domain: spi chim phys

text:
Second Harmonic Scattering from Mass Characterized 2D Graphene Oxide Sheets

Isabelle Russier-Antoine, a Hussein Fakhouri, a Srestha Basu, a Franck Bertorelle, a Philippe Dugourd, a Pierre-François Brevet, a Prajitha Velayudhan, b Sabu Thomas, b Nandakumar Kalarikkal b,c and Rodolphe Antoine* a

In this communication, we report the second harmonic scattering from mass characterized 2D graphene oxide sheets dispersed in an aqueous suspension, in the femtosecond regime at 800 nm laser excitation. Charge-detection mass-spectrometry, performing at the single sheet level, allows for an exhasutive molar mass distribution and thus concentration for these 2D nanomaterials samples. The orientation-averaged hyperpolarizability value is (1,36+/-0,15)x10 - 25 esu as determined by the concentration-dependent harmonic scattering signal. In addition, the multi-photon excited fluorescence spectrum is characterized by a broad band in the visible range between 350 and 700 nm centered at about 500 nm.

Two dimensional (2D) graphene has attracted considerable interest owing to its long-range  conjugation, leading for instance to extraordinary electronic and optical properties. 1,2 Also, large optical nonlinearities, including saturable absorption, four-wave mixing and two-photon absorption have been observed in pristine graphene. 3,4 However, solubility and processability of graphene come as the first issue for many perspective applications of graphene-based materials. Consequently, chemical functionalization of graphene has been focused on improving its solubility/processability in water and organic solvents. To date, the chemistry of graphene that has been reported mainly concerns the chemistry of graphene oxide (GO) 5 which has chemically reactive oxygen-containing groups including carboxylic acid and carbonyl groups at the edges of GO, epoxy and hydroxy groups on the basal planes. For GO, the unique chemical structure along with the heterogeneous electronic structure caused by the presence of sp 2 and sp 3 bonding confers intriguing properties that offer exciting prospects for the tailoring of NLO properties. 6,7 Liu et al. studied the nonlinear absorption in graphene oxide (GO) at visible wavelengths. 8 In particular, they revealed that both two-photon absorption and excited state absorption were responsible for nonlinear absorption process of GO in the case of nanosecond and picosecond pulses. Frequency conversion using 2D graphene based materials is a promising route for photonic materials due to their large optical nonlinearity. 9 Yet, for an isolated monolayer graphene, its centrosymmetry prevents electric-dipole allowed second-harmonic generation (SHG). However, SHG from this material can be obtained by breaking the inversion symmetry via an external excitation such as electrical fields, currents or charges, with dopants or structure variations or by placing them onto a substrate. 9,10 The chemically reactive oxygen-containing groups in GO can thus be viewed as doping groups breaking the inversion symmetry in 2D graphene. In addition, the oxidation leading to disruption of infinite large sp 2 domain induces the opening of bandgap that transforms the nonluminescent graphene into luminescent GO. 11 Even if the origin of luminescence in GO is still under debate, [12][13][14] GO nanosheets also exhibit multiphoton luminescence, electroluminescence, and chemiluminescence. 11 By an accurate characterization of the as-synthesized graphene oxide sheets, in particular with the measure of their molar mass, we have been able to determine for the first time their first hyperpolarizability at 800 nm laser excitation (see Fig. 1). The hyper Rayleigh scattering (HRS) technique examines concentration-dependent second harmonic generation (SHG) from colloidal dispersions to elucidate orientation-averaged hyperpolarizability <β>. Besides, polarization analysis investigates the nature of the SHG response. The benchmarking performed here permits to provide an absolute value of the hyperpolarizability for 2D graphene oxide sheets and can be used to understand the additional effect on NLO properties of targeting molecules 15 or decorating nanoparticles 16 on 2D graphene oxide platforms. 2D graphene oxide sheets were characterized by different techniques. We have obtained molar mass information for these 2D nanomaterials samples. Charge-Detection Mass-Spectrometry (CDMS) is a unique technique used to measure mass distribution. 17,18 It extends the limit of other MS devices by measuring molar weights in the range of mega-to gigadalton thus allowing the study of larger systems such as viruses, bio-particles and even NP assemblies. [19][20][21][22][23] Experiments were performed on a custom-built charge detection mass spectrometer with an electrospray ionisation (ESI) source (see details in Supplementary Information and Figs S1 andS2). This instrument is described in detail elsewhere. 17,24 The charge detection device was used in a single pass mode at the single particle level. Since graphene oxide has chemically reactive oxygen-containing groups, including carboxylic acid groups, epoxy and hydroxy groups, it is soluble in water and organic solvents and may be easily charged by the electrospray process. Charge detection mass spectrometry measures the mass of individual electro-sprayed ions thanks to the independent measurement of the m/z ratio and the charge z for thousands of individual ions. Fig. S3 in the supplementary information displays the raw data obtained by CDMS plotted in a twodimensional z vs m graph. These (z-m) maps illustrate the unique type of data that CDMS can provide. Mass histograms are built from the collection of a statistically relevant number of single mass measurements for each sample (>5000 typically).

A mass histogram from 2D graphene oxide sheets is depicted in Fig. 2 and shows an average mass of 70 MDa. The mass distribution is quite broad extending from ~10 MDa to ~1 GDa, illustrating the large polydispersity in GO samples obtained by the modified Hummers' method. 25 The average mass can be converted to an average size assuming a crude squared 2D form for the GO sheets. Knowing the atomic density of graphene, i.e. 38 atom/nm 2 , thus 2D GO samples exhibit an average size of ~390 nm in fair agreement with the dimensions observed for selected GO samples by TEM measurements (inset of Fig. 2 and Fig. S4). We would like to emphasize that the accurate and direct measurement of GO samples molar mass allows for a quantitative determination of their concentration, an essential requisite for absolute measurements of hyperpolarizability. Also, CDMS measurements allow us to have access to the entire distributions of all samples, which is not realistic with partial sampling by electron-based microscopy. Fig. S5 shows the C1s signal of the prepared graphite oxide powder obtained by x-ray photoelectron spectrometer (XPS). This signal was fitted by four components: C-C/C-H (67%,284.4 eV), C-O (18%, 286.5 eV), C=O (9.0%, 287.7 eV) and O=C-OH (6.0%, 288.8 eV) (see Table S2).

The estimated C/O ratio is ~2.6 (see table S1). Also SAED pattern from GO sheets leads to a spacing for GO planes of 0.22 nm (see Fig. S4) in accordance with previous studies. 26 The first hyperpolarizability of the as-prepared GO samples was obtained using the HRS technique (see details in Supplementary Information and Fig. S2). 27 The HRS intensity was recorded for several concentrations of the NPs dispersed in aqueous solutions (see Fig. 3a and Fig. S6) and short-range spectra were recorded on both sides of the HRS wavelength. The absolute concentration of GO was obtained by knowledge of the average molar mass of the sample (e.g. 70 10 6 g/mol). For the estimation of solubility values for GO in water, UV-vis spectroscopy was used. Using suspensions left over for more than 2 weeks, dispersion of GO (in g/l) in water using the calibration line built by measuring the absorbance at 660 nm of GO solutions with different low concentrations was obtained. 28 The linear dependence of the absorbance with concentration is given in Fig. S7. A final dispersion of GO (in g/l) in water was found to be 8.07 mg/L. Thus the initial concentration of the GO aqueous solution was 115 pM. The subtraction of the background coming from TPEF was performed by fitting the narrow band background spectra with a Gaussian function for the HRS line superposed on a linear function of the wavelength accounting for the broadband luminescence background. This subtraction procedure is allowed since the two processes, photoluminescence and HRS, are incoherent. The HRS intensity is then given by : 29 (1) where the subscript w stands for water, the reference solvent used in this experiment. Losses due to absorbance at the fundamental and harmonic frequencies were determined from separate UV-visible absorption measurements (see Fig. S8) and all data were corrected prior to the analysis. The first hyperpolarizability values <> in esu units at 800 nm laser excitation was found to be (1,36+/-0,15)x10 -25 esu. This value reported here for GO sheets is in the same order of magnitude than that reported for 2D WS2 monolayers under 1064 nm laser irradiation using paranitroaniline as the external reference. [START_REF] Forcherio | [END_REF]31 However, the SHG signal in 2D WS2 monolayers may find a different origin as compared to the SHG signal in GO because WS2 monolayers lack inversion symmetry and have therefore a non-vanishing first hyperpolarizability. Further analysis through DFT calculations have indicated that this large first hyperpolarizability in WS2 monolayers is due to resonance enhancement as well as the presence of a large joint density of states. 32 For GO sheets, the presence of reactive oxygencontaining groups, including carboxylic acid groups at the edges of GO and epoxy and hydroxy groups on the basal planes may be key factors leading to an enhanced first hyperpolarizability, as reported in this work. To go further in determining the origin of this nonlinearity, the SHG response of the GO samples was recorded as a function of the fundamental input beam polarization angle. The polar plots exhibit apurely dipolar contribution as evidenced by a perfect two-lobe pattern (see Fig. S9). Despite the size of the GO flakes, non negligible before the fundamental 800 nm wavelength, it is surprising that no retardation is observed. This result rather suggests a response stemming from the whole flake volume owing to a broken inversion symmetry rather than a response stemming from the edges only. Nevertheless, the sources to the nonlinearity seems to be distributed in orientation over the whole volume because the depolarization ratio, about 1/2, deviates from that of a single axis system. Finally, these conclusions must beconsidered in light of the flexibility of the GO flakes in solution and their possible wrinkling or wrapping, 33 geometries allowed by the observed experimental data. These geometries may lead to the appearance of non-negligible tensor elements otherwise forbidden by symmetry rules. To this end, in order to confirm the possibility of wrinkling and wrapping of GO flakes, TEM analysis was performed. Interestingly, the wrinkled and wrapped (folded) features of the GO flakes was clearly evidenced from the TEM images (see Fig. S10).

In addition to the characteristic monochromatic SHG line observed at 400 nm, emergence of a strong background is also observed at wavelengths shorter and longer than 400 nm. This background is the signature of a multiphoton excited fluorescence signal. 34 In particular, the appearance of the fluorescence at wavelengths shorter than 400 nm indicates that at least three photons are required in the excitation step. Fig. 3b presents the MPEF spectrum of GO samples recorded following a laser excitation at 800 nm. A broadband in the visible range between 350 and 700 nm and centred at about 500 nm was observed. The MPEF spectrum is similar to the one reported for aptamer conjugated graphene oxide. 15 The origin of MPEF signal for pure GO and GO composites may be due to several highly efficient intramolecular charge transfers between large -conjugated systems and the strong electron donating moieties carboxy and hydroxyl of water soluble graphene oxide sheets. Indeed, it has been shown by a computational approach that ideal graphene sheets decorated with functional groups such as hydroxyl, epoxy, carboxyl, or an atomic vacancy, which create distortions in the aromatic rings, cause a broad absorption spectra and also result in the absorption in the lower energy regions. 35 In summary, in this communication we report a nonlinear optical study of 2D graphene oxide sheets. By an accurate characterization of the as-synthesized graphene oxide sheets, and by measuring their molar mass distribution, we have been able to determine for the first time their first hyperpolarizability at 800 nm laser excitation. A large value of hyperpolarizability (e.g. (1,36+/-0,15)x10 -25 esu) is measured. This may open new avenues to applications of graphene-oxide based nanomaterials in second-order nonlinear optics. Furthermore, these data represent benchmarks for NLO properties of 2D graphene oxide sheets and shall be used in the future to understand the additional effects on the NLO properties of nanoparticles decorated 2D graphene oxide platforms. More work is needed in this area and is currently underway in our lab. The peak at 400 nm corresponds to SHG line.

Figure 2 :

 2 Figure 2: Mass histograms for graphene oxide samples obtained from charge detection mass spectrometry (CDMS) measurements. Inset. Selected TEM micrograph of a graphene oxide sheet.

Figure 1 :

 1 Figure 1: schematic showing the workflow used in this work. By an accurate physicochemical characterization of the as-synthesized graphene oxide sheets, in particular with the measure of their molar mass, concentration dependent nonlinear optical studies permit to determine their orientation-averaged hyperpolarizability value.

Figure 3 :

 3 Figure 3: (a) Plot of the HRS intensity of for GO as a function of concentration. The continuous lines correspond to the linear adjustment to eq (1). (b) multiphoton excited fluorescence spectrum of GO in aqueous solution (115 pM, blue curve) with excitation at 800 nm. The TPEF spectrum of fluorescein (10 µM in water) is also given (green curve). The peak at 400 nm corresponds to SHG line.

  International and Inter University Centre for Nanoscience and Nanotechnology and c. School of Pure and Applied Physics, Mahatma Gandhi, University, Kottayam 686560, Kerala, India.

a. Institut Lumière Matière, UMR 5306 CNRS, Université Claude Bernard Lyon 1, Domaine Scientifique de La Doua, Batiment Kastler, 10 rue Ada Byron, 69622 Villeurbanne CEDEX, France. † Footnotes relating to the title and/or authors should appear here. Electronic Supplementary Information (ESI) available: [Experimental details. 2D charge-mass image, TEM, XPS analysis, UV-vis spectrum of graphene oxide samples. HRS signal vs concentration and light polarization]. See DOI: 10.1039/x0xx00000x

Acknowledgments

The authors would like to thank Céline Brunon from Science et Surface (http://www.science-et-surface.fr) for XPS spectra and Nicholas Blanchard (iLM) for TEM measurements. This research was partially supported by the project STIM -REI, Contract Number: KK.01.1.1.01.0003, funded by the European Union through the European Regional Development Fundthe Operational Programme Competitiveness and Cohesion 2014-2020 (KK.01.1.1.01). One of the authors NK gratefully acknowledges the CNRS Visiting Professorship with Institut Lumière Matière, UMR 5306 CNRS, Université Claude Bernard Lyon 1, France. The authors PV, ST and NK also acknowledge DST-Nano Mission, Govt. Of India for projects funding.

Conflicts of interest

There are no conflicts to declare.


Martina Perić

Zeljka Sanader

Isabelle Russier-Antoine

Hussein Fakhouri

Franck Bertorelle

Pierre-François Brevet

Xavier Le Guével

Rodolphe Antoine

Vlasta Bonačić-Koutecký

Željka Sanader Maršić

Vlasta Bonačić- Koutecký

Ligand shell size effects on one-and two-photon excitation fluorescence of zwitterion functionalized gold nanoclusters

A. Introduction

In the past decade, gold nanoclusters (Au NCs) 1 have emerged as fascinating luminescent nanomaterials. 2,3 However, the photoluminescence quantum yield (QY) of most nanoclusters is still relatively low (generally of a few percent). Recent efforts focus on tailoring the emission properties, 4 including photoluminescence (PL) intensity and emission wavelength, the two basic characteristics of fluorescence. Some strategies such as surface ligand engineering, [5][6][7] aggregation-induced fluorescence, [8][9][10] silver doping, [11][12][13][14][15][16] and ligandshell rigidifying 17,18 have been achieved. In particular, the surface shell rigidification permits to decrease the energy loss due to intramolecular rotations and vibrations, thus increasing the PL intensity of nanoclusters. Introducing bidentate thiol zwitterionic ligands to cap the metallic kernel is another efficient approach to rigidify the surface structure of metal nanocluster. 19 Several groups have prepared bright fluorescent nanoclusters using this strategy. 20- 23 The PL intensity and emission wavelength of these nanoclusters can be easily tailored via controlling the functional groups of the zwitterionic ligands. 24 Indeed, due, to the zwiterionic form of the tails of ligands, strong intermolecular electrostatic interactions occur allowing for the formation of different ligand shells on the surface of the gold clusters. 24,25 Although it was found that the surface of Au NCs plays a major role in fluorescence generation, several fundamental issues are still not well understood. In particular, the extent to which the ligand shell and the metal core are coupled and how this coupling modulates the emission properties remain to be clarified. Therefore, theoretical explorations of the structural and electronic properties of various nanoclusters in the ground and excited states have been reported to address the origin of the photoluminescence enhancement. 26,27 The Aikens group investigated the geometric and electronic structural changes of gold nanoclusters upon photoexcitation using timedependent density functional theory (TD-DFT) method. [28][29][30] Moreover it was also shown that the inclusion of full ligands in calculations was needed to accurately describe the experimental optical spectra. 27,28,31 The combination of DFT with molecular mechanics within QM/MM approach appears to be appropriate and was recently used for structural elucidation of the clusters with explicit ligands. 32 Akola and coworkers 33,34 used QM/MM simulations to study the influence of aqueous solvent and explicit ligands (e.g. glutathione ligands) on the structural and electronic properties of thiolate-protected Au25(SR)18 - clusters. In this article, we show that the introduction of bidentate thiol zwitterionic ligands to cap the metallic kernel is another efficient approach to enhance nonlinear response in particular twophoton excitation fluorescence. In order to address better the ingredients responsible for their enhanced photo emission, we employ a QM/MM approach with explicit ligands. This approach permits us to address the linear and nonlinear optical regime of zwitterion functionalized gold nanoclusters (AuZw NCs) evaluating effects of ligand shell size. Optical properties including one-and twophoton absorption spectra obtained at the QM/MM level of theory are in qualitative agreement with optical measurements. Ligand shell rigidification should prevent excited state geometry relaxation through intramolecular motions, as well as through the associated non-radiative energy decay pathways. For this purpose we Please do not adjust margins

Please do not adjust margins investigated the influence of ligand shell size and the charge on linear and nonlinear properties of zwitterion functionalized gold nanoclusters. Also, we studied the penetration of water into AuZw NCs depending on the ligand shell sizes and their possible role as quencher for emission properties of AuZw NCs.

B. Results and discussion

Au NCs with thioctic-zwitterion ligand (Zw, C15H30N2O4S3, MW=399 g . mol -1 , see fig. 1) were synthesized following the protocol described elsewhere. 22 As-synthesized Au NCs were characterized by X-ray photoelectron spectroscopy (XPS) 19 and by electrospray mass spectrometry (ESI-MS). 22 By controlling the initial molar ratio of Au per Zw molecules : Au:Zw, three zwitterion stabilized Au NCs with different ligand shell sizes were synthesized (named in the following as Au:Zw 1:1, Au:Zw 1:2 and Au:Zw 1:5). XPS measurements 19 show that for AuZw 1:1; 1:2 and 1:5 the ratio Au 0 /Au + is ≈2. XPS measurements can also give a ratio between Au and sulfur atoms (~0.2). By coupling these results with mass spectrometer data, one can build AuZw clusters with a model "core" structure surrounded by a ligand shell of different size (e.g. different number of Zw molecules) (see supporting information for more details). Thus, possible molecular formula for AuZw 1:1 (≈11 kDa), AuZw 1:2 (≈ 17 kDa) and AuZw 1:5 (≈ 29 kDa) could be Au12Zw22, Au12Zw37 and Au12Zw66 respectively. In other words, the three AuZw nanoclusters can be built with a Au12Zw4 subunit surrounded by 18, 33 and 62 Zw molecules in the outer shell. Experimental optical spectra, i.e. one-photon absorption (OPA), oneand two-photon excited fluorescence, are given in supporting information (Figs. S1 andS2). One and two-photon quantum yields (1PQY and 2PQY), first hyperpolarizability values as well as twophoton absorption and excited fluorescence cross sections (TPA and TPEF) are collected in Table 1. The one-photon absorption spectra of the as-synthesized AuZw NCs are very similar and show a monotonic increase of intensity below ~500 nm. Fluorescence signals are Please do not adjust margins Please do not adjust margins strongly enhanced when Zw > Au as it was previously reported. 22 Furthermore, TPEF spectra for AuZw 1:5 display two bands in the blue and red region of visible range (See Fig. S2). Note that for AuZw 1:1 and AuZw 1:2 NCs, TPEF spectra display only one band in the blue with a weak intensity (data not shown). One and two-photon quantum yields of 1.4 % and 5x10 -3 % respectively are reported for AuZw 1:5 NCs. Experimentally, for AuZw 1:5, we found that the TPEF cross section at 800 nm excitation is 0.5 GM and the two-photon absorption (TPA) cross section 10000 GM. The first hyperpolarizability determined for an excitation wavelength of 800 nm for the AuZw NCs is found in the same range of the hyperpolarizability values reported for other protected gold nanoclusters. [35][36][37] The important point here is that linear absorption properties are not drastically changed by changing the ligand shell size and for the AuZw NCs surrounded by the largest ligand shell (i.e. AuZw 1:5), but dramatic enhancements in the one-and two-photon excited fluorescence signals are observed. The effects of ligand shell size on both the linear and nonlinear optical properties of zwitterion functionalized gold nanoclusters (AuZw NCs) have been investigated by performing QM/MM simulations. For the QM/MM study, the 12 gold atoms and the 4 zwitterionic ligands form the QM region. Ligands are either explicit zwitterions or modeled by S-CH2-CH2-CH2-S. The outer shell, surrounded by 18 or 62 explicit Zw molecules for AuZw ratios 1:1 and 1:5, are taken into account in the MM part (see computational section for more details). TDDFT QM/MM results obtained for OPA and TPA spectra for both systems are shown in Figure 1.

OPA spectra are located between 550 and 350 nm and are in qualitative agreement with experimental findings. They are of low intensities confirming that there is no explicit metallic core with delocalized electrons. In fact, structural properties of Au12Zw4 are characterized by 7 Au atoms forming two tetrahedral subunits connected by central Au atom. Note that 6 Au atoms are also part of the staple motifs (cf Figure 1). Calculated OPA spectrum for Au12Zw4 (given in Figs. S3 and S4 in supporting information) is similar to those calculated for NCs with the full ligand shell (compare fig. 1 and fig. S3). Due to the fact that the S1 state in OPA is located at ~525 nm, the lowest two-photon transition occurs approximately at ~1050 nm with a negligible value of cross section. The leading transitions for TPA involve ligand-core excitations. The calculations of additional TPA states give rise to S42 located at about 525 nm with a high value of TPA cross section. (cf Figure 1). Both OPA and TPA spectra are not dependent from the ligand shell size (compare spectra for AuZw 1:1 and 1:5) as observed experimentally for linear absorption spectra. Calculated spectra are also very similar to those obtained for Au12L4 with L modeled by S-CH2-CH2-CH2-S (see Fig. S3) confirming that linear absorption properties of S1 and S2 excited states are mainly due to excitations between central and neighboring Au atoms and therefore they do not depend on the lengths of ligands.

Our findings indicate that linear absorption properties of studied systems do not depend significantly from: i) ligand size (S-CH2-CH2-CH2-S vs explicit Zw ligand) and ii) ligand shell size (from 1:1 to 1:5). The question can be raised whether the charge has effect on optical properties. Therefore we introduced the positive charge (+1) placed near to central Au atom for system with modeled and explicit zwitterion ligands as show in Figures S5 andS6. The charge induces significant red shift of dominant features at experimentally accessible wavelengths. This is expected due to the lack of one electron. 38 An origin on the effect of ligand shell size on emission properties may be rigidification effect brought by the ligand shell on the gold kernel. However, already for AuZw 1:1, the number of ligands, the steric hindrance brought by the long-chain ligands as well as strong electrostatic interactions between permanent charges of terminal zwitterions may be enough to produce a string rigidification of the gold kernel. The addition of external ligand shells with AuZw 1:2 and AuZw 1:5 might have a moderate influence on the overall rigidity. Another origin may be due to a possible quenching effect due to water penetration propensity through the ligand shell. To better address this possible quenching effect due to water penetration propensity through the ligand shell, we investigated the density of water molecules within ligated gold clusters with AuZw ratio 1:1 and 1:5. Figure 2 illustrates that penetration of water is significantly higher for 1:1 than for 1:5 AuZw ratio. This suggests that quenching effects produced by the interaction of water with the gold kernel will be less with 1:5 ratio than with 1:1 ratio for NCs and might be responsible for measured larger emissive properties for AuZw 1:5 than in the case of AuZw 1:1. Interestingly, some analogy can be found with the ligand effects on the catalytic activities of polymerprotected gold nanoclusters. Okumura and co-workers. [39][40][41] investigated the effects of polymer type and size, and it turned out that the polymer of low active polymer-protected gold nanoclusters inhibit the penetration of H2O and reactants.

Table 1: Absolute magnitude of the first hyperpolarizability, two-photon absorption (TPA) and two-photon excited fluorescence cross-sections (TPEF) for AuZw clusters at 800 nm laser excitation wavelength. Of note, the measurements TPA and TPEF cross sections for AuZw 1:1 and AuZw 1:5 were not possible due to their poor photostability under laser irradiation (requiring higher concentrations for measurements as compared to HRS experiments).

AuZW

C. Experimental and computational details

Experimental Details. All chemical products were purchased from Sigma Aldrich (FRANCE). Synthesis of AuZw nanoclusters. lipoic acidbased sulfobetaine ligand (Zw, 399 g.mol-1 ) was synthesized following the procedure described elsewhere. 42 Au NCs were prepared with different molar ratios of Au per Zw (Au:Zw)= 1:1; 1:2, and 1:5 under basic conditions in the presence of sodium borohydride as a reducing agent. Briefly, in a standard experiment for the molar ratio Au:Zw 1:5, 12.5 mmol of Zw was added to the basic mixture of 50L of HAuCl4.3H2O (50 mM) in 10 mL of deionized water and 10L of NaOH (2M) under stirring for 5 min. Then, 100 L of freshly prepared NaBH4 (50 mM) was added to the solution and stirred for 15 hours. The solution changed quickly from colorless to brownish indicating the growth of Au NCs. Afterwards, the solution containing AuZw NCs was purified three times using 3kDa cut-off flters (Amicon ultra, Millipore) to remove the excess of free ligands.

The pH was adjusted to 7 and the AuZw NC solution was kept refrigerated until use. Instrumentation. UV-vis spectra in solution were recorded using an AvaSpec-2048 fiber optic spectrometer and an AvaLight-DH-S deuterium halogen light source. Fluorescence spectra were measured using a Fluoromax-4 Horiba fluorescence spectrophotometer. The light source for the present HRS and TPA experiments was a mode-locked femtosecond Ti:sapphire laser delivering at the fundamental wavelength of 800 nm pulses with a duration of about 140 femtoseconds at a repetition rate of 76 MHz, as described in ref. 43 . Two-photon excited fluorescence measurements were performed with a customized confocal microscope (TE2000-U, Nikon Inc.) in which the excitation light entrance has been modified to allow free-space laser beam input, instead of the original optical-fiber light input. The luminescence was excited at 780 nm with a mode-locked frequency-doubled femtosecond Er-doped fiber laser (C-Fiber 780, MenloSystems GmbH), as described in ref. 44 .

Computational Details. For the gold atoms the 19-e -relativistic effective core potential (19-e -RECP) from the Stuttgart group 45 taking into account scalar relativistic effects has been employed. For all atoms, split valence polarization atomic basis sets (SVP) have been used 46 . Coulomb-attenuated version of Becke's three-parameter non-local exchange functional together with the Lee-Yang-Parr gradient-corrected correlation functional (CAM-B3LYP) 47 have been employed to calculate optical properties. This approach has been used to determine OPA and TPA spectra (DALTON 48,49 program) for optimized structures of Au12L14 (L= S-CH2-CH2-CH2-S or zwitterionic ligand). In order to determine OPA and TPA for both systems with Au:Zw ratio 1:1 and 1:5 the two-layer quantum mechanics/molecular mechanics QM/MM approach 50 has been used. QM part includes Au12Zw4 (Zw= full ligand) and for the MM part the UFF force field (ref) has been employed. Polarizable embedding-quantum mechanics (PEQM) method with monopoles taken from UFF force field, implemented in Dalton package programs 48,49 has been used within the QM/MM approach for calculation of linear and nonlinear properties. One-and two-photon OPA and TPA absorption spectra were determined employing quadratic response properties within the time-dependent density functional theory (TDDFT). 48,49 For the calculation of TPA cross section (σ) 51 , the two-photon absorption probability (δ) is needed which can be obtained from two-photon absorption transition matrices from the ground to the excited state using either single residue or double residue quadratic response procedure 52,53 In order to simulate penetration of water molecules in both systems with Au:Zw ratios 1:1 and 1:5, 500 neutral water molecules have been included in MM part within QM/MM approach for the optimization of geometries. In addition, we formed the model by taking into account the distances from central Au atom to each water molecule in order to obtain radial distribution of the density of water molecules using R-studio software. 54

Conclusions

To summarize, the introduction of bidentate thiol zwitterionic ligands to cap the metallic kernel is another efficient approach to enhance nonlinear response in particular two-photon excitation fluorescence; The larger the ligand shell the higher the two-photon excitation fluorescence signal. Our QM/MM approach allowed for adequate description of key optical signatures of zwitterion functionalized gold clusters. These findings open the road for gaining insight into the origin of luminescence enhancement caused by different surface shell rigidification strategies such as introduction of bulky counterions or protein templating AuNCs, for which explicit templates at the molecular level are required.

Figure 1

 1 Figure 1 Comparison of OPA and TPA spectra obtained by QM/MM approach for Au12Zw4 (QM-TDDFT) with 1:1 and 1:5 ratio of Au/Zwitterions (MM)

4 |

 4 J. Name., 2012, 00, 1-3 This journal is © The Royal Society of Chemistry 20xx Please do not adjust margins Please do not adjust margins

Figure 2

 2 Figure 2 Density of water molecules as a function of radial distance from central Au atom within ligated gold clusters for ratio 1:1 (red) and 1:5 (blue)

This journal is © The Royal Society of Chemistry 20xx J. Name., 2013, 00, 1-3 | 3

Acknowledgments

This research was partially supported by the project STIM -REI, Contract Number: KK.01.1.1.01.0003, funded by the European Union through the European Regional Development Fund -the Operational Programme Competitiveness and Cohesion 2014-2020 (KK.01.1.1.01). We would also like to acknowledge the financial support received from the French-Croatian project "International Laboratory for Nano Clusters and Biological Aging, LIA NCBA". The authors would like to thank Christophe Moulin for his help with the confocal microscope.

Conflicts of interest

There are no conflicts to declare.


Estelle Porret

Muriel Jourdan

Béatrice Gennaro

Clothilde Comby-Zerbino

Franck Bertorelle

Vanessa Trouillet

Xue Qiu

Claude Zoukimian

Didier Boturyn

Niko Hildebrandt

Beatrice Gennaro

Rodolphe Antoine

Jean-Luc Coll

Xavier Le Guével

Influence of the Spatial Conformation of Charged Ligands on the Optical Properties of Gold Nanoclusters

établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

In the last decade, a new class of metal nanoparticles (NPs) composed of the assembly of few to hundreds of metal atoms (e.g., gold, silver, platinum, copper) [START_REF] Zhang | Metal nanoclusters: New fluorescent probes for sensors and bioimaging[END_REF][START_REF] Yuan | Synthesis of Highly Fluorescent Metal (Ag, Au, Pt, and Cu) Nanoclusters by Electrostatically Induced Reversible Phase Transfer[END_REF] has emerged. There are usually stabilized in solutions by ligands such as organic thiolate molecules, [START_REF] Ackerson | Thiolate Ligands for Synthesis of Water-Soluble Gold Clusters[END_REF][START_REF] Aldeek | Growth of Highly Fluorescent Polyethylene Glycol-and Zwitterion-Functionalized Gold Nanoclusters[END_REF] dendrimers, [START_REF] Zheng | Highly Fluorescent, Water-Soluble, Size-Tunable Gold Quantum Dots[END_REF] polymers, [START_REF] Duan | Etching Colloidal Gold Nanocrystals with Hyperbranched and Multivalent Polymers: A New Route to Fluorescent and Water-Soluble Atomic Clusters[END_REF][START_REF] Qu | Polyethyleneimine-Templated Ag Nanoclusters: A New Fluorescent and Colorimetric Platform for Sensitive and Selective Sensing Halide Ions and High Disturbance-Tolerant Recognitions of Iodide and Bromide in Coexistence with Chloride under Condition of High Ionic Strength[END_REF] DNA, [START_REF] Petty | DNA-Templated Ag Nanocluster Formation[END_REF][START_REF] Liu | Synthesis of DNA-templated fluorescent gold nanoclusters[END_REF] peptides, [START_REF] Negishi | Glutathione-Protected Gold Clusters Revisited: Bridging the Gap between Gold(I)-Thiolate Complexes and Thiolate-Protected Gold Nanocrystals[END_REF][START_REF] Wen | Peptide-Templated Gold Nanocluster Beacon as a Sensitive, Label-Free Sensor for Protein Post-translational Modification Enzymes[END_REF] or proteins, [START_REF] Xie | Protein-Directed Synthesis of Highly Fluorescent Gold Nanoclusters[END_REF][START_REF] Baksi | Protein-encapsulated gold cluster aggregates: the case of lysozyme[END_REF][START_REF] Wang | Construction of OVA-stabilized fluorescent gold nanoclusters for sensing glucose[END_REF]. These NPs are referred in the literature as metal nanoclusters (NCs), [START_REF] Jin | Atomically Precise Colloidal Metal Nanoclusters and Nanoparticles: Fundamentals and Opportunities[END_REF] and represent a link between metal complexes and plasmonic NPs. The common method to synthesize thiolated metal NCs in solution is based on the reduction of metal precursors in the presence of thiol ligands. Depending on the experimental conditions and the nature of the ligand, it can lead to i) NCs with atomically precise composition, [START_REF] Negishi | Glutathione-Protected Gold Clusters Revisited: Bridging the Gap between Gold(I)-Thiolate Complexes and Thiolate-Protected Gold Nanocrystals[END_REF] described by their formula M n SR m , where n and m are the respective numbers of metal atoms (M) and thiol ligands (SR), or to ii) NCs with broader size, corresponding to sub-nanometer NPs with a core size below 3 nm [START_REF] Zheng | Different sized luminescent gold nanoparticles[END_REF]. Due to their ultra-small size approaching to the Fermi wavelength of electrons, NCs exhibit discrete electronic states leading to tunable photoluminescence (PL) from the ultra-violet (UV) to the near infrared (NIR) wavelength regions. NCs could be produced with high colloidal stability in aqueous environment, [START_REF] Zheng | Different sized luminescent gold nanoparticles[END_REF] which makes them interesting candidates for bioimaging applications. [START_REF] Choi | Generation of luminescent noble metal nanodots in cell matrices[END_REF] These species exhibit a very high surface reactivity due to the high surface to volume aspect ratio. Several research groups demonstrated that the PL signal of gold NCs (Au NCs) is originated from the simultaneous contributions of the metal core [START_REF] Zheng | Highly Fluorescent, Water-Soluble, Size-Tunable Gold Quantum Dots[END_REF] and ligand coverage [START_REF] Wu | On the Ligand's Role in the Fluorescence of Gold Nanoclusters[END_REF][START_REF] Wang | NIR Luminescence Intensities Increase Linearly with Proportion of Polar Thiolate Ligands in Protecting Monolayers of Au 38 and Au 140 Quantum Dots[END_REF]. Despite extensive investigations over the past 10 years to identify the mechanisms involved in the PL of the Au NCs, [START_REF] Zhu | Correlating the Crystal Structure of A Thiol-Protected Au 25 Cluster and Optical Properties[END_REF] there are still some missing links to understand the exact role of the nature of the ligand on the optical properties of Au NCs. In this work, we investigate how increasing the quantity of charged ligands affect the growth, ligand shell organisation, colloidal stability and PL of Au NCs. Negatively charged Au NCs were synthesized using the tripeptide glutathione (SG) due to the presence of two carboxylic groups (acidic dissociation constant values, pKa~2). SG has been widely employed to synthesize atomically precise NCs. [START_REF] Negishi | Glutathione-Protected Gold Clusters Revisited: Bridging the Gap between Gold(I)-Thiolate Complexes and Thiolate-Protected Gold Nanocrystals[END_REF][START_REF] Negishi | Magic-Numbered Au n Clusters Protected by Glutathione Monolayers[END_REF] To generate neutral and positive Au NCs, we used a mixture of co-ligands during the growth of the Au NCs [START_REF] Aldeek | Growth of Highly Fluorescent Polyethylene Glycol-and Zwitterion-Functionalized Gold Nanoclusters[END_REF][START_REF] Yuan | Balancing the Rate of Cluster Growth and Etching for Gram-Scale Synthesis of Thiolate-Protected Au 25 Nanoclusters with Atomic Precision[END_REF] : SG and glutathione modified with one (SG-1Arg), two (SG-2Arg) or three (SG-3Arg) arginines. A set of 13 Au NCs with controlled charged was achieved by changing the ratio of glutathione and glutathione modified arginine peptides (SG/SG-XArg : 0/100, 65/25, 50/50, 25/75 and 100/0) during the synthesis of the Au NCs (Scheme 1). First, we analysed the surface charge and PL of the 13 types of Au NCs. Then, we selected the five different Au NCs, namely AuSG, AuSG-1Arg, AuSG-2Arg, AuSG/SG-3Arg (ratio 25/75 %) and AuSG-3Arg which presented the most significant changes to conduct a deeper understanding of the effect of arginine multivalence (X= 1, 2 or 3) and concentration on the structure, colloidal stability and PL properties of Au NCs, using a complete characterization by spectroscopic and physicochemical techniques. Increasing the amount of arginines during the synthesis of Au NCs led to PL enhancement and higher colloidal stability until to reach a threshold, at which growth of monodisperse Au NCs was not controlled anymore. Scheme 1. Synthesis route of Au NCs with SG and SG-XArg peptides.

MATERIALS AND METHODS

Materials

Arginine modified-glutathione peptides (SG-1Arg=C 16 H 29 N 7 O 7 S(CF 3 COO -), SG-2Arg=C 22 H 40 N 11 O 8 S (CF 3 COO -) 2 , SG-3Arg=C 28 H 53 N 15 O 9 S(CF 3 COO -) 3 with 10 % of H 2 O and purity ≥ 95%) were purchased from GenScript (USA). Gold(III)chloride trihydrate (HAuCl 4 •3H 2 O, reagent grade), reduced L-glutathione (C 10 H 17 N 3 O 6 S, ≥98%), sodium borohydride (NaBH 4 ), tetramethylammonium borohydride ((CH 3 ) 4 NBH 4 ), isopropyl alcohol (IPA, 99%) and 4-(Dicyanomethylene)-2-methyl-6-(4dimethylaminostyryl)-4H-pyran (DCM dye) were purchased from Sigma-Aldrich (France). Sodium hydroxide (NaOH, 98%) and hydrochloric acid (HCl, 35-37%), were purchased from Laurylab and VWR Chemicals BAH. Tris(hydroxymethyl)aminoethane (tris base, ≥99%), glycine (≥99%), acrylamide:bis-acrylamide (29:1) and agarose were purchased from Euromedex. Phosphate buffer (PBS, 10mM) were purchased from Gibco (Thermofisher, France) and formvar carbon grid from Agar scientific. All the chemicals were used as received without further purification. Water was purified using a Millipore Milli-Q system (Millipore, France).

Synthesis

Synthesis of Au NCs with co-ligands

The synthesis was achieved by adapting the protocol of Kyunglim Pyo et al. [START_REF] Pyo | Ultrabright Luminescence from Gold Nanoclusters: Rigidifying the Au(I)-Thiolate Shell[END_REF] on Au 22 SG 18 with our ligands. A set of 13 types of Au NCs was prepared by mixing glutathione (SG) and glutathione modified arginine peptides (SG-XArg, with X= 1,2 or 3) at five different molar ratios SG/SG-XArg : 100/0, 75/25, 50/50, 25/75 and 0/100 (%). Briefly for the SG/SG-1Arg (25/75 %), 12 mg of SG and 74.4 mg of SG-1Arg were dissolved in 3 mL of water, followed by the simultaneous addition of 5 mL of HAuCl 4 .3H 2 O (20 mM) and 95 mL of distilled water. Therefore, the molar ratio Au:SG/SG-XArg was 1:1.5. The solution was stirred for 3 min at 480 rpm before to adjust to pH 9 with NaOH. Then, 250 L of NaBH 4 (5 mM) freshly prepared in cold water was added by quantity of 25 L. The solution was stirred for 30 min and the pH maintained at 9. Reduction process was stopped by lowering to pH 2 with HCl. Finally, the solution was stirred during 4 hours at 350 rpm to complete the reaction. To purify the product, the solution was first rotary evaporated until nearly dry, before to be resolubilized in 10 mL of water. NCs was precipitated with IPA (around 12-15 mL) until the solution become turbid and then centrifugated (1 000 rpm, 2 min). Supernatant was centrifuged twice (2 000 rpm, 2 min on a HERAEUS Muktifuge X3R, Thermo Scientific) to recover more Au NCs. The precipitate was re-solubilised in water and adjust to pH 7 before to be lyophilized. The same procedure was applied to synthesize the others Au NCs by changing the ratio of ligands during the growth of the Au NCs.

Synthesis of Au 25 SG 18

Au 25 SG 18 were synthesized following the protocol in the Soleilhac et al. [START_REF] Soleilhac | Size Characterization of Glutathione-Protected Gold Nanoclusters in the Solid, Liquid and Gas Phases[END_REF] study. Briefly, HAuCl 4 and SG (Au:SG= 1:3) were reduced in a methanolic solution with tetramethylammonium borohydride ((CH 3 ) 4 NBH 4 ) in two steps. The product was collected after precipitation by adding methanol and centrifugate (3 times) before to be lyophilized. The physico-chemical and optical characterizations confirmed the production of Au 25 SG 18 (Figure S1). [START_REF] Negishi | Glutathione-Protected Gold Clusters Revisited: Bridging the Gap between Gold(I)-Thiolate Complexes and Thiolate-Protected Gold Nanocrystals[END_REF][START_REF] Zhu | Correlating the Crystal Structure of A Thiol-Protected Au 25 Cluster and Optical Properties[END_REF][START_REF] Wu | Probing the Structure and Charge State of Glutathione-Capped Au 25 (SG) 18 Clusters by NMR and Mass Spectrometry[END_REF] 1.3. Instrumentation

Chemical measurements

For the polyacrylamide gel electrophoresis (PAGE) experiments, separative and staking gels were prepared with total contents of acrylamide:bis-acrylamide of 25 and 2.5 % respectively. The eluting buffer consisted of 14.4 g of glycine and 3 g of tris(hydroxymethyl)aminoethane diluted in 100 mL of water and adjusted at pH 7. Samples were prepared by adding 2 L of glycerol to 20 L of Au NCs (4 mgAu/mL) before to deposit them in a well (15 L per well). The gel was run with a Mini PROTEAN Bio-Rad (Hercules, CA, USA) equipment at 150 V for 1 or 4 hours for positive and negative migrations respectively.

The same eluting buffer and samples preparation were used for the Agarose gel at 4%. The gel was run with GelXC Enduro (TM Labret International.Inc) equipment at 100 V for 30 min.

Zeta potential measurements of Au NCs solution were performed on a Zetasizer (Malvern). Measurements were performed in triplicate at 25°C. For water, 1.33 and 0.89 mPa.s were taken as refractive index and viscosity values.

Mass spectrometry measurements were collected with an electrospray ionization (ESI-MS) on a commercial quadrupole time-of-flight (micro-qTOF, BrukerDaltonics, Bremen, Germany). Isotoperesolved mass spectrum were recorded using the ultra-zoom scan mode of the instrument. The collision-induced dissociation (CID) was performed using helium gas at a normalized collision energy of 12% for 30 ms. External calibration was carried out with a set of synthetic peptides. Sample were diluted at a concentration around 0.5 mgAu/mL in H 2 O/MeOH (50/50%), electrospray at a flow rate of 10 L/min with a spray voltage of -3kV and a capillary temperature of 100°C. Other instrument settings were adjusted for each species to optimize the distribution of charges states observed in the mass spectrum. Each measurement was registered in negative and positive mode and was the summation of spectra over 5 min. If the distribution of charge states is too dense on the ESI-MS spectra, to estimate the mass of the Au NCs the signal could be deconvoluted using a multiplicative correlation algorithm :

f(m)= /g rms ∏ 𝑍1 𝑍 = 𝑍0 𝑔( 𝑚 𝑧 )
where f(m) is the intensity calculated in the spectrum for mass m, g(m/z) is the intensity in the massto charge spectrum, and g rms is the root-mean-square (rms) value of the signal measured.

High resolution transmission electron microscopy (HRTEM) images were measured on a JEOL 2010 LaB6 microscope operating at 200 kV with a 0.19 nm point-to-point resolution, after the deposition of the Au NCs (10 L, 4 mgAu/mL) on a formvar carbon grid. The images collected were analysed with ImageJ software.

Nuclear magnetic resonance spectroscopy (NMR) experiments were carried out at 298 K with a Bruker AVANCE III 500 MHz spectrometer equipped with a cryo-probe Prodigy. For each sample the concentration was ~2 mM in D 2 O and pH 7. Diffusion ordered NMR spectrocopy (DOSY) experiments were run using the standard "ledbpg2s" Bruker sequence with linear gradient stepped between 2% and 98%. 32 scans were recorded for each gradient step. Data processing were performed using the maximum entropy algorithm from Dynamics Center, a Bruker's NMR software to obtain the diffusion coefficient D. An average value of D was used for the hydrodynamic diameter (HD) calculation according to the Stokes-Einstein equation which assumes that molecules are spherical :

HD = k B T/ 3Dπη
where k B is the Boltzmann constant, T is the temperature, η is the viscosity of the solvent (η D2O = 1.232 10 -3 Pa.s at 298K). Standard 1D and 2D (NOESY, COSY, TOCSY and HSQC) spectra were recorded using standard presaturation on the water signal.

X-ray photoelectron spectroscopy (XPS) analysis was performed in a K-Alpha+ spectrometer (ThermoFisher Scientific) using a micro-focused, monochromated Al Kα X-Ray source (400 m spot size). Data acquisition and processing using the Thermo Avantage software is described elsewhere. [START_REF] Parry | ARXPS characterisation of plasma polymerised surface chemical gradients[END_REF] The K-Alpha+ charge compensation system was employed during analysis, using electrons of 8 eV energy, and low-energy argon ions to prevent any localized charge build-up. The spectra were fitted with one or more Voigt profiles (binding energy uncertainty +/-0.2 eV) and Scofield sensitivity factors were applied for quantification [START_REF] Scofield | Hartree-Slater subshell photoionization cross-sections at 1254 and 1487 eV[END_REF]. All spectra were referenced to the C 1s peak at 285.0 eV binding energy (C-C, C-H) and controlled by means of the well-known photoelectron peaks of Cu, Ag and Au, respectively. Measurements were performed at different locations of the solid and with different series of samples.

The powder product was characterized by elemental analysis (C, H, N) on an automatic analyser created by the SCA (Service Central d'Analyse) with detection limits in the range of 20-100%, 0.3-17% and 0.3-40% for the carbon, hydrogen and nitrogen respectively. Each experiment was repeated independently three times for each sample.

To remove the presence of salt that could perturbate the characterizations by elemental analysis and ESI-MS, Au NCs were further purified by diluted Au NCs in an ammonium acetate solution (1M) and filter several times to reach a final concentration of 10 nM by using Vivaspin column (Satorius, 10 kDa cutoff filters).

Optical measurements

Absorption spectra of Au NCs solution were recorded on a UV-visible spectrophotometer Lambda 650 (PerkinElmer) in the 400-900 nm range.

Fluorescence spectra of Au NCs solution were recorded on a Fluoromax-4 spectrometer (HORIBA Scientific) between 470 and 870 nm ( exc. = 450 nm).

With the aim to estimate the quantum yields (QY) of the Au NCs, fluorescence spectra of the reference DCM dye (4-(Dicyanomethylene)-2-methyl-6-(4dimethylaminostyryl)-4H-pyran) and Au NCs were recorded in methanol and in water respectively, between 430 and 800 nm, ( exc. = 410 nm). QY were calculated using the following formula: Fluorescence lifetime measurements were recorded with a fluorescence plate reader (Edinburgh Instruments) in the time correlated single photon counting (TCSPC) mode with 4096 detection bins.

QY = QY ref
Samples were excited at 405 nm (EPL-405 laser from Edinburgh Instruments) operating at 50 kHz and a dichroic long pass beam splitter of 552 nm and 660/14 nm optical bandpass filter (Semrock) were used to select the emission of Au NCs. Decay curves were fit using Fast (version 3.1.1) software from Edinburgh Instruments to triexponential decays, using the following equation:

I(t) = a i exp(--) ∑ 3 𝑖 = 1 𝐴 𝑡 𝑖
where τ i = decay times a = preexponential amplitude factor A i = amplitude fractions of the individual components (∑A i = 1) Intensity-averaged decay times were calculated using the following equation:

= i  i 〈 𝑎𝑚𝑝 〉 ∑ 3 𝑖 = 1 𝐴

RESULTS

Surface charge effects

The average charge of each 13 types of Au NCs can be predicted theoretically by using the pKa values of the carboxylic and amine groups of the different amino acids. We assumed the generation of Au NCs stabilized by 18 ligands because we used a similar protocol for the production of Au 22 SG 18 described by Pyo et al. [START_REF] Pyo | Ultrabright Luminescence from Gold Nanoclusters: Rigidifying the Au(I)-Thiolate Shell[END_REF], (see Figure S2 for the calculation details at pH 7-8). In agreement with the increased amount of arginine moieties containing amine groups (pKa~12), the Au NCs shifted from negatively to positively charged as shown in Figure 1A.

The evolution of the charge was experimentally estimated with agarose gel and eluting buffer at pH 7 under white and UV lights (Figure 1B). While Au NCs with solely SG ligand moved to the cathode indicating their negative charge, increasing the quantity of SG-XArg as co-ligand on Au NC surface led to a shift to the anode confirming the presence of positively charged NCs. This effect was more pronounced with co-ligand in the order SG-1Arg< SG-2Arg<SG-3Arg.

Surface charges of the Au NCs in water at different pH were measured by zeta potential (). As expected, the zeta potential values decreased when the pH increased from 2 to 12 (Figure 1C) due to the de-protonation of the carboxylic groups. At pH 7, the zeta potential values agreed with the agarose gel (Table 1). Indeed, Au NCs with a full coverage of SG or SG-1Arg were negatively charged while Au NCs with at least 75% of SG-2Arg or 75% of SG-3Arg were positively charged. We observed also different charge states in ESI-MS spectra, as displayed in Table 1. Clearly the average charge state <z> in positive mode of ionization increased with the number of arginines in the peptide, while in negative mode of ionization <z> slightly decreased until it was not possible to obtain any MS signal for AuSG-2Arg and AuSG-3Arg. We were not be able to deconvolute the signal coming from Au NCs with a mix of ligands (SG/SG-3Arg) in both ionization modes, due to the complexity of the multiplicative correlation algorithm. ESI-MS analysis again demonstrated that the zeta potential of Au NCs in solution were correlated with the average values of charge of Au NCs ionized by electrospray in gas phase. [START_REF] Colombé | Gold nanoclusters as a contrast agent for image-guided surgery of head and neck tumors[END_REF][START_REF] Ouadah | Correlation between the Charge of Polymer Particles in Solution and in the Gas Phase Investigated by Zeta-Potential Measurements and Electrospray Ionization Mass Spectrometry[END_REF] Page 7 of 31

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Optical properties

The absorption spectra of the Au NCs in water (pH 7.0; 0.4 mgAu/mL) illustrated in Figure 2A showed the typical profile of Au NCs with an exponential decay from the UV to the NIR region, with weak bands at 530 nm and 710 nm. The surface plasmon band at 520 nm of spherical gold nanoparticles (>10 nm) was absent of all the spectra. A slight overall increase in absorbance was observed for the sample AuSG-3Arg NCs. PL measurements of diluted Au NCs in water (pH 7.0) at the same optical density ( abs. = 450 nm, 0.04 OD) are illustrated in Figure 2B. The PL spectra showed broad emission between 570 and 860 nm with a maximum at  em. ~ 670 nm, similar to the PL spectrum of the Au 22 SG 18 described in literature [START_REF] Pyo | Ultrabright Luminescence from Gold Nanoclusters: Rigidifying the Au(I)-Thiolate Shell[END_REF][START_REF] Yu | Identification of a Highly Luminescent Au 22 (SG) 18 Nanocluster[END_REF].

The nature of the ligands clearly influenced the PL intensity and emission wavelengths of the Au NCs (Table 2). A global red shift (~30 nm) of the PL emission maxima was observed between AuSG ( em. = 662 nm) and AuSG-3Arg NCs ( em. = 693 nm). For Au NCs stabilized by a mix of SG/SG-1Arg or SG/SG-2Arg, the PL intensity progressively increased with the quantity of SG-XArg used during the synthesis.

A respectively 2-and 2.3-fold enhancement was observed between AuSG-1Arg and AuSG-2Arg NCs compared to AuSG. However, it was not the case for Au NCs stabilized by a mix of SG/SG-3Arg. The PL signal dropped (3-fold) between the ratio SG/SG-3Arg : 25/75 and 0/100 (%). Quantum yield (QY) measurements confirmed the modification of the PL intensity with the nature of the ligand (Table 2). This led to an increase of QY with the number of arginine up to 10.2% for AuSG-2Arg and then a fall to 2.5% for AuSG-3Arg. We obtained a QY of 3.4% for the AuSG NCs, which is lower than the values found for the Au 22 SG 18 in the literature between 7% [START_REF] Pyo | Ultrabright Luminescence from Gold Nanoclusters: Rigidifying the Au(I)-Thiolate Shell[END_REF] and 8% [START_REF] Yu | Identification of a Highly Luminescent Au 22 (SG) 18 Nanocluster[END_REF]. This could be explained by the use of a different dye reference, DCM dye [START_REF] Liu | Insulin-Directed Synthesis of Fluorescent Gold Nanoclusters: Preservation of Insulin Bioactivity and Versatility in Cell Imaging[END_REF] instead of Rhodamine B [START_REF] Pyo | Ultrabright Luminescence from Gold Nanoclusters: Rigidifying the Au(I)-Thiolate Shell[END_REF] or Rhodamine 6G [START_REF] Yu | Identification of a Highly Luminescent Au 22 (SG) 18 Nanocluster[END_REF].

The excitation spectra of the Au NCs were similar to the absorption spectra with two additional excitation peaks at 400 and 470 nm (Figure S3), which have been attributed to intra-and interelectronic transitions in the Au-core or between the ligands and the Au-core in the case of Au 25 SG 18. [START_REF] Zhu | Correlating the Crystal Structure of A Thiol-Protected Au 25 Cluster and Optical Properties[END_REF]. PL decay time measurements were recorded in water at  exc. = 405 nm (Table2, Figure S4). The decays curves were fit with three-exponential decays composed of two short decays with  1 ~63-78 ns (60%) and  2 ~469-640 ns (30%) and a smaller fraction of long decay with  3 ~2.09-2.53 s (10%). Increasing the number of arginine in the ligand led to an increase in the amplitude-averaged lifetime from 438 ns to 604 ns between AuSG and AuSG-2Arg, before to decrease at 410 ns for AuSG-3Arg NCs (Table 2).

Table 2. Optical properties of Au NCs stabilized by a full coverage of SG, SG-1Arg, SG-2Arg, SG-3Arg and the mix SG/SG-3Arg (25/75 %).

Colloidal Stability

As illustrated in Figure 3A, Au NCs dispersed in PBS at pH 7.0 under UV light confirmed the weak PL of the AuSG-3Arg and showed that Au NCs remained without any precipitation for at least 1 month at 4°C. The stability of these Au NCs in PBS in a wide pH range from 3 to 10 was also evaluated (Figure S5). For freshly prepared solutions, the main emission PL increased (Figure 3B) and blue shifted (~10 nm, Figure 3C) at higher pH, independent of the arginine content on Au NCs. After one month of storage time (Figure S6) Au NCs did not show significant PL changes at pH 7.0 (decrease of less than 30% for AuSG, AuSG-1Arg, AuSG-2Arg and AuSG/SG-3Arg and around 40% for AuSG-3Arg). Over time, the Au NCs stabilized by SG-XArg had similar behaviour and reached the same value independently of the pH conditions. The AuSG seemed less stable due to the higher difference of PL intensity in function of the pH after one month of storage.

Au NC size

The metal core sizes determined by HRTEM (Figure 4) showed diameters below 3 nm with an apparent semi crystalline structure. The metal core size was more monodisperse when stabilized by SG or SG-1Arg rather than SG-2Arg or SG/SG-3Arg. Interestingly the HRTEM images of AuSG-3Arg showed the presence of bigger species (>10 nm, NPs), that were probably at a concentration too low to be detected by absorption spectrometry. Acrylamide gels performed at pH 7 (Figure S7) enabled the discrimination of Au NCs present in each sample using Au 25 SG 18 as reference. For AuSG, a bright orange luminescence band was observed under UV light similar to Au 22 SG 18 in literature. [START_REF] Pyo | Ultrabright Luminescence from Gold Nanoclusters: Rigidifying the Au(I)-Thiolate Shell[END_REF][START_REF] Yu | Identification of a Highly Luminescent Au 22 (SG) 18 Nanocluster[END_REF][START_REF] Luo | From Aggregation-Induced Emission of Au(I)-Thiolate Complexes to Ultrabright Au(0)@Au(I)-Thiolate Core-Shell Nanoclusters[END_REF] The wider band for AuSG compared to Au 25 SG 18 suggested a broader size of AuSG NCs. AuSG-1Arg showed similar luminescence bands towards the anode indicating that these species present either higher molecular weight and/or stronger positive charge due to the addition of the arginines (Figure S7A). For positively charged Au NCs (SG-2Arg, SG/SG-3Arg and SG-3Arg) the bright orange luminescence band was still present with an additional weakly fluorescent brown band which migrates less into the gel. For the AuSG-3Arg NCs a sharp band stayed in the deposit well and was not detected under UV illumination probably related to the presence of few Au NPs in the sample (Figure S7B). This is furthermore in agreement with results obtained by HRTEM.

The average molecular weight of the Au NCs could be theoretically predicted assuming the synthesis of single species Au 22 SG x SG-XArg 18-x . Mass measurements of the Au NCs in water were obtained by ESI-MS in both positive and negative mode of ionization. Due to the zwitterionic ligands, a multiplicity of charge states was observed in the mass-to-charge spectrum, given rise to an "envelope" of peaks in the 800-3 000 or 1 000-4 000 m/z range after electrospray in positive or negative mode, respectively (see Figure S8 for all the spectra). For AuSG atomically precise NCs size could be identified, such as Au 15-20 SG 13-16 (6 937 -9 541 Da). A transition between atomically precise NCs size (Au 15-21 SG-1Arg 13-18 ; 8 967 -12 461 Da) and NCs with broader size dispersion occurred for AuSG-1Arg (see ESI-MS in positive in Figure S8). The set of peaks was too broad in the case of AuSG-2Arg and AuSG-3Arg. The use of a multiplicative correlation algorithm was required to deconvolute the signal with the aim to give an estimation of the Au NCs mass. For AuSG-2Arg and AuSG-3Arg the distribution was centred at 13 000 Da and 19 000 Da respectively. Since SG-XArg ligands present both acidic and basic components, it is possible that by increasing the number of Arg residue, few additional ligands could bond electrostatically on the surface of NCs. These additional ligands led to a broader range of cluster composition and thus a large mass (and thus size) dispersion. However, the average molecular weight of Au NCs obtained by mass spectrometry agreed with the predicted values (Table 3) and increased with the size of the ligand. Of note the calibration was optimized for the NCs and did not allow to detect the presence of NPs.

The size distribution of the five types of Au NCs measured by DOSY (diffusion ordered spectroscopy) in D 2 O showed an increase of the hydrodynamic diameter (HD) with the size of the ligands from 1.66±0.01 nm to 2.02±0.02 nm between AuSG and AuSG-2Arg (Table 3). An additional arginine in the peptide (SG-3Arg) did not further influence the HD. We also observed the existence of a second smaller species (~0.6 nm) that we attributed to the presence of free ligands. Regarding AuSG-3Arg, the concentration of bigger particles with size over 10 nm measured by HRTEM was probably too low to be detected by NMR.

Table 3. Size of the Au NCs stabilized by a full coverage of SG, SG-1Arg, SG-2Arg, SG-3Arg and SG/SG-3Arg (25/75 %) determined theoretically (* considering Au 22 SG x SG-XArg 18-x as main Au NCs population), by mass spectrometry ( unassigned peak in ESI-MS spectra, Figure S8) and by DOSY-NMR. The Journal of Physical Chemistry some arginine (see Figure 5 and S9-S12). These NOE peaks were totally absent in the spectra of the free ligands, which rather adopted a linear conformation (Figure S13, only data for AuSG-3Arg are presented as other spectra are similar). For AuSG-1Arg, weak NOE peaks were detected between H3 (less intense for H4) and the H and H protons of the Arg-c. For AuSG-2Arg the chemical shifts of the two H of the arginine Arg-c and Arg-t were clearly distinct and the NOE peaks were detected only between H3 (less intense for H4) and the H and H protons of the Arg-c. No NOE peaks were detected between H3 and H4 and Arg-t. For AuSG-3Arg and AuSG/SG-3Arg the Arg-t had an H chemical shift (and H and H  distinct from the two others and again NOE peaks were detected between H3 (to a less extent H4) and H H and H protons of Arg-c but we could not exclude that some NOE occurs with Arg-m as H H and H overlapped with those of Arg-c. However, because no NOE peaks were detected between H3 and Arg-t in AuSG-2Arg, we could reasonably hypothesize that no NOE occurs between H3 and Arg-m for AuSG-3Arg and AuSG/SG-3Arg. Thus, the terminal arginine chain Arg-t in the case of AuSG-2Arg (the two arginines chains Arg-m and Arg-t in the case of AuSG-3Arg and AuSG/SG-3Arg) are rather packed at the outer surface of the Au cluster. In contrary, the Arg-c is close to the glutathione part in all the clusters and forms the inner layer. It is noteworthy that we were not able to distinguish and specify if the NOE are observed between inter-or intra-ligands. Moreover, as the size of the Au-NCs increased (from AuSG-1Arg to AuSG-3Arg) the NOE peaks between the glutathione part (H3 and H4) and the arginine became more intense while levelling off and broadening for AuSG-3Arg and AuSG/SG-3Arg (Figure S9-S12). This indicates a tighter position between the ligands.

Ligand coverage

Therefore, from the ESI-MS and NOESY measurements, we could hypothesize the geometrical organization of the ligand shell stabilizing Au NCs in the following Scheme 2.

Scheme 2. Scheme of the hypothetical geometrical organisation of the ligands (SG and SG-XArg peptides) at the surface of the Au-core. Arg-t (in AuSG-2Arg), Arg-m and Arg-t (in AuSG-3Arg and AuSG/SG-3Arg) are flexible (illustrated by curved lines and arrows) and are excluded from the inner shell.

XPS measurements supports the presence of Au NCs since Au 4f 7/2 appeared at a binding energy of 84.6 eV, a higher value than for Au(0) layers (84.0 eV), like reported in the literature. [START_REF] Negishi | Glutathione-Protected Gold Clusters Revisited: Bridging the Gap between Gold(I)-Thiolate Complexes and Thiolate-Protected Gold Nanocrystals[END_REF] If we keep the hypothetical structure of Au 22 SG 18 described by Pyo et al. [START_REF] Pyo | Ultrabright Luminescence from Gold Nanoclusters: Rigidifying the Au(I)-Thiolate Shell[END_REF], the theoretical thiolate-to-gold ratio could be estimated to 0.82. Experimental values were found between 0.74 and 0.78 for the five Au NCs (Figure 6A). These values are significantly higher than for the species Au 144 SG 60 , Au 102 SG 44 , Au 38 SG 24 (0.42, 0.43 and 0.63 respectively) [START_REF] Negishi | Glutathione-Protected Gold Clusters Revisited: Bridging the Gap between Gold(I)-Thiolate Complexes and Thiolate-Protected Gold Nanocrystals[END_REF][START_REF] Jin | Atomically Precise Colloidal Metal Nanoclusters and Nanoparticles: Fundamentals and Opportunities[END_REF][START_REF] Yu | Identification of a Highly Luminescent Au 22 (SG) 18 Nanocluster[END_REF] but may correspond to smaller species, such as Au 25 SG 18 , Au 22 SG 17 , Au 22 SG 16 or Au 15 SG 13 with theoretical thiolate-to-gold ratios of 0.72, 0.77, 0.73 and 0.78 respectively [START_REF] Negishi | Glutathione-Protected Gold Clusters Revisited: Bridging the Gap between Gold(I)-Thiolate Complexes and Thiolate-Protected Gold Nanocrystals[END_REF][START_REF] Jin | Atomically Precise Colloidal Metal Nanoclusters and Nanoparticles: Fundamentals and Opportunities[END_REF][START_REF] Yu | Identification of a Highly Luminescent Au 22 (SG) 18 Nanocluster[END_REF]. This is furthermore in agreement with results obtained by ESI-MS. In addition, the presence of glutathione and arginine is proven by the peak at 400.1 eV stemming from the amide and amine groups as well as by the component at 401.8 eV attributed to the protonated nitrogen mainly due to the presence of protons in the solution (pH= 2). The nitrogen-to-thiolate ratio increased with the number of arginine introduced in agreement with the theoretical values (Figure 6B). We confirmed this trend by elemental analysis measurement, as well as the increase of concentration in carbon and hydrogen with the quantity of arginines used to synthesize the Au NCs (Figure 6C,D,E). Theoretical values were calculated assuming the production of Au 22 SG x SG-XArg 18-x (Figure S14). The Journal of Physical Chemistry

DISCUSSION

Despite evidences of the pivotal role of the surface ligands on the optical properties of Au NCs, the organization of the ligand shells has not been fully understood. In this study, we investigated the impact of ligand charges and organisation to their growth, colloidal stability, and optical properties by using the tripeptide glutathione (SG) and glutathione modified with 1, 2 or 3 arginines (SG-1Arg, SG-2Arg, SG-3Arg) as stabilizing agents. We synthesized 13 types of NCs with different charges by varying the ratio of co-ligands (SG/SG-XArg = 0/100, 65/25, 50/50, 25/75 and 100/0) during the growth of the Au NCs. We first characterized the 13 Au NCs using spectroscopic and physicochemical measurements before focusing on five representative types of Au NCs for an in-depth analysis to provide new insights on the ligand organization on the surface of Au NC.

Surface charge

Electrophoresis gels (agarose and acrylamide, respectively Figure 1B and S7), zeta potential (Figure 1C) and the average charge state <z> (Table 1) measurements confirmed the increase of positive charges on Au NCs with the amount of arginine in agreement with the theoretical values (Figure 1A andS2).

The evaluation of the impact of the Au NCs stability revealed an enhancement of the emission and a blue shift of the main emission with increasing pH (Figure 3B andC). Theoretical (Figure S2) and experimental values of the pKa of Au NCs as a function of the amount of arginine clearly indicated that we could tailor precisely the overall surface charge of Au NCs.

Ligand effect on NC's size

The use of different amounts and multivalency of arginine during Au NC synthesis had a significant impact on the growth of the Au NCs. Clearly AuSG NCs (expected to be Au 22 SG 18 ) displayed mass spectra that were typical of NCs obtained at atomic precision, such as our reference (Au 25 SG 18 ). On the other hand, AuSG-2Arg and AuSG-3Arg displayed mass spectra with a quite broad molecular weight distribution. And AuSG-1Arg NCs displayed mass spectra that combines both behaviours (Figure S8). Such broad molecular weights distribution in mass spectra were already reported for zwitterionliganded NCs, [START_REF] Colombé | Gold nanoclusters as a contrast agent for image-guided surgery of head and neck tumors[END_REF][START_REF] Musnier | High photoluminescence of shortwave infrared-emitting anisotropic surface charged gold nanoclusters[END_REF][START_REF] Shen | Zwitterion functionalized gold nanoclusters for multimodal near infrared fluorescence and photoacoustic imaging[END_REF] and generally attributed to the extra additions of ligands (bond electrostatically) on the surface of NCs. In our case, this few additional ligands that occurred between AuSG-1Arg and AuSG-2Arg led to a broad range of cluster size. HRTEM (Figure 4) and acrylamide gel (Figure S7) confirmed the presence of more monodisperse Au NCs stabilised by SG and SG-1Arg rather than SG-2Arg, SG/SG-3ARg and SG-3Arg. It should be noted that using a peptide with high number of arginines (SG-3Arg) led to bigger species, most probably related to the difficulty of controlling Au NCs involved in multiple electrostatic interactions. All these results suggested the presence of more than one species for each Au NCs synthesized in our study, which is in agreement with the absence of specific absorption bands of atomically precise NCs (Au 15 SG 13 , Au 18 SG 15 , Au 22 SG 18 ) [START_REF] Negishi | Glutathione-Protected Gold Clusters Revisited: Bridging the Gap between Gold(I)-Thiolate Complexes and Thiolate-Protected Gold Nanocrystals[END_REF] in the absorption spectra (Figure 2A). For AuSG, AuSG-1Arg, AuSG-2Arg and AuSG/SG-3Arg, a metal core size (<3 nm) evidenced by HRTEM and the thiol-to-gold ratio determined by XPS (Figure 6A) indicated both that Au NCs are probably stabilized by almost the same number of ligands. However, the overall size of the ligand increased with the number of arginine as followed SG<SG-1Arg<SG-2Arg. This suggests an enhancement of the density within the organic shell.

In order to get a deeper insight into the effect of the amount and multivalency of arginine on the organisation of the ligand shell, we conducted a NMR study. The presence of inter and/or intra ligand spatial proximity could be clearly identified by the detection of NOE peaks between the H3 (and to a lesser extent H4) of the glutathione and H and H protons of the Arg-c (Figure 5, Figure S9-12). The Arg-c had limited freedom at the Au NCs surface compared to the Arg-m and Arg-t. Therefore, the Arg-c could form an inner rigid layer whereas the Arg-m and Arg-t could form an outer flexible layer on the Au NCs surface leading to the different Au NCs structures observed between SG-1Arg and SG-2Arg, SG/SG-3Arg or SG-3Arg. Thanks to ESI-MS and NMR experiments, we thus could propose a reasonable organisation of the ligands on the Au-core surface (Scheme 2).

Ligand charge and organisation affect NCs' photoluminescence

All Au NCs displayed PL in the orange-red wavelength range with a maximum between 662 and 693 nm and a large Stokes shift (> 300 nm). Thiolate-to-gold ratio and electrophoresis mobility properties showed similar patterns to the ones obtained with Au 22 SG 18, [START_REF] Pyo | Ultrabright Luminescence from Gold Nanoclusters: Rigidifying the Au(I)-Thiolate Shell[END_REF][START_REF] Yu | Identification of a Highly Luminescent Au 22 (SG) 18 Nanocluster[END_REF][START_REF] Luo | From Aggregation-Induced Emission of Au(I)-Thiolate Complexes to Ultrabright Au(0)@Au(I)-Thiolate Core-Shell Nanoclusters[END_REF] despite the presence of more than one species for each Au NC.

We observed a significant enhancement of the intensity and a red-shift of the PL emission at pH 7.0 with the amount of arginine on Au NCs with QYs increasing from 3.8 % for AuSG to 10.2 % for AuSG-2Arg (Figure 2B and Table 2). Based on the relatively good colloidal stability of the AuSG-XArg over a month of storage in PBS at pH 7 (Figure S5, S6), we could exclude that this enhancement of PL originated from Au NC aggregation. A PL enhancement related to the metal core size, so-called quantum confinement phenomenon (Au-Au transitions), is also unlikely to happen in our case due to the obtention of similar Au NCs core size determined by HRTEM. [START_REF] Zheng | Different sized luminescent gold nanoparticles[END_REF] The HD of the Au NCs increased with the number of arginine per ligands before reaching a maximum with AuSG-2Arg (Table 3). Thus, PL enhancement was most likely not induced by the formation of supramolecular assembly. [START_REF] Guevel | Ligand effect on the size, valence state and red/near infrared photoluminescence of bidentate thiol gold nanoclusters[END_REF] Indeed, the SG-to-Au molar ratio (1.5:1) used to synthesize the Au NCs is too low to enable the formation of a second ligand shell.

In our case, the densification of the ligand shell with an increasing amount of arginine might induce a rigidification that could limit the intramolecular vibrations and rotations of the ligands and enhance the steric hindrance for the foreign species (H 2 O and O 2 molecules). By this way, the stability of the Au NCs should increase and the probability of nonradiative relaxation should decrease, which would result in PL enhancement. This hypothesis is confirmed by the fact that AuSG-XArg NCs were more stable than AuSG, after one month of storage in PBS buffer at pH increasing from 3 to 10 (Figure S5,S6). This result was in agreement with a study from X. Yuan et al. [START_REF] Yuan | Enhancing stability through ligand-shell engineering: A case study with Au25(SR)18 nanoclusters[END_REF] which showed that using a mix of ligands with similar length but opposite charge could efficiently improve the stability of Au NCs. The storage time in aqueous condition of Au 25 MPA 11 Cyst 7 NCs (~120h) was 40 times longer than that of Au 25 MPA 18 (~3h). Indeed, the use of protecting ligands with a mix of charges amplifies the rigidification of the ligand shell by eventually inducing coordination among the surface ligands via inter-ligand electrostatic interaction (i.e., -COO-···-NH 3 + bond). Similar effects were observed for the Au NCs stabilized by SG-XArg ligands. The NMR results clearly identified the presence of inter-and/or intra-ligands spatial proximity and the broad molecular weight distribution in ESI-MS spectra suggested the addition of few ligands that bond electrostatically on the surface of the Au NCs (Scheme 2). Therefore, the main explanation for the PL and stability enhancement of the Au NCs is the formation of a compact SG-XArg shell that decreases the probability of nonradiative relaxation and protects the Au core from solvent molecules.

In addition, several studies [START_REF] Wu | On the Ligand's Role in the Fluorescence of Gold Nanoclusters[END_REF][START_REF] Wang | NIR Luminescence Intensities Increase Linearly with Proportion of Polar Thiolate Ligands in Protecting Monolayers of Au 38 and Au 140 Quantum Dots[END_REF][START_REF] Goswami | Luminescent Metal Nanoclusters with Aggregation-Induced Emission[END_REF] have reported that the nature of the ligands strongly influences the PL of the Au NCs by charge transfer from the ligand to the metallic core via the Au-S bonds, so called ligand-to-metal-charge transfer (LMCT). Another transfer, the LMMCT go further by implied aurophilic interactions (Au(I)---Au(I)) when the distance between gold atoms are less than 3.6A°. [START_REF] Cha | Preparation and Photoluminescent Properties of Gold(I)-Alkanethiolate Complexes Having Highly Ordered Supramolecular Structures[END_REF] The effects of the electron donation capability of electron rich atoms (N, O) in the ligand were also studied. For a given core size of Au 38 (SPhX) 24 initially stabilized by (HSPhC 2 ), a 5-fold emission enhancement was observed when some ligands were replaced by HSPhNO 2 rather than HSPhOCH 3 . [START_REF] Wang | NIR Luminescence Intensities Increase Linearly with Proportion of Polar Thiolate Ligands in Protecting Monolayers of Au 38 and Au 140 Quantum Dots[END_REF] Shortening the distance between the electron rich group and the gold core seemed also important to improve the PL. Indeed, the PL of Au 140 [S(CH 2 ) 5 CH 3 ] 53 was greatly improved after ligand exchange of just 3 MPA (3-mercaptopropionic acid, HS(CH 2 ) 2 CO 2 H but no effect was observed for 7 MUA (mercaptoundecanoic acid, HS(CH 2 ) 10 CO 2 H). We also observed a PL enhancement with increasing amounts of arginine, which contains electron rich atoms (O, N). Based on the previous examples, the electron delocalisation of rich atoms, thanks to LMCT and LMMCT, might also be responsible of this PL increase at a lower degree, due to the presence of the first rigid layer.

In the case of PL decrease for AuSG-3Arg NCs, it could be related to the quenching effect due to the presence of Au NPs observed by TEM and PAGE.

CONCLUSION

In this study, we prepared a library of charged Au NCs and investigated the influence of the ligand shell charge and organisation to the growth, colloidal stability, and PL properties of Au NCs. Using co-ligands glutathione and glutathione modified arginine peptides at different ratios we were able to generate, in one step, a series of Au NCs with controllable number of reactive groups (carboxylic or amine) leading to overall negative, neutral or positive charge at physiological pH. We observed an improvement of the colloidal stability over time of the Au NCs by selecting ligands with opposite charges. Mass spectrometry measurements revealed a transition between the formation of atomically precise NCs to broad molecular weight distribution NCs with the increasing numbers of arginine on the ligand used to stabilize the Au-core. An enhancement of the PL intensity and bathochromic shift of the maximum emission peak was correlated to the formation of a compact ligand shell via inter and/or intra ligand spatial proximity, as demonstrated by NOESY experiments. This compact shell limited the intramolecular vibrations and rotations of the ligands and protected the Au NC from the environment, which decreased the probability of nonradiative relaxation and could be the main explanation for the stability and PL enhancement of the Au NCs. The electron delocalisation from the ligand to the gold core (LMCT and LMMCT) could also contribute to the improvement of PL, at a lower degree. This work highlights the importance of precisely controlling the particle surface to synthesize Au NCs for dedicated applications. Due to their fluorescence in the NIR region (~ 670 nm), QY (up to 10%), relatively long excitation lifetimes (> 400 ns), good stability in PBS at pH 7.0, and various charges these Au NCs appear as promising tool for (bio)imaging or sensing.

SG-1Arg

  quantum yield of the reference DCM dye (QY = 44%,  em . = 615 nm, in methanol), η = refractive index of H 2 O (1.333) η ref = refractive index of MeOH (1.326) I = integration of the emission spectra for the sample I ref = integration of the emission spectra for the DCM dye A = optical density (0.05 OD at λ exc . = 410 nm for the sample and the reference).

Figure 1 .

 1 Figure 1. (A) Theoretical charge values of the Au NCs at pH 7-8. (B) Agarose gel at pH 7 of concentrated Au NCs solution (4 mgAu/mL) under UV ( exc. = 366 nm; above) and white (below) lights. (C) Zeta potential measurements of diluted Au NCs solution (0.04 mgAu/mL) in PBS buffer at pH from 2 to 12.

Figure 2 .

 2 Figure 2. (A) Absorbance spectra of Au NCs in water (0.4 mgAu/mL, pH 7.0) between 400 and 900 nm. (B) PL spectra of diluted Au NCs in water (0.04 mgAu/mL, pH 7.0) at the same optical density (0.04 OD) between 470 and 870 nm ( exc. = 450 nm).

Figure 3 .

 3 Figure 3. Evaluation of the optical stability of Au NCs in PBS (0.4 mgAu/mL) at different pH (3-10) stabilized by a full coverage of SG, SG-1Arg, SG-2Arg, SG-3Arg and the mix SG/SG-3Arg (25/75 %). (A) Photos of Au NCs freshly prepared or after one month of storage at pH 7.0 and 4°C under white (left) or UV ( exc. = 366 nm; right) lights. (B) Evolution of the PL intensity ( max. ) and (C) shift of the PL emission peak of Au NCs freshly prepared as a function of the pH from 3 to 10.

Figure 4 .

 4 Figure 4. HRTEM of Au NCs stabilized by a full coverage of (A) SG, (B) SG-1Arg and (C) SG-2Arg (scale bars: 20 nm) or (D) SG/SG-3Arg (25/75 %) and (E) SG-3Arg (scale bars: 50 nm).

Figure 5 .

 5 Figure 5. Representation of observed NOE (red arrows) between protons of the SG part (H3 and H4) and protons of arginine (H and H) when the ligands are covalently bound to the Au-core. The light red arrow represents NOE that could not be excluded but that are highly unlikely.

Figure 6 .

 6 Figure 6. Theoretical value (red) and data extracted from XPS spectra (black) of the Au NCs stabilized by a full coverage of SG, SG-1Arg, SG-2Arg, SG/SG-3Arg and SG-3Arg for thiolate-to-gold ratios (A) and nitrogen-to-thiolate ratios (B). 5% of the measurement was taken as error bar. Theoretical values (red) and data extracted from elemental analysis (black) of concentration (%) in carbon (C), hydrogen (D) and nitrogen (E) in Au NCs samples stabilized by a full coverage of SG, SG-1Arg, SG-2Arg, SG-3Arg and SG/SG-3Arg (25/75 %). Standard deviation was taken as error bar.

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Table 1 :

 1

The average charge state (<z>) extracted in positive and negative modes of ionization by ESI-MS and zeta potential values associated (H 2 O, pH 7). <z> is calculated from the observed charge state distribution in ESI-MS spectra (plotted in m/z).

ACKNOWLEDGEMENTS

XLG would like to thank Cancéropôle Lyon Auvergne Rhône-Alpes (CLARA), Plan Cancer (C18038CS) and ARC (R17157CC) for their financial support. EP would like to thank GDR or-Nano for their financial support, Martine Fayolle for elemental analysis and Laetitia Rapenne for the HRTEM measurements, the CERMAV laboratory and the DCM laboratory CIRE for sharing the equipments. The authors also acknowledge support from the ICMGFR 2607 NMR platform, and Labex ARCANE and CBH-EUR-GS (ANR-17-EURE-0003).