560 nm corresponds to the surface plasmon band (SPB) of AuNP-1, and the SPB of AuNP-2 overlaps with the ferricenium band, which is deduced from the comparison with the SPB band of AuNP-Both methanol and THF were suitable solvents in the one-step preparation of AuNPs-1 and AuNPs-2.

Figure 2 . 1 (

 21 Figure 2. UV-vis. spectra revealing the characteristic band of a) the Fc group in AuNP-1 (321 nm, 430 nm) and b) the ferricenium group in AuNP-2 (627 nm) (both were recorded in ethanol solution).

Figure 3 .

 3 Figure 3. TEM images of AuNP-1 (d = 5 nm) and AuNP-2 (d = 4.4 nm) prepared using two distinct methods.

4 -

 4 NP reduction to triazole-stabilized AuNPs of close size stabilized by other ligands such as thiolate and citrate due to the labile character of the AuNP-triazole bonds.

Figure 4 . 3 .

 43 Figure 4. Plots of the decreasing rate of 4-NP [-ln(C/C 0 )] vs. the reaction times using two distinct amounts of catalyst AuNP-2 (0.5% cat. in black curve and 1% cat.in red curve).

Figure 5 .Scheme 1 .

 51 Figure 5. Fluorescent emission spectra of Cou-trz (0.2 mM) and Cou-trz-capped AuNP-3, (both in DMSO/H 2 O solution. λ ex = 405 nm, recorded at 22 o C).

Figure 6 .

 6 Figure 6. Fluorescent emission spectra of AuNP-3 mixed with various amount of homocysteine (1 eq. analyte = 0.05 mM in the solution, λ ex = 405 nm, standard quartz cuvette: path length = 1 cm, recorded at 25 o C).

  thiol per AuNP-5. The AuNP-5 retained its core size and monodispersity during the reaction in organic solvent, which resulted in a AuNP-cored dendrimer containing about 140 ± 25 thiol dendrons, i.e. 1260 ± 180 Fc termini and displayed chemical and electrochemical reversibility together with significant adsorption in its cyclic voltammogram (Supporting Information).33 voltammetry. Easy trz ligand substitution has been shown to usefully apply to dendrimer synthesis, biothiol fluorescence "turn-on" sensing assay and nitrophenol reduction by NaBH 4 . Finally, concerning the synthetic aspect, facile AuNP-cored dendrimer construction is demonstrated upon triazole substitution by a thiol dendron.These remarkable applications show the versatility and applicability of the trz-AuNP in organic media. The strategy that consists in stabilizing stable AuNPs by easilysynthesized 1,2,3-triazoles is conclusively powerful due to the complementarity between the stability of these AuNPs and the multiple applications resulting from facile triazole substitution at the AuNP-surfaces.EXPERIMENTAL SECTIONGeneral dataAll solvents and chemicals were used as purchased. Dialysis was performed with a Spectra/Por 6 dialysis membrane. NMR spectra were recorded at 25 °C with a Bruker 300 (300 MHz) spectrometer. All the chemical shifts are reported in parts per million (δ, ppm) with reference to Me 4 Si for the1 H and 13 C NMR spectra. The infrared (IR) spectra were recorded on an ATI Mattson Genesis series FT-IR spectrophotometer. UV-vis. absorption spectra were measured with Perkin-Elmer Lambda 19 UV-vis. spectrometer. Elemental analyses were recorded on a PAR 273 potentiostat under nitrogen atmosphere. The DLS measurements were made using a Malvern Zetasizer 3000 HSA instrument at 25 °C at an angle of 90°. Fluorescence emission spectra were recorded by Spex FluoroLog 2 Spectrofluorometer. Cyclic voltammetry (CV) measurements: All electrochemical measurements were recorded under nitrogen atmosphere at 25 °C . Synthesis of the 1,2,3-triazoles. Synthesis of Fc-trz: trimethylsilyl azide (0.23 mL, 1.8 mmol) was added to a DMF and MeOH solution (3 mL, 9:1) of CuI (7.5 mg, 0.04 mmol) and propargyloxymethylferrocene (300 mg, 1.18 mmol) under N 2 in a pressure vial. The reaction mixture was stirred at 100 o C for 12 h. The mixture was cooled to room temperature (r.t.) and then filtered and concentrated. The residue was purified by silica gel column chromatography (n-hexane/EtOAc, 5:1 to 2:1) to afford Fc-trz in 60%

AuNP- 2 :AuNP- 3 :

 23 Fc-trz (11.5 mg, 0.04 mmol) was dissolved in 20 mL EtOH in a Schlenk flask, a HAuCl 4 (4.3 mg, 0.012 mmol) EtOH solution (10 mL) was then injected into the flask under a N 2 atmosphere with stirring. The solution was further stirred for 1 h, purified by dialysis against a large volume of EtOH before analysis. AuNPs were prepared in MeOH or THF with the same procedure. Cou-trz (30 mg, 0.06 mmol) and HAuCl 4 (4.3 mg, 0.012 mmol) was dissolved in 20 mL EtOH in a Schlenk flask that is refilled with N 2 . A NaBH 4 (2.5 mg, 0.06 mmol) EtOH soltion (10 mL) was then added dropwise into the flask under a N 2 atmosphere with stirring. The solution was further stirred for 1 h. AuNP-3 was purified by precipitation in EtOH and redissolution in DMSO three times.

Scheme 1 .Scheme 2 .

 12 Scheme 1. η 5 -C 5 H 5 Fe + -Induced Nona-allylation of Mesitylene Yielding the Nona-allylated Core 1

Scheme 3 .

 3 Scheme 3. Synthesis of PEG-Branched Dendrimers DEND550 (n = 11, 12) and DEND2000 (n = 40-44) by "Click" Reactions

Figure 1 . 1 H

 11 Figure1.1 H NMR spectrum of DEND2000 (in red) and1 H NMR spectra of DEND2000 after titration of HAuCl 4 (to form a trz/Au(III) ratio of 1:1 (in blue) and 1:2 (in black), individually). All the samples were prepared in D 2 O, and a few drops of acetone-d 6 were added in the case of 1:2 DEND2000/Au(III) in order to solubilize all the components.

Figure 2 .

 2 Figure 2. Dendrimer-encapsulated small AuNP (left) and large AuNP surrounded by several dendrimers (right).

Figure 3 .

 3 Figure 3. TEM images of Au-DEND550-1 (d = 3.2 nm) and Au-DEND2000-1 (d = 1.8 nm).

Figure 4 .

 4 Figure 4. Plots of the consuming rate of p-nitrophenol [-ln(C/C 0 )] vs the reaction times with various AuNPs as catalysts: (a) Au-DEND550-1, (b) Au-DEND550-5, (c) Au-DEND2000-1, (d) Au-DEND2000-5.

Figure 5 .

 5 Figure 5. Bar graph showing the kinetic constant (k value) of pnitrophenol reduction with various AuNP catalysts.

  Gold nanoparticles as electron reservoir redox catalysts for 4-nitrophenol reduction: a strong stereoelectronic ligand influence † Roberto Ciganda, ab Na Li, a Christophe Deraedt, a Sylvain Gatard, a Pengxiang Zhao, ac Lionel Salmon, d Ricardo Herna ´ndez, b Jaime Ruiz a and Didier Astruc* aThe stereoelectronic properties of the stabilizing ligands of gold nanoparticles (AuNPs) are shown to play a considerable role in their catalytic efficiency for 4-nitrophenol reduction by NaBH 4 , consistent with a mechanism involving restructuration of the AuNP surface that behaves as an ''electron reservoir''.

Fig. 1

 1 Fig.1Various AuNPs under study. See the diameters (D) in Table1. The ligands of 7 and 8 are respectively HS-PEG400 and HS-PEG2000.

Li

  Na was born in Ningxia province, China. She studied chemical engineering with Prof. Shiqiang Yan at Lanzhou University in Lanzhou, China. She then joined the research group of Prof. Didier Astruc at the University of Bordeaux, France, in 2011, for PhD study. Her research interests are the functionalization and engineering of gold nanoparticles for applications in nanomedicine. Pengxiang Zhao was born near Chengdu, China. He studied chemistry and engineering with Prof. Wuyong Chen at Sichuan University in Chengdu. He completed his PhD in 2012 in the group of Prof. Didier Astruc at the University of Bordeaux on the application of gold nanoparticles for docetaxel delivery. He is currently an independent researcher position at the Science and Technology on Surface Physics and Chemistry Laboratory at Mianyang, Sichuan, China. His research interests concern gold nanoparticles.

Figure 1 .

 1 Figure 1. Seed-mediated synthesis of AuNRs by Murphy and co-workers. From Ref. [30]. Copyright 2001 Wiley-VCH.

Figure 2 .

 2 Figure 2. TEM image of branched gold nanocrystals and the corresponding electron diffraction pattern. From Ref. [40]. Copyright 2003 American Chemical Society.

Figure 3 .

 3 Figure3. Three-step seed-mediated growth approach for making gold and silver nanorods with a controlled aspect ratio. From Ref.[45].Copyright 2005 American Chemical Society.

Figure 4 .

 4 Figure 4. TEM images of gold NRs with different positions of the plasmon bands. Scale bar = 50 nm. From Ref. [47]. Copyright 2003 American Chemical Society.

Scheme 1 .

 1 Scheme 1. Mechanisms for the formation of AuNRs. a,c) Reproduced from Refs. [45] and [60] with permission. Copyright 2005 American Chemical Society and 2006 American Chemical Society, respectively. b) Reproduced from Ref. [57] with permission. Copyright 2004 Wiley-VCH.

Scheme 2 .

 2 Scheme 2. TiO 2 -catalyzed photochemical synthesis of a) Au platelet-like nanorods and b) six-star AuNPs. Reprinted from Ref. [70] with permission. Copyright 2010 American Chemical Society.

Figure 5 .

 5 Figure 5. HRTEM images recorded from a [110]-oriented Au nanorod and the corresponding positions of the projected atom rows. From Ref. [79]. Copyright 2000 American Chemical Society.Figure 6. a,b) TEM images, c) HRTEM image, d) SAED pattern, and e) XRD pattern of gold nanobelts synthesized by a sonochemical method. From Ref. [90]. Copyright 2006 Wiley-VCH.

Figure 6 .

 6 Figure 5. HRTEM images recorded from a [110]-oriented Au nanorod and the corresponding positions of the projected atom rows. From Ref. [79]. Copyright 2000 American Chemical Society.Figure 6. a,b) TEM images, c) HRTEM image, d) SAED pattern, and e) XRD pattern of gold nanobelts synthesized by a sonochemical method. From Ref. [90]. Copyright 2006 Wiley-VCH.

Figure 7 .

 7 Figure 7. a) Formation of a nanoshell around a silica nanoparticle core, and b) the TEM image of an Au nanoshell. From Ref. [111]. Copyright 2002 American Chemical Society.

Figure 9 .

 9 Figure 9. Principle of the formation of gold nanorods by surface functionalization with biotin disulfide as well as the corresponding TEM images. Scale bars: a) 100 nm, b) 20 nm, c) 100 nm, and d) 500 nm. From Ref. [159]. Copyright 2003 American Chemical Society.

Figure 10 .

 10 Figure 10. TEM images of the aligned Au nanorods. From Ref.[171].Copyright American Chemical Society.

Figure 11 .

 11 Figure 11. Synthetic strategy for preparing nanostructure arrays with OWL. From Ref.[196]. Copyright 2011 American Chemical Society.

Figure 12 .

 12 Figure 12. SEM image of AuNPs in the form of A) rhombic dodecahedra and B) bipyramids. Scale bars: 500 nm (main images) and 100 nm (insets). From Ref.[210]. Copyright 2011 American Chemical Society.

Figure 13 .

 13 Figure 13. Formation of hot spots by end-to-end self-assembly of gold NRs in chains. From Ref. [212]. Copyright 2011 American Chemical Society.

Figure 14 .

 14 Figure 14. a) STEM images of the self-assembled chains of AuNRs from Figure 13. Scale bar = 40 nm. b) Variation of the extinction properties of NRs during the course of their self-assembly into chains (LSPR shifts from 754 to 812 nm). From Ref. [212]. Copyright 2011 American Chemical Society.

Scheme 3 .

 3 Scheme 3. Mechanism for the formation of Cu-Au alloy nanotubes. Reprinted from Ref. [231] with permission. Copyright 2012 American Chemical Society.

Figure 15 .

 15 Figure 15. a) Equilibria between DMAP, elemental Au, and protons on Au surfaces. b) SEM images of free-standing NTs. From Ref. [236]. Copyright 2010 American Chemical Society.

Figure 16 .

 16 Figure 16. a,b) SEM images and c,d) high-magnification SEM images of gold nanobelts. From Ref. [90]. Copyright 2006 Wiley-VCH.

Figure 17 .

 17 Figure 17. SEM images of Au nanobelts. From Ref. [245]. Copyright 2008 American Chemical Society.

Figure 18 .

 18 Figure 18. a) TEM image of gold nanoplates. b,c) TEM images of gold nanoplates arranged into chainlike structures. Scale bar = 200 nm. d) TEM image of a single triangular gold nanoplate and e) the corresponding electron diffraction pattern. Scale bar = 50 nm. f) TEM image of a single quasihexagonal gold nanoplate and g) the corresponding electron diffraction pattern. Scale bar = 50 nm. From Ref. [255]. Copyright 2006 American Chemical Society.

Figure 19 .

 19 Figure 19. A) TEM and B) AFM images of tadpole-shaped AuNPs. C,D) HRTEM images of the C) head and D) tail of a gold nanotadpole. Insets: the corresponding Fourier transform patterns. From Ref. [268]. Copyright 2004 American Chemical Society.

Figure 20 .

 20 Figure 20. TEM micrographs of Au nanorods: a) initial AuNRs, b) grown in the absence of KI, c-g) grown in the presence of KI and with the amount of KI decreasing from (c) to (g). h) Electron tomography 3D reconstruction of a single Au dumbbell from the sample shown in (g). From Ref. [273]. Copyright 2008 Wiley-VCH.

Figure 22 .

 22 Figure 22. SEM images of Au nanodendrites grown in mixed DTAB/b-CD solution. From Ref. [293]. Copyright 2009 American Chemical Society.

Figure 23 .

 23 Figure 23. Images and spectra of nanocups. a) SEM images from above (left) and tilted (right). The yellow curves highlight the geometries of the nanocups. Scale bar = 100 nm. b) Theoretical extinction spectra of these nanocups. The dashed red line indicates the wavelength of the incident light in SHG experiments. c) Surface-charge distribution on a nanocup (08) and the magnetic field on its symmetry plane one-quarter cycle later showing the magnetic dipole mode. From Ref. [303]. Copyright 2011 American Chemical Society.

Figure 24 .

 24 Figure 24. a-c) TEM images, X-Y computational slices, X-Y computational slices assembly (i-viii), and d) the 3D surface rendering of the hollow spherical gold nanoparticle superstructures. e) Synthesis of the AuNP superstructures. From Ref. [317]. Copyright 2010 American Chemical Society.

Figure 25 .

 25 Figure 25. A) Principle of the formation of an AuNC from an Ag nanocube, and then an Au nanoframe. 1) Formation of Au/Ag alloy nanocage, 2) selective removal of the Ag atoms from the nanobox with an aqueous etchant, 3) complete removal of Ag from the nanocage as more etchant is added. B-E) Corresponding TEM and SEM images (insets) of the Ag nanocube, Au nanobox, Au nanocage, and Au nanoframes, respectively. From Ref. [321]. Copyright 2007 American Chemical Society.

Figure 26 .

 26 Figure 26. Schematic view of an AuNP under reaction conditions for the oxidation of CO (1 vol % CO in air (100 Pa) at room temperature). An AuNP with {111} and {100} facets has a polygonal interface with the TiO 2 support. Possible catalytically active sites are highlighted in green. From Ref. [343]. Copyright 2012 Wiley-VCH.

Figure 27 .

 27 Figure 27. a) SEM image of Au/Ag-Mo nanorods, b) TEM image of Au/ Ag-Mo nanorods, c) TEM image of an Au/Ag-Mo nanorod, and d) HRTEM image of an Au/Ag-Mo nanorod. From Ref. [349]. Copyright 2012 Royal Society of Chemistry.

Figure 29 .

 29 Figure 29. Principle of the identification of pathogenic fungal DNAs by a patterned nanowire-on-film SERS sensor coupled with an exonuclease III assisted target recycling reaction. From Ref. [233]. Copyright 2011 Wiley-VCH.

Figure 30 .Figure 31 .

 3031 Figure 30. Cancer diagnostics using AuNR-enhanced light scattering. Optical dark-field microscopy of normal HaCaT cells and cancerous HSC and HOC cells incubated with anti-EGFR antibody conjugated gold nanospheres (top panels, left to right). Bottom, as above, but with gold nanorods. Anti-EGFR-conjugated gold nanoparticles specifically bound to cancer cells, thereby resulting in strong scattering under dark-field microscopy and thus enabling detection of malignant cells. From Ref. [367]. Copyright 2006 American Chemical Society

Figure 32 .

 32 Figure 32. Synthesis protocol for the formation of hybrid nanomaterials and the principle for the imaging and destruction of cancers. From Ref. [366]. Copyright 2012 Royal Society of Chemistry.

Figure 33 .

 33 Figure 33. Principle of light-controlled release of ssDNA from Au nanoshells. From Ref. [391]. Copyright 2009 Elsevier.

Figure 34 .

 34 Figure 34. Principle of the aptamer-target interaction for the bioresponsive controlled-release of AuNCs. From Ref. [397]. Copyright 2012 Royal Society of Chemistry.

( 1 )

 1 A. Franç ois, A. Laroche, N. Pinaud, L. Salmon, J. Ruiz, J. Robert, D. Astruc Encapsulation of docetaxel into PEGylated gold nanoparticles for vectorization to cancer cells and in vitro results ChemMedChem 2011, 6, 2003-2008.

2. 3 .Scheme 1 .

 31 Scheme 1. Synthesis of gold nanoparticles and encapsulation of docetaxel.

Fig. 1 .

 1 Fig. 1. TEM images of aggregates of AuPEG550 (A-B) and AuPEG550Fol (C-D). Bar = 100 nm.

  [A] test /[A] control × 100, in which [A] test is the absorbance of the tested sample and [A] control is the absorbance of control sample (Braydich-Stolle et al., 2005).

Fig. 2 .

 2 Fig. 2. TEM images showing non-lyophilized AuPEG550Fol (A-B), AuPEG2000 (C-D) and AuPEG2000Fol (E-F). Bar = 100 nm.

Fig. 3 .

 3 Fig. 3. Optical microscopy images of LnCaP cells after 144 h of contact with: (A) control without treatment; (B) cells incubated with AuPEG550Fol; (C) cells incubated with free DOC; (D) cells incubated with AuPEG550FolDOC. In (C) and (D) the DOC concentration was 60 M. The bar represents 50 m.

3. 1 .

 1 Characterization of AuPEG550 and AuPEG550Fol

Fig. 1

 1 Fig. 1 shows TEM images of AuPEG550 before click functionalization (A and B) and AuPEG550Fol (C and D), both freeze-dried. Fig. 2 shows AuPEG550Fol, AuPEG2000 and AuPEG2000Fol in aqueous suspension without freeze-drying.The AuNPs prepared by the Schiffrin-Brust(Brust et al., 1994) method showed individualized particles with a gold core diameter of 7 ± 3 nm for the nanoparticles containing PEG 550 and 10 ± 5 nm for the nanoparticles containing PEG 2000 with a narrow polydispersity, as determined by TEM (Fig.2). The freeze-drying process seems to provoke the aggregation of some nanoparticles because even after 30 min of treatment in an ultrasound bath, the TEM images showed aggregates with irregular shapes up to 400 nm (Fig.1). This phenomenon has already been described in the literature (Franc ¸ a et al., 2010).

Fig. 5

 5 Fig. 5 summarizes the cell viability measured by the MTS test after treatment with free DOC or the different AuNPs formulations containing DOC. The results are expressed as a percentage of the appropriate control (DMSO or empty nanoparticles).Fig.5Ashows LnCaP viability after a 24-h exposure to DOCloaded gold nanoparticles with PEG 550 chains, with or without folate, compared with free DOC. The presence of folate did not affect the cytotoxicity of the loaded nanoparticles at any concentration.Free DOC produced a statistically significantly greater effect at the two lower concentrations than the loaded NPs. However, above 6.10 -7 M docetaxel, the difference decreased making the effects of all treatments statistically equivalent.Fig.5Bshows the results of LnCaP viability after 24 h of exposure to DOC-loaded gold nanoparticles with longer PEG chains(PEG 2000). At the lowest concentration, the AuPEG2000DOCFol were less effective than AuPEG2000DOC; however this trend was not reproduced at other concentrations, where the two formulations yielded similar results. Unlike AuNPs with PEG 550, the effect of free DOC was statistically significantly greater than that of loaded AuNPs at all concentrations.Fig.5Cshows the viability after 48 h of exposure to free docetaxel and AuNPs bearing PEG 550. The effect of AuPEG550DOC was concentration-dependent. AuDOCPEG550Fol showed a less evident effect of concentration. There was no statistically significant difference between the effects of free DOC and the loaded AuNPs at any concentration.Fig.5Dshows cell viability after 48 h of exposure to free DOC and AuNPs(PEG 2000). Like the AuNPs with shorter PEG, AuPEG2000DOCFol demonstrated a statistically significant effect on cell viability for the two most concentrated suspensions compared with the control. The AuNPs without folate only had significant activity at 6.10 -5 M. At all concentrations, treatment with AuPEG2000DOCFol resulted in significantly lower viability than the same AuNPs without folate, except at 6.10 -9 M.

Fig. 4 .

 4 Fig. 4. Mitochondrial enzyme activity of LnCaP after 24 h (A, B), 48 h (C, D) and 144 h (E, F) exposure to free DOC or to unloaded nanoparticles containing PEG 550 (A-C-E) and PEG 2000 (B-D-F). Results are expressed as a percentage of untreated control. Values are the means ± SD of three experiments.

Fig. 5 .

 5 Fig. 5. Mitochondrial enzyme activity of LnCaP after 24 h (A, B), 48 h (C, D) and 144 h (E, F) exposure to free DOC or to loaded nanoparticles containing PEG 550 (A-C-E) and PEG 2000 (B-D-F). Results are expressed as a percentage of untreated control. Values are the means ± SD of three experiments.

137

 137

Fig.

  Fig.5Eshows the cell viability after a longer exposure time(144 h) for the loaded AuNPs with the shorter PEG chains (PEG 550). The DOC-loaded nanoparticles produced a significant reduction in the cell viability at all concentrations. There was no difference between AuNPs with and without folate. Similar results were obtained with the AuNPS with longer PEG chains at 144 h (Fig.4F). At this exposure time, the free drug showed a marked dose-response, with significant reductions in viability at all concentrations except the lowest concentration tested (6.10 -11 M).In general, free DOC showed significant cytotoxicity toward LnCaP cells at all exposure times and concentrations except after 144 h at the lowest concentration, confirming the sensitivity of this cell line to DOC. The decrease in the efficacy of the lower concentrations of DOC at long incubation times could be due to precipitation of this very hydrophobic molecule in the culture medium.Compared with free DOC, the loaded nanoparticles were less effective against LnCaP cells over the first 48 h. After 144 h of exposure the nanoparticles containing DOC were in general more effective than the free drug suggesting that there is a slower release of the incorporated drug and that its association with the AuNPs prevented its precipitation in the medium. An effect of the PEG chain length could only be observed after 48 h of exposure, when the AUNPS bearing shorter chains (PEG 550) had more effect on viability than those with longer chains(PEG 2000). This may indicate that the higher molecular weight PEG chains retained the DOC in the AuNPs longer and it was therefore less available to cells.The DOC-loaded nanoparticles reduced the mitochondrial activity by 50% or more at concentrations of 6.10 -9 M and above after 144 h of treatment (Fig.4E and F). However, no significant difference was observed between folate-bearing AuNPs and non modified ones. This might indicate that the folate residues are not orientated correctly for binding to PSMA, or that the non modified PEG chains cause steric hindrance, especially since these chains were longer than those carrying the folate. This hypothesis was confirmed by surface plasmon resonance measurements made with the Biacore instrument (data not shown). AuNPs bearing folate showed no specific interactions with folate-binding protein (FBP). A similar observation had been made concerning liposomes bearing PEG terminated with folate residues and was considered to indicate steric hindrance from the PEG chains(Botosoa et al., 2011).The decrease in the number of cells after contact with DOCloaded nanoparticles was confirmed by observation with an optical microscope. Fig.3shows far fewer cells after 144 h of treatment with free or AuNP-associated DOC compared with control cultures or cultures treated with unloaded nanoparticles, where the cells can be seen to be growing as aggregates typical for this line. Cells incubated with AuPEG2000DOC and AuPEG2000FolDOC (data not shown) showed similar changes in the appearance of the cell monolayer to AuPEG550DOC (Fig.3B) and AuPEG550FolDOC (Fig.3D), respectively.

  has prepared Au 25 (SCH 2 CH 2 Ph) 18 by a modified version of the Brust-Schiffrin procedure. In this synthesis, AuCl 4 -was phase-transferred from water to toluene, reacted with HSCH 2 CH 2 Ph, and then reduced by adding aqueous NaBH 4 . The crude product Au m (SCH 2 CH 2 Ph) n (between 1 and 2 nm) was purified by precipitation. For instance, the clusters Au 25 (SCH 2 CH 2 Ph) 18 and Au 140 (SCH 2 CH 2 Ph) 53 were isolated from the crude product by precipitation in acetonitrile, Au 25 (SCH 2 CH 2 Ph) 18 being soluble in acetonitrile unlike Au 140 (SCH 2 CH 2 Ph) 53 . The formation of [oct 4 N + ][Au 25 (SCH 2 CH 2 Ph) 18 -

Table

Table 2

 2 Microbe-stabilized AuNPs.

	Microbe type	Microbe	Localization	Size	Reference
	Bacteria	Sulfate-reducing bacteria	Intracellular	<10 nm	[315]
		Shewanella algae	Intracellular	10-20 nm	[316]
		Plectonema	Intracellular	10 nm	[317]
		Rhodopseudomonus capsulata	Extracellular	50-400 nm	[318]
	Fungi	Verticillium sp.	Intracellular	20 ± 8 nm	[319]
		Fusarium oxysporum	Extracellular	20-40 nm	[320]
	Actinomycete	streptomyces viridogens strain HM10	Intracellular	18-20 nm	[321]
		Thermomonospora sp.	Extracellular	8 nm	[322]
	Yeast	Extremophilic Yeasts	Intracellular	30-100 nm	[323]
		Yarrowia lipolyrica NCIM 3580	Intracellular	15 nm	[324]

  clusters Au 144-n Ag n (SR) 60 (SR = SCH 2 CH 2 Ph, SC 6 H 13 or SC

Table 1 .

 1 Ligand-Substitution Reaction of Azido-Terminated AuNPs-2a, AuNPs-2b, and AuNPs-2c

	azido-	percentage	reaction time	feed ratio a	yield
	AuNPs	(-N 3 )	(day)	(azido/alkane)	(%)
	AuNPs-2a	50%	5	1: 1	90
	AuNPs-2b	33%	2	1: 1	95
	AuNPs-2c	80%	3 + 3 + 3	2: 1 b	80

a Ligand ratio of azidoundecanethiol to dodecanethiolate ligand bound to AuNPs.

b

The feed ratio was 2: 1 in each time.

Table 2 .

 2 Core Size, Ligand Ratio, and Calculated Number of Carborane or PEG Ligands of Carborane-AuNPs

	AuNPs	core diameter (nm)	ligand ratio a	number of carborane b	number of PEG c
	AuNP-3a	3.42	1:1:0	86	0
	AuNPs-3b	5.18	4:1:1	65	65
	AuNPs-3c	4.75	1:1:4	55	222
	AuNPs-4a	3.43	0:3:4	74	99
	AuNPs-4b	7.59	0:1:2	282	565

a Molar ratio of dodecanethiolate: carborane: PEG. b Calculated amount of carborane per AuNP. c Calculated amount of PEG per AuNP.

  H NMR (CDCl 3 , 300 MHz), δ ppm = 7.36-7.24 (5H, H Ar ), 3.69 (28H, -CH 2 CH 2 O-), 3.61 (3H, -OCH 3 ), 3.51 (2H, CH 2 -triazole), 3.41 (2H, CH 2 -carborane), 2.73 (2H, -CH 2 SH), 2.20 (3H, carborane-CH 3 ), 1.30 (22H, -CH 2 CH 2 CH 2 -), 0.91 (3H, -CH 2 CH 3

  H NMR (CDCl 3 , 300 MHz): δ ppm = 7.81-7.80 (2H, CH Ar ), 7.71 (1H, CH triazole ), 7.33: (4H, CH Ar ), 7.25-7.24 (2H, CH Ar ), 5.59 (2H, CH 2 -triazole), 4.36 (2H, CH 2 -N 3 ), 3.47 (2H, CH 2 -carborane), 2.16 (3H, carborane-CH 3 ). 13 C NMR (CDCl 3 , 75 MHz): δ ppm = 147.60, 136.21, 134.94, 134.78, 130.86, 130.34, 128.91, 128.49, 125.86, 119.97, 75.04, 54.27, 53.85, 40.95, 23.69. 11 B NMR (CDCl 3 , 96 MHz): δ ppm = -4.19, -5.80, -10.53 (typical for o-carborane). MS (ESI, M+Na + ): calcd. for 484.3 m/z, found in 484.3 m/z. IR: ν N3 band at 2098 cm -1 , σ B-H at 2583.1 cm -1 . Anal. Calcd. for C 20 H 28 B 10 N 6 : C 52.18, H 6.13, N 18.25; found: C 51.62, H 5.99, N 18.07.

  CDCl 3 , 75 MHz): δ ppm = 194.9, 151.55, 136.45, 133.80, 109.25, 79.23, 78.42, 75.94, 75.26, 60.34, 57.09, 33.65, 30.34. MS (ESI, M+Na + ): calcd. for 351.3 m/z, found in 351.1 m/z. IR: σ CC band at 2121 cm -1 . Anal. Calcd. For C 18 H 16 O 4 S: C 65.84, H 4.91, S 9.76; found: C 65.04, H 4.98, S 9.65.

H NMR (CDCl 3 , 300 MHz): δ ppm = 6.09 (2H, CH Ar ), 4.74 (4H, CH 2 -C CH), 4.70 (2H, CH 2 -CCH), 4.07 (2H, CH 2 S-), 2.51 (2H, HC C-), 2.45 (1H, HCC-), 2.36 (3H, CH 3 -CO). 13 C NMR (

  H NMR (CDCl 3 , 300 MHz): δ ppm = 7.8-7.7 (12H, CH triazole and CH Ar ), 7.19-7.17 (12H, CH Ar ), 6.53 (2H, CH Ar in the focal point), 5.52-5.28 (12H, Ar-CH 2 -triazole), 5.01 (6H, -CH 2 -O-), 3.92 (2H, CH 2 -S-), 3.42 (6H, CH 2 -carborane), 2.28 (3H, CH 3 -CO), 2.12 (9H, CH 3 -carborane). 13 C NMR (CDCl 3 , 75 MHz): δ ppm = 195.36, 152.19, 147.77, 144.32, 136.81, 135.77, 135.56, 135.50, 135.42, 135.14, 134.22, 131.07, 130.43, 129.00, 128.90, 128.85, 128.78, 128.70, 126.03, 123.77, 120.58, 120.37, 120.17, 108.82, 75.22, 66.38, 63.24, 54.04, 54.39, 41.12, 33.72, 30.62, 23.90. 11 B NMR (CDCl 3 , 96 MHz): δ ppm = -6.38, -10.54 (typical for o-carborane). MS (MALDI-TOF, M+Na + ): calcd. for 1733.1 m/z, found in 1732.7 m/z. IR: disappearance of ν N3 band at 2098 cm -1 , σ B-H at 2583 cm -1 . Anal. Calcd. for C 78 H 100 B 30 N 18 O 4 S+H 2 O: C 54.21, H 5.95, N 14.59, S 1.86; found: C 54.00, H 5.98, N 14.24, S 2.14.

  H NMR (CDCl 3 , 300 MHz): δ = 7.8-7.7 (4H, CH triazole and CH Ar ), 7.3-7.21 (6H, CH Ar ), 6.72-6.65 (2H, CH Ar in the focal point), 5.6-5.4 (4H, Ar-CH 2 -triazole), 5.33 (2H, -CH 2 -O-), 4.77-4.76 (2H, -CH 2 -O-CCH), 4.74-4.73 (2H, -CH 2 -O-CCH), 4.10 (2H, CH 2 -S-), 3.42 (2H, CH 2 -carborane), 2.55 (1H, HCC-), 2.49 (1H, HCC-), 2.38 (3H, CH 3 -CO), 2.20 (3H, CH 3 -carborane). MS (ESI, M+Na + ): calcd. for 812.4 m/z, found in 812.4 m/z. IR spectroscopy: σ C-O-C

  H NMR (CDCl 3 , 300 MHz): δ ppm = 7.72-7.69 (6H, CH triazole and CH Ar ), 7.27-7.16 (6H, CH Ar ), 6.72-6.65 (2H, CH Ar in the focal point), 5.54-5.48 (6H, Ar-CH 2triazole), 3.61-3.50 (176H, -CH 2 CH 2 O-), 3.42 (2H, CH 2 -carborane), 3.34 (3H, CH 3 -CO), 2.12 (3H, CH 3 -carborane). 13 C NMR

  H NMR (300MHz, CDCl 3 ) δ ppm = 7.66 (1H, CH trz ), 4.65 (2H, CH 2 -trz), 4.35 (2H, CH 2 -Fc), 4.24 (2H, CH Cp ), 4.15 (2H, CH Cp ), 4.13 (5H, CH Cp ). 13 C NMR (75 MHz, CDCl 3 ) δ ppm = 145, 122.2, 82.7, 69.1, 68.9, 62.4, 50.2. IR: ν Cp at 819 cm -1 , disappearance of σ C≡C band at 2100 cm -1 . MS (ESI, m/z) [M + Na + ]: Calcd. for C 14 H 15 FeN 3 O, 320.12; found 320.0. Analysis (calcd., found for C 14 H 15 FeN 3 O): C Fc-trz (19 mg, 0.06 mmol) and NaBH 4 (2.5 mg, 0.06 mmol) was dissolved in 20 mL EtOH in a Schlenk flask, a HAuCl 4 (4.3 mg, 0.012 mmol) EtOH solution (10 mL) was then injected dropwise into the flask in a N 2 atmosphere with vigorous stirring. The solution was further stirred for 1 h, purified by dialysis against a large volume of EtOH before analysis. AuNPs were prepared in MeOH or THF with the same procedure.

	(56.59, 56.70), H (5.09, 5.34), N (14.14, 13.87) .
	Coumarin-triazole (Cou-trz) was synthesized according to the literature. 28
	Synthesis of the AuNPs
	AuNP-1:

  Two new water-soluble 1,2,3-triazole-containing nona-PEGbranched dendrimers are obtained with nine intradendritic 1,2,3-triazoles (trz). Addition of HAuCl 4 in water to these dendrimers quantitatively leads to the intradendritic formation of AuCl 3 (trz) moieties subsequent to complete Cl - substitution by trz on Au(III), whereas the analogous complexation reaction of AuCl 3 with a linear PEG trz ligand forms only an equilibrium between trzcoordinated Au(III) and Au(III) that is not coordinated to trz. Reduction of the dendrimer-Au(III) complexes to Au 0 by NaBH

	4.1 Introduction
	Part A. Dendrimers are classic branched single macromolecules that have been demonstrated
	to have extraordinary ability to hold hydrophobic guests by noncovalent bonding. A ligand
	site, such as triazole ring in the interior of dendrimers, enhances the interaction between the
	host and the guest. PEG-terminated dendrimers have a hydrophilic periphery that provides
	water solubilization of dendrimer-stabilized materials. With variious lengths of PEG
	branches, dendrimers have significant influence in stabilization of AuNPs and catalytic
	activity of 4-nitrophenol reduction reaction.
	that is 70% of thiolates on the surface of AuNP-5 are dodecanethiolate ligands (about
	400 ± 70 dodecanethiolates per AuNP). Meanwhile, approximately 140 ± 25 Fc-
	dendrons in average are located on each AuNP surface. The calculations indicate that
	there are in average about 1260 ± 180 ferrocenyl units surrounding every single
	AuNP-5 (Supporting Information).

1

H NMR (CDCl 3 , 200 MHz) δ = 7.61 (9H, CH in triazole),

6

.71 (8H, H Ar ), 5.

28, 5.16

(24 H, Ar-O-CH 2 -), 4.91 (18H, -CH 2 -Cp), 4.24-4.12 (81H, CH Cp ), 1.83, 1.59. 1.24 (22H, CH 2 -in alkyl chain), 0.86 (3H, CH 3 -CH 2 -); UV-vis. spectroscopy: plasmon band at 535 nm. DLS: 16.3 ± 3 nm. Using Leff's method,

32

the number of AuNP atoms in AuNP-5 and the number of ligands, 540 thiol ligands on the surface of AuNP-5, were determined. From the integration of H-1 (CH 3 in dodecanethiolate) and H-2 (CH in Cp) in the

1

H NMR spectrum (Supporting Information, Figure

S14

) of AuNP-5, the ratio of the numbers of the two ligands (Fc-dendron/dodecanethiolate) was calculated to be about 1:2.8, Part B. Among the transition metal-catalyzed redox reactions, reduction of nitroaromatics is one of the most crucial ones. Indeed, 4-nitrophenol (4-NP) is anthropogenic, toxic and inhibitory in nature. Its reduction product, 4-aminophenol (4-AP), finds applications as a very important substrate in industry and medicine. It has reported that surface functional groups of NPs influenced the catalytic behavior. We compared catalytic activities of different ligands (triazole, citrate or thiolate) capped AuNPs in uniform size in 4-NP reduction. The weak bonding of the trz ligands, compared to the stronger bonding of thiolate and citrate ligands, is responsible for their easy displacement from the AuNP surface by substrates. All the 1,2,3triazole (trz)-stabilized AuNPs that are examined here are much more efficient catalysts than standard AuNPs that are stabilized by the formally anionic thiolate and citrate ligands. This part was essentially conducted by Dr. Roberto Ciganda with whom we collaborated, with contributions from Christophe Deraedt and Sylvain Gatard. "Click" Synthesis of Nona-PEG-branched Triazole Dendrimers and Stabilization of Gold Nanoparticles That Efficiently Catalyze p-Nitrophenol Reduction Na Li, † María Echeverría, ‡ Sergio Moya, ‡ Jaime Ruiz, † and Didier Astruc* , † † ISM, UMR CNRS 5255, University of Bordeaux, 351 Cours de la Libeŕation, 33405 Talence Cedex, France ‡ CIC biomaGUNE, Unidad Biosuperficies, Paseo Miramoń no. 182, Edif. "C", 20009 Donostia-San Sebastiań, Spain * S Supporting Information

ABSTRACT: 4 then leads to stabilization of gold nanoparticles (AuNPs) in water. The sizes of the AuNPs stabilized by the dendritic macromolecules are further controlled between 1.8 and 12 nm upon selecting the stoichiometry of Au(III) addition per dendritic trz followed by NaBH 4 reduction. With a 1:1 Au/trz stoichiometry, the AuNP size depends on the length of the PEG tether of the dendrimer; small dendrimer-encapsulated AuNPs are formed with PEG2000, whereas large AuNPs are formed with PEG550. With Au/trz stoichiometries larger than unity, Au(III) is reduced outside the macromolecule, resulting in the formation of large interdendritically stabilized AuNPs. The formation of very small and only mildly stabilized AuNPs by neutral hydrophilic triazole ligands offers an opportunity for very efficient p-nitrophenol reduction by NaBH 4 in water at the AuNP surface.

Table 1 .

 1 TEM and SPB Data for the Dendrimer-Stabilized AuNPs

	AuNPs	SPB (nm)	TEM (d = nm)	Au atoms/NP 25
	Au-DEND550-1	516	3.2	1000
	Au-DEND550-5	535	7.3	12 000
	Au-DEND550-10	537	9	22 000
	Au-DEND550-20	538	12	53 000
	Au-DEND2000-1		1.8	180
	Au-DEND2000-5	526	5.7	5700
	Au-DEND2000-10	529	8.7	20 200
	Au-DEND2000-20	538	11.4	45 000

Table 1

 1 Catalytic AuNP activity in the reduction of 4-NP at 20 1C

	AuNPs
	1	3	Fast a	0 -	24
	2 3 4 5 6 7 8 9	3 6 6 3.5 3.6 3.5 13.5 13.5	Fast b 1.4 Â 10 À2 6.7 Â 10 À3 7.5 Â 10 À3 1.1 Â 10 À2 7 Â 10 À4 4 Â 10 À4 6 Â 10 À4	0 -0 4.3 Â 10 À2 0 2 Â 10 À2 0 1.2 Â 10 À2 0 1.9 Â 10 À2 900 1 Â 10 À3 2100 3 Â 10 À3 1200 4 Â 10 À4	29 37 40 --132 --

stabilized D (nm) k app (s À1 ) t 0 (s) k 1 (L s À1 m À2 ) E a (kJ mol À1 ) a At 13 1C: k app = 1.2 Â 10 À2 s À1 , k 1 = 1.7 Â 10 À2 L s À1 m À2 . b At 13 1C: k app = 9.6 Â 10 À3 s À1 , k 1 = 1.4 Â 10 À2 L s À1 m À2 . 10128 | Chem. Commun., 2014, 50, 10126--10129

This journal is © The Royal Society of Chemistry 2014

  ; Niidome et al., 2006; Pissuwan et al., 2006), cancer diagnosis (Lee, 2007; Llevot and Astruc, 2012; Zeng et al., 2011), tumor imaging (Copland et al., 2004; Kim et al., 2010,) and 0378-5173/$ -see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.05.031

	drug delivery
	131

  H NMR (CDCl 3 , 300 MHz) 3.60 (40H, CH 2 CH 2 O ), 3.33 (3H, OCH 3 ), 2.68 (2H, HSCH 2 ).2.2.2.3. Synthesis of FA-NHS. The synthesis of FA-NHS (Folate-NHS) was carried out as described previously(Zhang et al., 2010). Folic acid (FA) and anhydrous triethylamine were added to anhydrous DMSO. To this solution, N-hydroxy succinimide of dicyclohexyl carbodiimide were added and the reaction mixture was stirred for 48 h. After filtration, the filtrate was poured into ethyl acetate. The yellow precipitated powder was collected by filtration and washed twice with ethanol. The crude product was further purified by precipitation in ether, with a yield 77%.1 H NMR (DMSO-d6, 300 MHz) 8.60 (1H, pyrazine ring), 7.61 (1H, arom ring), 6.97 (1H, CH 2 NH arom ring), 6.63 (1H, arom ring), 4.64 (2H, CH 2 NH arom ring), 2.82 (4H, NOC CH 2 CH 2 CON ), 2.29 (2H, CH 2 CH 2 COO ), 1.99 (2H, CH 2 CH 2 CH 2 COOH). 2.2.2.4. Synthesis of AuPEGNPs. Thiolate-mPEG (Mw = 550 mg; 55 mg, 0.1 mmol) and thiolate-PEG-NH 2 HCl (Mw = 490; 49 mg, 0.1 mmol) were dissolved in 10 mL MeOH, and this solution added into a solution of HAuCl 4 (100 mg) in 30 mL (MeOH:H 2 O 1:1). After stirring for 5 min, 1 mL of a freshly prepared NaBH 4 (100 mg) aqueous solution was added dropwise and stirred vigorously for another hour. Then, MeOH was evaporated under reduced pressure, and the water phase was salted into 30 mL CH 2 Cl 2 with a minimum amount of NaCl. The organic phase was separated and dried over Na 2 SO 4 .

  H NMR (CDCl 3 , 300 MHz) 3.58 (40H, CH 2 CH 2 O ), 3.33 (3H, OCH 3 ), 3.17 (2H, CH 2 NH 2 HCl), UV-vis: Plasmon band at 530 nm.2.2.2.5. Synthesis of AuPEGNPs bearing folate residues. 20 mg of AuPEGNPs from the above synthesis was dissolved in 4 mL CH 2 Cl 2 , and 0.02 mmol FA-NHS was dissolved in 4 mL DMSO and added, 0.2 mL triethylamine (TEA) was added to the mixture and stirred overnight at room temperature. After evaporating CH 2 Cl 2 under vacuum, 50 mL ether was poured into the solution, and then the precipitate was collected and dissolved into CH 2 Cl 2 , and filtered over paper. The filtrate was evaporated to obtain the product as 14 mg of deep red crystals.1 H NMR (DMSO-d6, 300 MHz) 6.97(1H, CH 2 NH arom ring), 6.63 (1H, arom ring), 3.58 (40H, CH 2 CH 2 CO ), 3.33 (3H, OCH 3 ), 2.29 ( CH 2 CH 2 COO ), 1.99 (2H, CH 2 CH 2 CH 2 COOH). UV-vis: Plasmon band at 530 nm. Four different gold nanoparticles were synthesized using the methodology described above: AuPEG550 -Gold nanoparticles modified with PEG 400 and PEG 550 AuPEG550Fol -Gold nanoparticles modified with PEG 400 with folate and PEG 550 AuPEG2000 -Gold nanoparticles modified with PEG 400 and PEG 2000 AuPEG2000Fol -Gold nanoparticles modified with PEG 400 with folate and PEG 2000.

P. Zhao et al. / Coordination Chemistry Reviews 257 (2013) 638-665

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(a) M.-C. Daniel and D. Astruc, Chem. Rev.,

2004, 104, 293; (b) X. Chen and S. S. Mao, Chem. Rev., 2007, 107, 2891; (c) Y. Xia,

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Acknowledgments

Financial support from the China Scholarship Council (CSC) of the People's Republic of China (Ph.D. grants to Na Li and Pengxiang Zhao), the Université Bordeaux 1, the Centre National de la Recherche Scientifique (CNRS) and the Agence Nationale pour la Recherche (ANR) is gratefully acknowledged.

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

Financial support from the China Scholarship Council (CSC) (Ph.D. grants to N.L. and P.Z.), the Universities Bordeaux 1 and Toulouse III, and the CNRS is gratefully acknowledged. N.S.H. gratefully acknowledges the support by grant from the National Science Foundation (CHE-0906179) and NIU for an Inaugural Board of Trustees Professorship and for granting a Leave of Absence.

Acknowledgements

Financial support from the China Scholarship Council (CSC) (Ph.D. grants to N.L.), the Universities of Bordeaux and Toulouse III, the CNRS, and L'OrØal is gratefully acknowledged. Keywords: click chemistry • fluorescence • gold • nanoparticles • reduction • sulfur-mustard analogues Financial support from the University Bordeaux, China Scholarship Council (CSC, PhD grants to L.N. and P.Z.), Science and Technology on Surface Physics and Chemistry Laboratory (Mianyang), CNRS, IUF (D.A.), Nanosolution European consortium, and LOrØal are gratefully acknowledged. Acknowledgments We thank the Bergonié Institut, Bordeaux, France for the LnCaP cells; Ludivine Houel-Renault from the Electronic Microscopy platform -CNRS Orsay, France and Claire Boulogne from the Imagif platform -CNRS Gif-sur-Yvette, France for the TEM images, Magali Noiray from the CIBLOT platform for the Biacore analysis, the Brazilian Ministry of Education (CAPES Nanobiotec Project 04/CII-2008, 23038.019135/2009-63 and CAPES PDSE Program n • 05560/12 for a PhD grant to ROO, the University of Bordeaux 1 and the Chinese Scholarship Council (CSC) for a PhD grant to PZ and NL.

Financial support from the China Scholarship Council (CSC) (Ph.D. grants to N.L.), the University of Bordeaux, and the CNRS is gratefully acknowledged.

ABSTRACT

Monofunctional triazoles linked to ferrocene, ferricenium or coumarin, easily synthesized by copper catalyzed azide alkyne (CuAAC) "click" reactions between the corresponding functional azides and trimethylsilylacetylene followed by silyl group deprotection, provide a variety of convenient neutral ligands for the stabilization of functional gold nanoparticles (AuNPs) in polar organic solvents.

These triazole-AuNPs are very useful towards a variety of applications to synthesis, sensing and catalysis. Both ferrocenyl (Fc) and isostructural ferricenium linkedtriazoles give rise to AuNP stabilization, although by different synthetic routes. Indeed, the first direct synthesis and stabilization of AuNPs by ferricenium is obtained by reduction of HAuCl 4 upon reaction with a ferrocene derivative, AuNP stabilization resulting from a synergy between electrostatic and coordination effects. The ferricenium/ferrocene triazole-AuNP redox couple is fully reversible as shown by cyclic voltammograms that was recorded with both redox forms. These triazole-AuNPs are stable for weeks in various polar solvents, but in the same time the

CONCLUSION

In this work, the first trz-AuNPs that are soluble in organic media have been synthesized in a size range of 4.4 to 6.3 nm with narrow dispersities in ethanol with ferrocene, ferricenium and coumarin groups linked to the trz ligands. The first ferricenium-stabilized AuNPs have been synthesized by direct redox reaction between the trz-linked Fc and HAuCl 4 without any other reductant. The excellent stabilization of these AuNPs results from the synergistic stabilization by both the electrostatic effect and trz coordination. This excellence of stabilization is confirmed by the slower catalysis of 4-NP reduction by NaBH 4 than with other trz-AuNP catalysts. In addition the retention time characterized here was not found with other trz-AuNPs, indicating a higher reorganization energy at the AuNP surface than with neutral trz ligands. The two Fe II and Fe III redox forms of the trz-AuNPs with closely related AuNP core sizes were independently synthesized and shown to reversibly interconvert by cyclic

ASSOCIATED CONTENT

Supporting Information

Characterization and data of compounds and AuNPs. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author E-mail: d.astruc@ism.u-bordeaux1.fr

ACKNOWLEDGEMENT

Applications Conclusion and perspectives

In this thesis, we have investigated the synthesis, functionalization, properties of AuNPs and their applications in various fields.

The first chapter demonstrated the development of various syntheses of AuNPs and their morphology variations. These numerous synthetic methods lead to property and morphology differences of AuNPs, indicating a wide range of applications. Two classic methods of synthesis are presented: "bottom up" and "top down" approaches, each strategy having its limitations. The "bottom up" approach was comparatively preferred in terms of morphology control and further functionalization of AuNPs. This investigation gives us ideas of the preparation, and in particular the categories of stabilizers. In this chapter, the significance of novel ligand design and morphology control of AuNPs are emphasized.

The Brust-Schiffrin method leads to stable monodispersed AuNPs of small size due to the relatively strong Au-S partially polarized covalent bond. The ligand-substitution process allows the introduction of azido-terminated thiol access to the AuNPs surface and provides opportunities to graft functional groups via "click" reactions in the presence of a Cu(I)-tren catalyst. Carborane derivatives were successfully grafted on the periphery of AuNPs either by the above-mentioned "click" process or one-step stabilization of AuNPs with pre-functional carborane-branched thiol dendrons. The combination of PEG contributed to the solubility in water and the bio-compatibility of carborane-functionalized AuNPs (Chapter 2). The bifunctional AuNPs that were obtained provide a biocompatible platform in therapeutical BNCT investigation.

A family of triazole linear molecules that contain PEG and (or) another functional fragment were synthesized and utilized in the stabilization AuNPs. This new general strategy that involves the use of common "click" chemistry to stabilize AuNPs with triazole ligands in aqueous or organic media opens promising applications toward sensing, catalysis, supramolecular encapsulation and synthesis of highly functionalized thiolated-AuNPs (Chapter 3).

ANNEX Synthesis and in vitro Studies of AuNPs

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