domain: chim sdv

text:
Nathalie Dupont

Milosevic Irena

Barbey Carole

Rinnert Emmanuel

Lidgi-Guigui Nathalie

Guenin Erwann

La Chapelle

Marc Lamy

Marc Lamy De La Chapelle
email: marc.lamydelachapelle@univ-paris13.fr

Investigation of aromatic hydrocarbon inclusion into cyclodextrins by Raman spectroscopy and thermal analysis Tijunelyte

Keywords: Raman spectroscopy, Thermogravimetric analysis, Cyclodextrin, Polyaromatic hydrocarbons, PAH, Host-guest complex

Among various cavitand molecules, cyclodextrins are extensively studied due to their ability to form host-guest complexes with small hydrophobic molecules. Aiming to explore cyclodextrin implementation on the scopes related to the environmental pollution monitoring or remediation, extensive studies for understanding the cyclodextrin-based host-guest complex formation with selected targeted substances are conducted. In this context, two polycyclic aromatic hydrocarbons, naphthalene and fluoranthene as well as toluene as a member of volatile organic compounds, were studied regarding their ability to encapsulate into cyclodextrin cavities. Synthesised complexes were examined by thermogravimetric analysis combined with Raman spectroscopy. The obtained results demonstrated that the size between targeted molecules and the cyclodextrin cavities strongly correlates with its ability to engage in complexation. Thus, this latter parameter plays an important role in the inclusion complex formation as well as in the strength of the interaction between the molecules.

Introduction

Environmental water pollution by organic compounds is a worldwide concern due to its continuous emission in both natural [START_REF] Kvenvolden | Natural seepage of crude oil into the marine environment[END_REF] and anthropogenic [START_REF] Readman | Oil and combustion-product contamination of the Gulf marine environment following the war[END_REF] ways. The contaminants, consisting of two or more fused aromatic rings known as polycyclic aromatic hydrocarbons (PAHs) are a group of pollutants which have received considerable attention because of their documented significant toxicity and carcinogenicity (Diggs et al. 2011;[START_REF] Harris | Benzo(a)pyrene modulates fluoranthene-induced cellular responses in HT-29 colon cells in a dual exposure system[END_REF][START_REF] Lienig | Polycyclic Aromatic Hydrocarbons in the Aquatic Environment[END_REF][START_REF] Varanasi | Chemical carcinogenesis in feral fish: uptake, activation, and detoxication of organic xenobiotics[END_REF].

Therefore, constant monitoring of these water contaminants at the trace level is of first importance regarding the latest European Water Framework Directives (2000/60/EC, 2006/118/EC and 2006/11/EC).

Within this scope many efforts have been devoted for the development of an analytical tool capable of sensitive and rapid detection of chemical water bodies with minimal sample handling [START_REF] Pejcic | Environmental Monitoring of Hydrocarbons: A Chemical Sensor Perspective[END_REF]). In the course of last decades, Surface Enhanced Raman Spectroscopy (SERS) based pollutant detection has attracted more and more interest [START_REF] Bantz | Surface-enhanced Raman scattering detection and discrimination of polychlorinated biphenyls Vibrational[END_REF][START_REF] Carron | Octadecylthiol-modified surface-enhanced Raman spectroscopy substrates: a new method for the detection of aromatic compounds[END_REF][START_REF] Costa | Chemical analysis of polycyclic aromatic hydrocarbons by surface-enhanced Raman spectroscopy[END_REF][START_REF] Guerrini | Sensing Polycyclic Aromatic Hydrocarbons with Dithiocarbamate-Functionalized Ag Nanoparticles by Surface-Enhanced Raman Scattering[END_REF][START_REF] Halvorson | Surface-Enhanced Raman Spectroscopy (SERS) for Environmental Analyses[END_REF]. SERS is based on the exploitation of the optical properties of metallic nanostructures and especially on their ability to provide a tremendous enhancement of the electromagnetic field at the nanostructure vicinity. Any molecules close to the nanostructure surface can benefit from this enhancement and scatter a Raman signal magnified by a factor up to 10 10 . This highly sensitive method allows observing chemical compounds at the trace level as low as the single molecule sensitivity [START_REF] Chang | Surface molecular imprinting onto silver microspheres for surface enhanced Raman scattering applications[END_REF][START_REF] Yang | Ultrasensitive SERS Detection of TNT by Imprinting Molecular Recognition Using a New Type of Stable Substrate[END_REF]) and can provide quantitative and qualitative multi-analysis in complex environment [START_REF] Pfannkuche | The use of surface-enhanced Raman scattering (SERS) for detection of PAHs in the Gulf of Gdansk (Baltic Sea)[END_REF]. However, in SERS configuration, the molecules that are close to the nanostructure surface will experience the high field enhancement and the Raman spectrum will be then composed of all the spectroscopic features of these molecules including the non-targeted ones.

To have a specific detection, it is then necessary to pre-concentrate the targeted molecules at the nanostructure surface. For this, a specific molecular layer that exhibits higher affinity to the targeted molecules has to be used. Among different strategies to design a SERS-based nanosensor able to pre-concentrate aromatic compounds, supramolecular cage systems as cyclodextrins or calixarenes appear to be particularly promising [START_REF] Guerrini | Functionalization of Ag Nanoparticles with Dithiocarbamate Calix[]arene As an Effective Supramolecular Host for the Surface-Enhanced Raman Scattering Detection of Polycyclic Aromatic Hydrocarbons[END_REF][START_REF] Hill | Investigation of the adsorption of gaseous aromatic compounds at surfaces coated with heptakis(6-thio-6-deoxy)-beta-cyclodextrin by surface-enhanced Raman scattering[END_REF][START_REF] Shi | Trace analysis of polycyclic aromatic hydrocarbons using calixarene layered gold colloid film as substrates for surface-enhanced Raman scattering[END_REF][START_REF] Xie | Selective SERS detection of each polycyclic aromatic hydrocarbon (PAH) in a mixture of five kinds of PAHs[END_REF][START_REF] Xie | Sensing of polycyclic aromatic hydrocarbons with cyclodextrin inclusion complexes on silver nanoparticles by surface-enhanced Raman scattering[END_REF].

Cyclodextrins (CDs) are natural cyclic oligosaccharides. The three commercially available CDs: α-CD, β-CD and γ-CD are consisting of six, seven or eight α-D-glucose units, respectively, linked though α-1,4 glycosidic bonds (figure 1a)). Torus-shaped structure of CDs possesses a relatively hydrophobic internal cavity which is known to provide binding sites for wide variety of molecules [START_REF] Valle | Cyclodextrins and their uses: a review Process[END_REF]. Mainly for this reason CDs are applied in food [START_REF] Astray | A review on the use of cyclodextrins in foods[END_REF], pharmaceuticals [START_REF] Loftsson | Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization[END_REF], cosmetics [START_REF] Buschmann | Applications of cyclodextrins in cosmetic products: A review[END_REF], bioconversion [START_REF] Dufosse | In situ detoxification of the fermentation medium during gamma-decalactone production with the yeast sporidiobolus salmonicolor[END_REF], textile industries [START_REF] Cireli | Application of cyclodextrin to the textile dyeing and washing processes[END_REF] as well as in environmental research [START_REF] Viglianti | Removal of polycyclic aromatic hydrocarbons from aged-contaminated soil using cyclodextrins: Experimental study[END_REF]. Host-guest complex formation between CDs and organic molecules have been widely studied by different techniques, such as thermo-analytical methods, microscopy, chromatography and spectroscopy [START_REF] Singh | Characterization of cyclodextrin inclusion complexes-a review[END_REF]. For solid state analysis, Raman [START_REF] Amado | Metasubstituted styrene molecules included in cydodextrins: A Raman spectroscopic study[END_REF][START_REF] Amado | Selection of substituted benzaldehyde conformers by the cyclodextrin inclusion process: a Raman spectroscopic study[END_REF] and Infra-Red (García-Zubiri et al. 2004;[START_REF] Nagao | Infrared spectra of monosubstituted toluene derivatives in cyclodextrin: Orientation of guest molecules in included complexes[END_REF] spectroscopies are the most used techniques to collect important evidences on the complex formation, thanks to changes in the molecular vibrational modes (frequency and relative intensity) due to the molecular interaction between the host and the guest molecules.

However, literature reveals that complexes formed between CDs and non-polar aromatic compounds are still undergoing difficulties in spectroscopic based analysis [START_REF] García-Zubiri | Infrared Study of Solid Dispersions of β-Cyclodextrin with Naphthalene Derivitaves[END_REF]) due to very weak molecular interactions. In the present work, three forms of CDs were employed in host-guest complexes formation with toluene (TOL), naphthalene (NAP) and fluoranthene (FL). Complexes in solid state were synthesised and studied by Raman spectroscopy and thermogravimetric (TG) analysis. We investigated the molecular affinity comparing the size of the guest with the diameter of the host cavity. We also studied the changing in vibrational modes of the complexes in order to subsequently use enhanced spectroscopy techniques for the detection of those compounds in the wild aquatic environments.

Experimental

Materials

Pharmaceutical grade CDs (α, β and γCD) were purchased from Wacker Chemie (Germany) and used without further purification. Aromatic compounds: TOL, NAP and FL, and ethanol were obtained from Sigma-Aldrich (France) at high purity grade and were used as received.

Inclusion complex preparation

Following the procedure described in literature [START_REF] Sardo | Inclusion compounds of phenol derivatives with cyclodextrins: a combined spectroscopic and thermal analysis[END_REF], based on co-precipitation method, saturated aqueous solutions of αCD, βCD and γCD were mixed with aromatic compounds (TOL, NAP and FL) dissolved in ethanol at the concentration of 50 mM. The addition of the pollutant solutions into CDs, were made drop-wise using strong agitation at ambient temperature. This process led to immediate formation of white precipitants. Mixed solutions were then kept in shaker for 2 hours and precipitants were collected using centrifuge. To make sure that there were no non-included aromatic compounds precipitants were briefly washed with ethanol/water solution 50/50 (v/v), and dried for 2 days at room temperature.

To obtain additional information on complex formation extensive study was performed on βCD:NAP complexes. For this NAP dissolved in ethanol was mixed with saturated βCD solution at different molar ratios (2βCD:1NAP, 1βCD:1NAP and 1βCD:2NAP). Further steps of sample collection, washing and drying were performed as previously described.

For analysis of CDs based selectivity towards the pollutant molecules the complexes were prepared as follows: TOL, NAP and FL were dissolved together in ethanol at 50 mM concentration and added in saturated aqueous solutions of each: βCD and γCD. Ethanol solution containing both TOL and NAP at 50 mM concentration was added into αCD solution. Further steps of sample collection, washing and drying were performed as previously described.

Thermogravimetric analysis

For TG analysis LabSys evo instrument (SETARAM) was used. Each sample (8-12 mg) was kept for 1 hour at 30 °C in alumina pan before heating over the temperature range from 30 to 600 °C under argon atmosphere with the flow rate of 20 mL/min. The empty sealed alumina pan was used as reference. For data processing Calisto software was used.

Raman spectroscopy measurements

Raman spectra of α, β and γCD, TOL, NAP and FL as well as of their inclusion complexes in solid powder were recorded using Xplora ONE (Horiba scientific) microspectrometer. Laser excitation wavelength of 785 nm with a power of 8 mW was focused on the sample with x100 objective (N.A. 0.9). Integration time for signal collection was set to 60 s for CDs and their complexes and to 5 s for aromatic compounds. Each spectrum is the average of two repetitive measurements. Applied grating of 1200 grooves/mm has ensured spectral resolution of less than 1 cm -1 . It is important to note, that the studied aromatic molecules adsorb in the UV range [START_REF] Koban | Absorption and fluorescence of toluene vapor at elevated temperatures[END_REF][START_REF] Maeda | Absorption and fluorescence spectroscopic properties of 1-and 1,4-silyl-substituted naphthalene derivatives Molecules[END_REF][START_REF] Saranya | Optical Absorption and Emission Properties of Fluoranthene, Benzo[k]fluoranthene, and Their Derivatives[END_REF]. Thus, the energy of the near infrared laser could not match with any electronic transitions of these compounds and as a consequence the measured Raman scattering is not in resonant condition.

Computational

All calculations were carried out using the Gaussian09 suite of programs (Frisch et al. 2009).

Following full geometry optimization of FL with C2v symmetry using the density functional three-parameters hybrid model B3LYP [START_REF] Becke | Density-functional thermochemistry. IV. A new dynamical correlation functional and implications for exact-exchange mixing[END_REF][START_REF] Lee | Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density[END_REF]) with the 6-311G (d,p) basis set, vibrational frequencies and Raman activities were calculated. The B3LYP method with the 6-311G (d,p) basis set is frequently used in predicting vibrational spectra of poly aromatic molecules. Raman intensities were computed from activities with GaussSum 3.0 [START_REF] O'boyle | cclib: A library for package-independent computational chemistry algorithms[END_REF]) using an excitation wavelength of 785 nm. The calculated harmonic frequencies of CH stretching vibrations were scaled by a factor of 0.96 and all the other frequencies by 0.98 to allow a good agreement with observed ones for FL alone within almost 10 cm -1 on the whole spectra. Gaussview 5 [START_REF] Dennington | Polycyclic Aromatic Hydrocarbons and Digestive Tract Cancers: A Perspective[END_REF]) was used to visualize the participation and direction of atoms in vibrational normal modes in order to make bands assignments easier.

Results and discussion

Study of complexes formed between β-CD and variable concentration of NAP

Aiming to determine the relevant parameters that describe the host-guest interaction, we first studied the ratio between the host and the guest in the complex βCD:NAP. For this purpose different molar ratios (2βCD:1NAP, 1βCD:1NAP and 1βCD:2NAP) between the host and the guest were used to form the complexes. Two techniques: TG analysis and Raman spectroscopy were then employed to analyze the complexes in solid state.

Thermogravimetric analysis

Thermal analysis is a powerful tool to study the complex formation between guest molecules and CD and to evaluate the inclusion compound stoichiometry [START_REF] Giordano | Thermal analysis of cyclodextrins and their inclusion compounds[END_REF]). Among the various thermo-analytical techniques, TG analysis is frequently used to determine whether the guest exhibits some changes in its phase transition temperatures indicating inclusion complex formation.

The thermograms and the derivatives of the three prepared stoichiometries regarding molar ratio (2βCD:1NAP, 1βCD:1NAP and 1βCD:2NAP), as well as the thermograms of pure compounds are presented in figure 2.

Isolated CD thermal degradation profile consists of three parts. In the first stage, a gradual loss of hydrated water from ambient to 120°C corresponding to the dehydration process can be recorded. During the second stage, in a temperature range between 270 and 350 °C single step weight loss is observed, corresponding to the thermal breaking of CD ring and further glucosidic ring decomposition [START_REF] Russell | FT-IR and raman spectral evidence for metal complex formation withβ-cyclodextrin as a first sphere ligand J Inclusion Phenom[END_REF]. Remaining 30-40% of residue is then showing loss in mass at a temperature range upon 350 °C and is assigned to a slow degradation of char [START_REF] Trotta | Thermal degradation of cyclodextrins[END_REF]. On the other hand, pure aromatic compounds decompose at much lower temperature. In the case of NAP, the thermal decomposition occurs in one step between 80 and 160 °C temperature range.

The TG curves of the complexes are quite different from those of pure components. Worth to remind, that complexes were prepared in aqueous solution and should be considered as ternary water-guest-CD systems. In themograms, the initial weight loss observed until 120°C is attributed to the release of water molecules. However, it is noticeable that the number of water molecules is continuously decreasing within the complexes with growing number of the guest (table 1). This change in number of water can be contemplated as a first proof of the complex formation since the inclusion process is considered as the replacement of the internal water molecules by a guest. Additionally, a peak around 62 °C can be observed on the derivatives of TG analysis curves for some complexes. This peak might be assigned to the ethanol molecules remaining after complexes formation. However, since the evaporating temperature is close to boiling point of the ethanol, we assume that the ethanol does not form complexes with the CD molecule. Further analysis of the thermograms revealed no peak corresponding to free NAP molecules decomposition after the complex formation. Instead, a small endothermic peak centred at around 268 °C appears for 2βCD:1NAP and 1βCD:1NAP complex systems. Considering comparable mass loss in the complexes (table 1) the peak around 268 °C could be assigned to a thermal decomposition of NAP placed inside the CD cavity. Indeed, since this temperature is intermediate between the ones observed for individual CD and for individual NAP, we assume that it should be assigned to third process corresponding to the decomposition of NAP included inside the CD. Similar shift in guest decomposition temperature have been already observed by other groups [START_REF] Nolasco | Spectroscopic and thermal studies on the inclusion of trans-cinnamic acid and a number of its hydroxyl-derivatives with α, β and γ-cyclodextrins molecules[END_REF][START_REF] Sardo | Inclusion compounds of phenol derivatives with cyclodextrins: a combined spectroscopic and thermal analysis[END_REF]). The shift in decomposition temperature between free NAP and the one embedded into host indicates that the host-guest interaction protects the pollutant molecules from early volatilization. For the system with higher concentration of NAP (1βCD:2NAP), the latter peak is found slightly shifted to 270 °C. Moreover, a new peak at lower temperature, centred at around 186 °C can be observed and assigned to the NAP molecules placed outside the cavities. However, the higher decomposition temperature of those NAP molecules in comparison with free NAP signifies that they are in interaction with the complex.

Finally, the weight loss in temperature range from 270 °C to 350 °C was observed and assigned to the CD decomposition. A slight shift of 10 °C to lower degradation temperature can be noticed when comparing with the free CD decomposition. This slight shift could be induced by the engaged guest which disorders the hydrogen bonded supramolecular structure of CD [START_REF] Trotta | Thermal degradation of cyclodextrins[END_REF].

The quantification of the mass losses upon temperature increase provides the information on the molar ratio between CD, NAP and H 2 O in the systems. Calculated stoichiometries of the complexes are summarized in table 1.

Fig.2

Top: thermograms of the reference molecules: NAP and βCD presented as dot and dash lines respectively, and of the inclusion complexes formed mixing different molar ratios: 2βCD:1NAP (red curve), 1βCD:1NAP (blue curve), 1βCD:2NAP (violet curve). Bottom: derivatives of the TG curves.

As explained previously, the studied complexes are actually ternary βCD-NAP-H 2 O systems.

Nevertheless, an increase of the initial guest concentration leads to an increase of the NAP molecules included into CD cavities. On the contrary, the number of water molecules per CD decreases in agreement with the increasing number of engaged NAP molecules, as already mentioned proving the successful replacement of the water molecules. The cavity of βCD is generally considered large enough to accommodate aromatic guest molecule [START_REF] Köhler | The β-cyclodextrin/benzene complex and its hydrogen bonds -a theoretical study using molecular dynamics, quantum mechanics and COSMO[END_REF]. The 1βCD:1NAP stoichiometry was observed to be in good agreement with the predicted one, as well as in case of 2βCD:1NAP formed inclusion complex where 1 molecule of NAP is between 2 βCD molecules. Unusual host-to-guest stoichiometry was calculated for the last complex with the highest NAP concentration. Calculated stoichiometry of 3 NAP molecules per 2 βCD was found. Similar stoichiometry has been already observed and documented for a complex formed between βCD and 1,10-phenanthroline [START_REF] Liu | Interaction between β-cyclodextrin and 1,10-phenanthroline: uncommon 2:3 inclusion complex in the solid state[END_REF]). With the help of X-ray diffraction authors have observed that two 1,10-phenanthroline molecules were individually included into two βCD cavities and stabilized by the third guest molecule located in the interstitial space between two head-to-head CD molecules. The observed stoichiometry of 2βCD:3NAP should have similar structure. In the complex two NAP molecules which were found to decompose at 270 °C are included into 2 βCD individually. These 1βCD:1NAP complexes are then stabilized by a third NAP molecule placed outside the CD. NAP molecules located in the interstitial space between two complexes decompose at around 186 °C as observed in TG analysis.

Raman spectroscopy based analysis

The complexes synthesised mixing different molar ratios between βCD and NAP were then studied by Raman spectroscopy. Figure 3 presents the Raman spectra of the native βCD, of the NAP and of the three inclusion complexes: 2βCD:1NAP, 1βCD:1NAP, 1βCD:2NAP. Peaks corresponding to naphthalene were observed in the spectra of all the complexes.

Moreover, the proof of molecular inclusion and interaction with the CD cavities was clearly noticeable by comparing the relative intensities of isolated NAP peaks with the ones of NAP included in the complexes. Significant differences in intensities can be observed for several peaks (figure 4). First, the one located at 761 cm -1 , corresponding to ring breathing mode and C-C central stretch, appears as the most intensive peak in the NAP spectrum whereas it is remarkably diminished in the spectra of complexes. On the contrary, the peak at 1379 cm -1 : assigned to a C-C stretching and ring deformation is becoming the most intense mode when the NAP is embedded into the cavity of the CD. Other modes which are varying after the complex formation are less obvious in the presented graph thus their relative intensities and positions are summarized in table 2. The peak located at 1379 cm -1 was used as reference for the relative intensities calculation given in the table 2. An increase of the relative intensity is clearly observed for the C-C-C bending at 508 cm -1 . On the contrary, the C-H out of plane stretching mode at 3050 cm -1 as well as in plane C=C stretching at 1575 cm -1 are less visible when NAP is included into CD. The observed change indicates that the stretching modes inducing a potential interaction of H atoms with the CD as well as the radial mode or the C-H stretching exhibit a lower intensity compared to the other modes; that is an evidence of the inclusion of the NAP in the βCD cavity. Some changes were also examined on CD vibrational modes after inclusion complex formation. For example, a change in the relative intensities of the doublet at 927-943 cm -1 was observed as well as for the bands located between 1050 and 1200 cm -1 . To have a better insight on such changes, the figure 5 shows the subtractions of the isolated CD spectrum from normalized 2βCD:1NAP, 1βCD:1NAP, 1βCD:2NAP complexes spectra. The observation of several positive and negative peaks superimposed with the NAP modes indicates that a large number of the CD vibrational modes are strongly modified by the interaction with the NAP molecule. The most impacted vibrational modes are related with skeletal vibrations (478 cm -1 , 850 cm -1 , 927 cm -1 ) (Egyed 1990), O-H wagging (562 cm -1 ) [START_REF] Yao | Inclusion complexes of cypermethrin and permethrin with monochlorotriazinyl-beta-cyclodextrin: A combined spectroscopy, TG/DSC and DFT study[END_REF]), C-O stretching ( 943 cm -1 ,1051 cm -1 , 1076 cm -1 , 1124 cm -1 ) [START_REF] Yao | Inclusion complexes of cypermethrin and permethrin with monochlorotriazinyl-beta-cyclodextrin: A combined spectroscopy, TG/DSC and DFT study[END_REF]) and C-H stretching or wagging (1345 cm -1 , 2914 cm -1 , 2966 cm -1 ) [START_REF] Yao | Inclusion complexes of cypermethrin and permethrin with monochlorotriazinyl-beta-cyclodextrin: A combined spectroscopy, TG/DSC and DFT study[END_REF]. We observed that the spectral modifications become more visible when the number of naphthalene molecules increase compared to CD ones, since the intensities of the negative or positive bands increase. Thus, the host/guest complex behaves as a global system that induces large variation on the vibrational modes of both compounds.

Fig. 5 Normalized Raman spectra obtained after β-CD spectrum subtraction from 2βCD:1NAP (black spectrum), 1βCD:1NAP (red spectrum), 1βCD:2NAP (blue spectrum) inclusion complexes spectra. The bottom Raman spectrum corresponds to the isolated NAP.

Study of complexes between α, β, γCD with TOL, NAP and FL

Further investigations on CDs ability to form the complexes were realized by mixing selected aromatic compounds: TOL, NAP and FL dissolved in ethanol, with the saturated aqueous solutions of each CD (αCD, βCD and γCD). This study was performed to determine the ability of complex formation between the host and the guest in regards to their sizes.

Thermogravimetric analysis

In this part, a study of the inclusion compounds of the three types CD (αCD, βCD and γCD)

is presented using TOL, NAP and FL molecules as guests. Decomposition temperatures were found for each pollutant molecule: total mass loss of TOL was recorded from ambient till 113 °C, NAP (as mentioned in the section 1.1) decomposes from 80 to 160 °C and FL thermal decomposition occurs from 125 to 280 °C. For the pure CD, no important dissimilarity in the degradation temperature regarding to the cavity size was found.

Generally, an increasing number of guest molecules involved in the complex was expected (with an increasing cavity size (αCD < βCD < γCD)). The γCD cavity is the wider and may be able to accommodate two guest molecules [START_REF] Sardo | Inclusion compounds of phenol derivatives with cyclodextrins: a combined spectroscopic and thermal analysis[END_REF]). On the contrary, αCD is the smaller host so its cavity is not large enough to accommodate one aromatic guest. Usually, one guest is stacked between two αCDs [START_REF] Nolasco | Spectroscopic and thermal studies on the inclusion of trans-cinnamic acid and a number of its hydroxyl-derivatives with α, β and γ-cyclodextrins molecules[END_REF].

As shown in the first part, the inclusion of the guest into the cavity of the CD permits the guest to withstand higher temperature comparing to free molecules. For instance, toluene which is known to have a boiling point at 111°C, was found to decompose at more than 270°C when it is in contact with the αCD.

Table 3 shows the calculated stoichiometries for the host/guest complexes. As expected, in the case of TOL, complexes are composed of one guest molecule between two αCDs (1:2 stoichiometry), whereas one molecules of TOL can be included in one molecule of both: βCD and γCD (1:1). As a consequence, in the system of 2αCD:1TOL a shift towards higher decomposition temperature was found, showing higher thermal stability.

Concerning NAP, one guest per one host molecule was calculated regardless to the cavity dimension. Unexpectedly, we observed that thermal stability of the NAP molecules is also independent on the size of the host. This behaviour could be related with different orientation of guest molecule inside the cavity of guest.

Finally, FL molecules formed a complex only with γCD. Indeed, in this case, calculation revealed that two γCD molecules were engaged with one molecule of FL.

Raman spectroscopy based analysis

Raman spectroscopy measurements were performed on complexes in order to observe spectral evidence of guest-host interactions. It has already been observed that small shifts and variation in intensities for some bands in Raman spectra of the guest molecule appear upon formation of inclusion complexes [START_REF] Amado | Metasubstituted styrene molecules included in cydodextrins: A Raman spectroscopic study[END_REF][START_REF] Amado | Selection of substituted benzaldehyde conformers by the cyclodextrin inclusion process: a Raman spectroscopic study[END_REF]. The moieties of guest molecules that suffer from intermolecular interactions can be identified by assigning the shifted bands using DFT calculation. Starting from the nature of vibrations modified upon complexation, it becomes also possible to discuss about the orientation of the guest molecules in the inclusion complexes.

Quantum chemical computations predicting harmonic frequencies and spectral intensities are essential for interpretation of experimental spectra, particularly for large molecules. Accurate computation of IR and Raman intensities is difficult because of their dependence on dipole moment and polarizability derivatives. For this reason, it is invariably the relative, rather than absolute, band intensities that are used since experimental determination of absolute band intensities, especially for Raman bands, is difficult. Accordingly, the intensity of some bands is enhanced in experimental data in comparison with the calculated ones, especially for the more intensive bands, but the order of magnitude is kept on the whole spectra [START_REF] Zvereva | Ab Initio and DFT Predictions of Infrared Intensities and Raman Activities The[END_REF] In experimental IR absorption spectra of inclusion complexes, bands due to CD are prominent [START_REF] Nagao | Infrared spectra of monosubstituted toluene derivatives in cyclodextrin: Orientation of guest molecules in included complexes[END_REF]) while it is not so obvious in Raman spectra. Hence, it becomes easier to observe Raman bands attributed to the guest molecules.

The figure 6 shows the Raman spectra of complexes formation of CDs with TOL and NAP respectively. Both graphs A and B are composed of isolated pollutant spectrum (a) and the spectra of guests engaged in complexes with αCD (c), βCD (e) and γCD (g). The native CDs spectra (b), (d) and (f) for α, β, γCD respectively are also given.

As demonstrated previously by study based on TG analysis, TOL and NAP form complexes with all three kinds of CD. Some weak peaks attributed to TOL vibrations were observed in Raman spectra, confirming the TOL contribution in complexes formation. Unexpectedly, TOL vibrational modes are just weakly impacted by interaction with host molecule. Subtle changes can be noticed in the position of peaks assigned to in plane C-C stretch and ring deformation at 516 cm -1 , methyl group stretch at 781 cm -1 and in plane C=C stretching at 1026 cm -1 (vibrational modes assignment was found in literature [START_REF] Tasumi | Some thoughts on the vibrational modes of toluene as a typical monosubstituted benzene[END_REF])). Worth noting, that latter peak was absent in the spectrum of the αCD:TOL complex. The more impacted vibrations related with C-H stretching modes were observed. Unfortunately, analysis of C-H stretching mode belonging to methyl group at 2921 cm -1 is rather impossible due to strong signal of CDs in this region. However, other peak representing later mode in the ring presented at 3058 cm -1 was found to remarkably decrease after complex formation. The comparison of shift in positions of the peaks belonging to isolated TOL and TOL engaged to the complexes with CDs can be found in Online Resource table 6.

The interactions with TOL have caused more steric hindrance on αCD molecules than on the ones having larger diameter of cavity. Comparable effect on αCD vibrations was observed in the case of interaction with NAP. In contrary, just small disturbance on γCD vibrations can be noticed after hosting these two aromatic guests. The analysis of NAP peaks in inclusion complexes spectra revealed similarities with those discussed previously (1.1 section). The most impacted modes by the complexation are related with the ring breathing, ring deformation, C-C central stretching and C-H in plane stretching located at 761 cm -1 , 1379 cm -1 , 3050 cm -1 respectively (figure 4). Furthermore, considering relative intensities of these peaks, some differences were observed regarding the diameter of host cavity. The C-H symmetric stretch is supposed to be more constrained if NAP is inserted in horizontal or diagonal orientations into the CDs cavity. These orientations can be achieved in 1βCD:1NAP and 1γCD:1NAP systems. Calculated relative intensities of the peak at 3050 cm -1 showed less intensity in latter systems confirming guest diagonal orientation. Contrary, in complex with αCD decrease in C-H stretching mode intensity is less important, suggesting the vertical guest orientation. Latter NAP orientation can be further confirmed analyzing peak at 1379 cm -1 . This vibrational mode is less impacted when NAP is included vertically into the smallest CD. Worth noting, that a slight shift of this vibration was observed. However, for the complexes with βCD and γCD it shifted to a lower frequency by 3 cm -1 and in the case of complex with αCD it is moved by 2 cm -1 to a higher frequency, again indicating different guest interaction with the host molecule. Furthermore, comparing relative intensities of this peak was noticed that in NAP interaction with αCD case it increases two times and in case of interaction with other two hosts this vibration is three times more intense. Collation of calculated NAP vibrational modes with the relative intensities as well as of the experimentally achieved isolated NAP and NAP engaged in complexes is presented in Online Resource table 7. To find relative intensities of NAP peaks, spectra of CDs with included NAP and the spectra of the isolated CDs were normalized regarding the peak at 2879 cm -1 (corresponding to the in plane C-H stretching vibration). Then the spectra of isolated CDs were subtracted from the complexes ones and compared with NAP spectrum regarding the peak at 1025 cm -1

(corresponding to symmetric in plane C-C stretching motion).

In our case, shifts in frequency and intensity of the guest peaks are mostly observed for the complex between γCD and FL demonstrating that intermolecular interactions involved in its formation are stronger than those involved in the association of TOL and NAP in the hosts.

As mentioned before, FL is compatible just with γCD diameter of cavity for inclusion complex formation. TG analysis study has revealed that FL embedded into CD has a stoichiometry of two FL molecules in complex with one molecule of host. FL structure includes the five member cycle connecting naphthalene and benzene units (figure 1b)) and most likely latter units would be responsible for an interaction with the γCD cavities. Raman spectroscopy based analysis confirmed this arrangement, since these units have few isolated non-overlapping vibrational modes which can be studied independently. With the help of literature [START_REF] Manal | Theoretical Study of Vibration Spectroscopy for Neutral and Charged Fluoranthene Molecule[END_REF], we have calculated the FL vibration frequencies and Raman scattering intensities of all the 72 normal modes. We present them in Online Resource table 8 together with the achieved experimental values of isolated FL and of its complex with γCD. stretching or wagging modes (1341 cm -1 , 2875 cm -1 , 2934 cm -1 ) [START_REF] Yao | Inclusion complexes of cypermethrin and permethrin with monochlorotriazinyl-beta-cyclodextrin: A combined spectroscopy, TG/DSC and DFT study[END_REF].

The CDs selectivity for complex formation. Test of specificity

The CDs selectivity discussed in previous section was herein tested by mixing each CD with a solution of the three pollutants (TOL, NAP, FL). Experiments were performed by drop-wise addition of the ethanol solution containing three pollutants into saturated aqueous solutions of each CD. A different procedure was applied for αCD: in this case FL was absent in mixture of pollutants. Figure 9 presents achieved spectra of complexes formed between guests and αCD a), βCD c) and γCD e). They were normalized regarding the band at 2879 cm -1 corresponding to a C-H stretching vibration of CD and compared to CD native forms: b), d), f) for αCD, βCD, γCD respectively as well as the pollutants spectra: g), h), i) for TOL, NAP and FL respectively.

To determine the selective interaction of the CD with specific pollutants, we have calculated the ratio between the Raman intensity of the pollutants involved in the CD complex for both experiments: individual pollutant and mixture of pollutants. This ratio has been calculated as follow:

Guest %=𝐼𝑔𝑢𝑒𝑠𝑡 𝑝𝑒𝑎𝑘𝑠 𝐼𝐶𝐷, 𝐶-𝐻 𝑚𝑜𝑑𝑒𝑖𝑛𝑑𝑖𝑣𝑖𝑑𝑢𝑎𝑙 * 𝐼𝐶𝐷, 𝐶-𝐻 𝑚𝑜𝑑𝑒𝐼𝑔𝑢𝑒𝑠𝑡 𝑝𝑒𝑎𝑘𝑠𝑚𝑖𝑥𝑒𝑑 * 100%

The first term corresponds to the relative intensity of the individual pollutant in the CD complex whereas the second term corresponds to the relative intensity of the pollutant in the CD complex in the case of the mixture. Both relative intensities were normalised by the intensity of the C-H vibration mode of the CD in order to use the CD as a reference. This ratio is then independent on the Raman cross section of the pollutant modes and allows comparing directly the proportion of pollutants forming a complex with the CD.

Raman spectra of precipitants collected after mixing αCD with TOL and NAP shows the appearance of peaks belonging to both pollutants (roman numerals I and II indicated peaks from TOL and NAP respectively). Further calculation of the relative intensities belonging to TOL peak located at 1029 cm -1 and NAP peak at 761 cm -1 revealed that 62 ± 7 % of complexes in this system are formed between TOL and αCD. This latter result demonstrates higher affinity of the αCD to the TOL then to the NAP. On the contrary, 92 ± 5 % of NAP was found in the complex formed between βCD and mixture of pollutants. Calculation of relative intensities of the peaks belonging to NAP and further comparison with the values presented in table 1 confirmed 1βCD:1NAP stoichiometry in the system. Raman spectrum of precipitants collected after mixing γCD with three pollutants, showed peaks coming from NAP and FL (roman numerals II and III indicated peaks from NAP and FL respectively).

Calculation of relative intensities of guests vibrational modes (peaks at 761 cm -1 for NAP and at 669 cm -1 for FL) revealed that FL dominates in this system forming 70 ± 9 % of all complexes with γCD. The explanation of why no inclusion of TOL in βCD and γCD occurred can be related with the competitive exchange in parallel reactions. Another reason contributing more in γCD case is that the size of the cavity is not adapted to a small TOL molecule. As a consequence, a molecule smaller than the cavity will have less interactions (hydrophobic, van der Waals, etc) and thus less affinity with the CD. Since TOL is the smallest molecule in this reaction and γCD shows better affinity to bigger molecules, it is possible that interaction between TOL and the large cavity of CD is too low and can be easily replaced.

Fig.9

Raman spectra of complexes formed mixing aromatic molecules and αCD, βCD, γCD are presented in graph a), c) and e) respectively. The spectra b), d), f) correspond to αCD, βCD, γCD respectively and g), h), i) correspond to TOL, NAP and FL respectively.

Conclusion

In this study, results of TG analysis, Raman spectroscopy and DFT calculations provide information on the complexes formation between organic pollutants (TOL, NAP and FL) and three forms of CDs (α, β, γCD) in the solid state. In the first part of this study, three stoichiometries of βCD:NAP system have been analyzed. The obtained results permit to rationalize the host-guest complex formation in regards to different stoichiometry. Second part of the study was dedicated to description of complexes formed between all selected pollutants with three kind of CD. Data collected from TG analysis suggested that mainly stoichiometry of one guest per one host was formed with the exception in αCD:TOL and γCD:FL systems, where one guest is interacting with two host molecules. It is noteworthy, that FL was found to form complex just with γCD due to its larger size.

Using Raman spectroscopy, the most impacted vibrational modes after complexes formation, Finally, all achieved spectral evidences opened the way for analysis of more complexed systems. Test of CDs specificity regarding the guest size showed considerable affinity in the systems of αCD:TOL, βCD:NAP and γCD:FL.

All achieved information could be exploited in the CDs applications related to the environmental pollution monitoring and remediation.

Fig. 1

 1 Fig.1 Illustrated scheme of (a) molecular and 3D structures of three forms of CDs, with indications of diameters of the cavities, (b) molecular structure of the three selected pollutants with their sizes.

Fig. 3

 3 Fig.3 Raman spectra of the βCD (a), of the NAP (b) and of the inclusion complexes (c), (d), (e) obtained after mixing βCD with NAP in molar equivalents of 2:1, 1:1 and 1:2 respectively.

Fig. 4

 4 Fig.4 Three vibrational modes of NAP which are the most impacted by the interaction with βCD.

Fig. 6

 6 Fig.6 Raman spectra of complexes formed based on CDs with TOL and NAP are presented in graph A and B respectively. In each graphs a), b), d), f) spectra are indicating vibrations of isolated pollutant, αCD, βCD and γCD respectively; Raman spectra of the inclusion complexes between pollutants and αCD, βCD and γCD are given in b), d) and f) respectively.

Fig. 7

 7 Fig.7Raman spectra of FL calculated and experimentally obtained are presented in graph a) and b) respectively. The spectrum c) corresponding to a free γCD and spectrum d) corresponds to the complex formed between FL and γCD.

  were analysed. In the case of TOL a strong decrease in relative intensity of ring C-H stretching mode was observed. Other subtle changes were found for the motions related to the ring deformation, C=C stretching and methyl group symmetric deformation. The study of NAP peaks assigned to the ring breathing, ring deformation, C-C central stretching and C-H in plane stretching gave the possibility to discuss of potential guest orientation inside the host cavity. Due to differences in the relative intensities of these modes we assumed that vertically orientated NAP is inserted into αCD cavity. With the bigger hosts NAP is most likely interacting in diagonal or horizontal orientations. The spectroscopic evidences of a guest molecule orientation and intermolecular interaction were found especially obvious in the FL case. Three classes of mostly impacted vibrations were found and proposed as the fingerprint for a FL engaged in complex.

  Figure 1

Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9

 456789 Figure 4

Table 1

 1 TG analysis based calculation on stoichiometry for inclusion complexes

	System	Theoretical	% (wt)	% (wt)	Calculated	Water per
		stoichiometry	(Guest)	(H 2 O)	stoichiometry	CD
	2βCD:1NAP	2:1	9.2	4.1	2:1	10
	1βCD:1NAP	1:1	5.1	7.2	1:1	7
	1βCD:2NAP	1:2	10.6	4.6	2:3	5

Table 2

 2 Positions of the vibrational modes and their calculated relative intensities for free NAP and NAP included in the βCD. Assignment of isolated NAP vibrational modes found in literature[START_REF] Srivastava | Theoretical and experimental studies of vibrational spectra of naphthalene and its cation Indian[END_REF]

	NAP	2CD:1NAP	1CD:1NAP	1CD:2NAP	Assignment
	Peak Rel.Int	Peak	Rel.Int	Peak	Rel.Int	Peak	Rel.Int
	508	0.2	508	0.39	508	0.41	508	0.44	CCC bend
	762	3	757	0.77	758	0.83	758	0.87	Ring breathing
	1379	1	1375	1	1376	1	1376	1	CC str.; ring def.
	1575 0.65	1575	0.12	1576	0.13	1576	0.15	C=C str.
	1627 0.05	1627	0.04	1628	0.05	1628	0.05	C=C str.
	3050 0.82	3055	0.06	3055	0.12	3055	0.18	CH str.

Table 3

 3 TG analysis based calculation on stoichiometry for inclusion complexes

	System	°C	% (wt)	% (wt)	Calculated	Water per
		(Guest)	(Guest)	(H 2 O)	stoichiometry	CD
	αCD : TOL	273	4	7.3	2:1	6
	αCD : NAP	268	9.9	7.5	1:1	6
	βCD : TOL	268	3.5	8.8	1:1	9
	βCD : NAP	269	5.1	7.2	1:1	7
	γCD : TOL	265	3.5	8.2	1:1	9
	γCD : NAP	270	2.7	7.4	1:1	9
	γCD : FL	272	5.1	8.2	2:1	9

Table 4

 4 Comparison of FL peaks shifts and relative intensities (free FL and engaged in complexes with γCD). Numbers correspond to assignment in Online Resource table 8 The main normal modes of FL vibrations mostly impacted by the complexation (C-H asymmetrical stretching modes are not represented). Numbers correspond to assignment in tables 4 and Online Resource table 8.Finally, the changes in γCD vibrations have appeared due to FL inclusion. Mostly they are related with C-O stretching modes (945 cm -1 ,1059 cm -1 , 1069 cm -1 )[START_REF] Yao | Inclusion complexes of cypermethrin and permethrin with monochlorotriazinyl-beta-cyclodextrin: A combined spectroscopy, TG/DSC and DFT study[END_REF]) and C-H

	FL		Complex	Assignment
	Peak	Rel.Int.	Peak	Rel.Int
	560	1.40	559	0.62	Ν°14 δring (δCCC) A breathing
	669	2.73	669	0.65	Ν°19 δring (δCCC) B breathing
	1018	1.25	1016	0.35	Ν°37 δring B (CH rock. and CC str.)
	1101	1.00	1102	1.00	Ν°40 δring (CCH and CCC bending)
	1409	1.71	1410	0.67	Ν°52 δring (CH rock. and CC str.)
	1608	1.72	1610	1.14	Ν°60 δring A (CH rock. and CC str.)
	3022	0.17	3037	0.05	Ν°63-65 δCH Asym. Str.
	3034	0.09	3051	0.24	Ν°66 δCH Asym. Str.(ring B)
	3048	0.83	3059	0.05	Ν°67-69 δCH Str.
	γ out plane of the molecule
	δ in plane of the molecule

Graphical illustration of vibrational modes which were found to be the most affected when FL is engaged in the complexes with γCD, is presented in figure

8

. Fig. 8

Acknowledgements

The authors would like to acknowledge the Remantas project (ANR-11-ECOT-0010), supported by French National Research Agency (ANR) for a financial support.

Conflict of interest

The authors declare no conflict of interest.


Colas Florent

Lamy De La Chapelle

M Lamy De La Chapelle
email: marc.lamydelachapelle@univ-paris13.fr

Nanoplasmonics tuned "click chemistry"

Nanoplasmonics is a growing field of optical condensed matter science dedicated to optical phenomena at the nanoscale level in metal systems. Extensive research on noble metallic nanoparticles (NPs) has emerged within the last two decades due to their ability to keep the optical energy concentrated in the vicinity of NPs, in particular, the ability to create optical near-field enhancement followed by heat generation. We have exploited these properties in order to induce a localised "click" reaction in the vicinity of gold nanostructures under unfavourable experimental conditions. We demonstrate that this reaction can be controlled by the plasmonic properties of the nanostructures and we propose two physical mechanisms to interpret the observed plasmonic tuning of the "click" chemistry.

Introduction

Nanoplasmonic effects are governed by coherent oscillations of the free electron gas of metal nanoparticles (NPs) at the incident electric field frequency. The excitation of these modes, called localized surface plasmons (LSP) 2,3 , can be further enhanced if the frequency of the incoming light matches the LSP resonance (LSPR) frequency 4 . The unique optical properties of the metal NPs induced by LSP excitation are of great interest in the fields of physics 5,6 and biology 7,8 . It may also be exploited in chemistry 9 . This latter field can significantly benefit from the application of nanoplasmonics as an efficient source of electrons, light and heat. The control of chemical reactions at the metallic surfaces is a crucial parameter for many applications. For instance, a two-photon photopolymerization of a thin photo-resist layer SU8 has been achieved locally inside the nanogaps between lithographic gold nanostructures due to the near-field enhancement induced by LSP excitation 10 . Galloway et al. have also demonstrated light assisted protein immobilization in the nanogap between two gold nanoantennae within a nanometric accuracy 11 . In their study, the authors exploited the cleavage of disulfide bridges in the proteins resulting in their subsequent attachment to the gold nanoantennae in the regions of the highest near-field enhancement. Another important parameter to control the speed and yield of chemical reactions is the temperature generated by LSP. Thermoplasmonics is a recent field of research, which has proved to be highly applicable for many chemical approaches. Cao et al. have used the local heat generated by metallic NP through laser illumination to control the position and growth direction of a single semiconductor nanowire 12 . In this paper, we demonstrate that the plasmonic properties of metallic NPs can be exploited to induce "click" chemistry reactions. Introduced less than two decades ago, this set of reactions is described as simple, widely scoped, modular, generating high yields, and producing only harmless byproducts that can be isolated by nonchromatographic methods 13 . In particular, a copper-catalysed azide/alkyne click reaction has received considerable attention in organic and polymer chemistry 14 but also in nanosurface chemistry 15 . Another reaction: thiol-ene coupling, fulfills the click chemistry concept as well. This reaction known for over 100 years 16 and recently rediscovered 13 , consists in grafting thiol-bearing molecules to a carbon double bond. The applications of this reaction have had significant impact in polymer synthesis and material science 17,18 and has attracted growing interest in surface functionalisation and modifications 19,20 . Generally, this reaction is performed through UV light or thermal activation, with the presence of a radical initiator. In this contribution, we demonstrate that the thiol-ene coupling can be simply initiated in the visible range using nanoplasmonic effects. The kinetics of this reaction was monitored in situ by Surface Enhanced Raman Scattering (SERS). By investigating the reaction rates on different diameters of gold nanocylinders (NCs) made by electronbeam lithography (EBL) we claim that this reaction can be tuned by controlling the NCs plasmonic properties.

Experimental

Materials 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH, 97%), 2-mercaptoethanol (BME, 99%), ethanol (99.8%), 4-pentenoyl chloride (98%), 4-nitroaniline (99%), 1,4-butanedithiol (97%) and thiophenol (thiophenol, 99%) were purchased from Sigma-Aldrich (France) and allyl mercaptane (2-propene-1-thiol, 70%) was obtained from Acros Organic (France). All reagents were used without further purification. Deionized water with resistance of 18.2 M.cm was used in all described experiments as the reaction medium.

SERS active substrates

Gold nano-cylinder (NCs) arrays on glass produced by electron beam lithography and lift-off techniques 34,35 were used for this study. The substrates were designed to contain the variable diameters of NCs: 110 nm, 140 nm and 200 nm. The height of the cylinders was set at 60 nm evaporated on 3 nm of chromium for better gold adhesion on glass. The gap between two NCs was kept constant at 200 nm to avoid any effect of near-field coupling.

General procedure for chemical reactions on gold surface

Prior to each experiment, gold NCs were cleaned as follows: substrates were immersed in ethanol for 10 min, dried using nitrogen and treated by ozone under intense 185 nm and 254 nm ultraviolet light (PSD Standard UV-Ozone Cleaner, Novascan), ensuring the removal of organic contaminants. Substrates were then once again immersed in ethanol for 10 min, dried and investigated by Extinction and Surface Enhanced Raman Spectroscopies in aqueous conditions using the microscopic side with the mould (Carl Roth).

Thiophenol grafting by thiol-ene click chemistry

The substrate was incubated in allyl mercaptan solution of 0.08 M in ethanol for 12 hours ensuring self-assembled monolayer (SAM) like coating formation to block the gold surface. As-prepared gold NCs based substrate was then ready to be introduced to a reaction mixture containing thiophenol (0.25 mM) and AAPH (0.25 mM) dissolved in deionized water. The sample was placed on a microscopic slide where the mould was filled with 100 µL of reaction mixture. The thiol-ene click reaction was then monitored by SERS.

Experiment C1: Thiophenol interaction with allyl mercapthan functionalized gold NCs

The substrate was functionalized with allyl mercaptan and then placed on a microscopic slide containing 100 µL of aqueous thiophenol solution (0.25 mM). Contrary to the experiment above, the radical initiator was not included here. Thiophenol interaction with double bonds decorated surface was then monitored by SERS.

Experiment C2: thiophenol interaction with mercaptoethanol functionalized gold NCs

The substrate was incubated in mercaptoethanol solution of 0.1 M in ethanol for 12 hours to block the free gold surface. The functionalized substrate was then orientated onto a microscopic slide with a mould filled with 100 µL of the aqueous solution containing thiophenol (0.25 mM) and AAPH (0.25 mM). Thiophenol interaction with mercapthoetanol was then monitored by SERS.

Experiment C3: thiophenol interaction with non-functionalized gold NCs

The cleaned substrate with gold NCs arrays was directly placed on a microscopic slide with the mould filled with 100 µL of solution containing thiophenol (0.25 mM) and AAPH (0.25 mM) in deionized water. Thiophenol interaction with non-functionalized gold surface was then monitored by SERS.

Pentenoyl-nitroaniline interaction with butanedithiol functionalized gold NCs

The substrate was first functionalized using butanedithiol (10 mM in ethanol) by incubating it in solution for 12 h. The substrate was then washed with ethanol and placed on a microscopic slide containing 100 µL of aqueous solution with dissolved pentenoyl-nitroaniline (0.2 mM) and AAPH (0.2 mM). Pentenoyl-nitroaniline was chemically synthesized from 4-pentenoyl chloride and 4-nitroaniline, using a nucleophilic addition reaction. As in the experiments above, molecular grafting by thiol-ene reaction was then monitored by SERS.

Raman spectroscopy measurements

SERS and extinction measurements were recorded using Xplora ONE (Horiba scientific) spectrometer. For SERS, a laser of 660 nm excitation wavelength with the power of 0.45 mW was focused on the sample with x60 objective (N.A. 0,70). Integration time for signal collection was set at 15s. Each spectrum was the average of two repetitive measurements. Applied grating of 600 grooves/mm ensured a spectral resolution of 3 cm -1 .

Nuclear Magnetic Resonance (NMR) measurements

Nuclear Magnetic Resonance (NMR) measurements were performed in deuterium oxide using Bruker Avance III 400 MHz instrument.

Results and discussion

Thiol-ene "click" reaction involves the addition of a thiol to an alkene group via a free-radical mechanism 21 (Figure 1). Briefly, in the initiation step the thiol group is converted to a thiyl radical thanks to the interaction with a photo or thermal radical initiator exposed to UV light 22,23 or to a specific temperature 24 , respectively. This thiyl radical can then react with the C=C bond to form a carbon centred radical. From this step the initiation is turned to a propagation step resulting in the formation of a thio ether as a reaction product and a new thiyl radical subsequently involved in the reaction chain. In our case, the click reaction was performed using 2,2′-Azobis(2-methylpropionamidine)dihydrochloride (AAPH) as radical initiator, allyl mercaptan as double bond substrate and thiophenol as reactant. The AAPH is known to be decomposed into cationic radicals under UV light (365 nm) or temperature above 60 °C (See supplementary information). The reaction was initiated at the surface of the gold NCs (figure 2a) in aqueous environment using a 660 nm laser beam (figure 2b). The allyl mercaptan was grafted onto the NCs surface through the S-Au bond whereas the AAPH and the thiophenol were free in solution (figure 2c). Since EBL allows the fine control of the size and shape of the nanostructures at the nanometer scale, the substrates benefit from the reproducibility of both: LSPR position and SERS signal 25,26 . Arrays of NCs (figure 2d) with different diameters: 110, 140 and 200 nm were fabricated to investigate whether the thiol-ene click reaction can occur due to plasmonic effects. As shown by the extinction spectra of the NCs in aqueous environment (figure 2e), the 110 nm diameter NCs were found to have a LSPR position close to 660 nm (excitation laser wavelength) whereas 140 nm NCs had a LSPR position red-shifted compared to 660 nm. For the 200 nm NCs, the excitation wavelength was nearly out of resonance, resulting in a largely less intense electromagnetic near-field. The reaction kinetics were evaluated for the three diameters in order to observe changes in reaction performances depending on the mismatch between the LSPR position and the laser wavelength.

In situ thiol-ene click reaction

To initiate the reaction, the solution of AAPH and thiophenol was deposited on a microscopic slide with a cavity and the substrate with NCs decorated with grafted allyl mercaptan was then placed above the slide with the NCs inside the solution. Local reaction at the microscale level was implemented using the excitation laser focused with a 60x objective (N.A. 0.70) through the NCs substrate. The reaction kinetics were then monitored by SERS in real time using the same laser wavelength. SERS spectra were first recorded on the 110 nm NCs: one spectrum each minute for a total duration of 30 min. The process was then reproduced on the NCs of 140 nm and finally on the NCs of 200 nm (figure 2f).

The peaks observed in the SERS spectra correspond to the thiophenol vibrations. Due to method limitations (including placing the substrate in liquid and laser focusing procedures) the NCs were exposed to the solution for about 4 min before the first SERS measurement was made. For this reason the SERS signal of thiophenol was not strictly equal to zero on the first SERS spectrum indicating a spontaneous thiophenol interaction with non-blocked gold areas. To follow the reaction kinetics, the intensity of the peak at 1572 cm -1 assigned to an aromatic C=C vibration was plotted versus reaction time (figure 3a) for all NCs diameters. The observed SERS intensity of the thiophenol increases and reaches a plateau, when the gold surface is saturated. Reaction times of less than 10 min were observed for the 110 nm and the 140 nm diameters whereas for the 200 nm, saturation was not achieved even after 40 min of reaction. 10 min is very fast for this thiol-ene reaction since it has been estimated to be of several hours in normal conditions (i.e. without NCs and with UV excitation) 27 . To determine precisely the reaction time, the experimental data were fitted with a first order Langmuir isotherm assuming that during the reaction a single layer of thiophenol was formed:

𝛉 = 𝛉 𝐬𝐚𝐭 [𝟏 -𝐞𝐱𝐩 (-𝐭 𝛕 ) ] ( 1
)
where θ is the surface coverage, θsat is the coverage saturation, t is the time and τ is the reaction time constant. Figure 3b shows the reaction time constant, , that corresponds to the time required to reach 63% of SERS signal saturation versus the shift between the LSPR position of NCs and the laser excitation wavelength, =LSPR-exc. It is clearly visible, that the reaction time is strongly dependent on the LSPR position. A twenty times faster reaction was observed on highly resonant 110 nm NCs compared to 200 nm NCs. Unexpectedly, 140 nm NCs with a 54 nm difference between the LSPR position and the excitation wavelength reveals a slightly lower reaction efficiency compared to the one measured on 110 nm NCs.

In order to demonstrate that our experiments actually correspond to the thiol-ene "click" chemistry, the experimental conditions were modified to perform negative controls, since the grafting of thiophenol on the NCs can be attributed to two other mechanisms: spontaneous grafting or competitive replacement. On one hand, it is known that short molecules with the thiol group -like allyl mercaptan -form non-homogeneous self-assembled monolayers (SAM) due to the lack of intermolecular interactions 28 . This leaves non-blocked active gold sites for spontaneous thiophenol grafting. On the other hand, competitive molecular displacement could occur directly on the gold surface, inducing the replacement of allyl mercaptan by thiophenol 29 . Thus, the SERS signal measured in the thiol-ene experiments could then also include contributions from these two thiophenol-gold interactions. For this concern, three negative controls (experiments C1 to C3) were designed to analyze the thiophenol grafting rate via side reactions (figure 4). The first negative control (experiment C1) was designed to duplicate the thiol-ene reaction without a radical initiator. The second negative control (experiment C2) was dedicated to observe the competitive thiophenol adsorption by replacing molecules grafted onto the gold surface. Herein, mercaptoethanol was used instead of allyl mercaptan to avoid any reaction between thiophenol and the allyl group. The last negative control was devoted to the monitoring of the spontaneous adsorption of thiophenol on non-functionalized NCs (experiment C3). All these experiments were performed in the same manner as previously described for the thiolene reaction. The reaction kinetics of the three negative controls recorded on the 110 nm NCs are shown in figure 4 and are compared to the kinetics of the thiol-ene reaction.

The SERS signal for the experiments C1 and C2 are nearly constant with time. This suggests that in the latter experimental conditions, thiophenol was interacting spontaneously by replacing the short thiols previously deposited on the surface. Since this mechanism is a competitive one, equilibrium was reached very rapidly (less than a few minutes) resulting in a constant SERS signal.

Figure 4: SERS intensities of the integrated peak at 1572 cm -1 for the thiol-ene reaction and for all three negative controls recorded on 110 nm NCs (black dot: thiol-ene reaction, blue dots: C1 negative control, green dots: C2 negative control, red dots: C3 negative control). The schemas of the different reactions are given on the right of each curve.

However, the measured signal is significantly lower than the one recorded during the thiol-ene reaction, meaning that the high level of the thiophenol signal observed with the thiolene reaction cannot be explained by a simple competitive mechanism. In fact, the non-zero SERS signal observed prior to the thiol-ene reaction start (figure 3a and figure 4 black circles) might be assigned to the competitive replacement mechanism. The fast increase in the SERS signal was then induced by the initiated thiol-ene reaction. Furthermore, the comparison of the thiol-ene reaction signal to the one achieved during experiment C1 suggests that AAPH is essential to initiate the thiol-ene reaction.

The SERS signal recorded in experiment C3 is relevant to the kinetics of thiophenol SAM formation. Two main observations can be made from the comparison between the experiment C3 and the thiol-ene reaction. Firstly, thiolene kinetics is faster than the spontaneous grafting of thiophenol suggesting the strong contribution of the "click" chemistry process. Secondly, the signal at saturation in experiment C3 is comparable to the one observed in the thiol-ene reaction. This means that the same NCs coverage has been achieved using either the thiol-ene or the simple thiol reaction. By comparing the different reaction kinetics associated with each negative control experiment (C1 to C3), it is evident that thiophenol grafting onto NCs under conditions demonstrated in figure 3 is favored by the thiol-ene "click" reaction acting as a dominant mechanism.

The contribution of plasmon to the thiol-ene reaction can then be elaborated. To provide evidence, we compared the SERS intensities obtained after surface saturation for the thiol-ene reaction and for experiment C3 on the three NCs diameters (figure 3b). During experiment C3, the grafting of thiophenol should not be dependent on the plasmonic properties of the NCs since the thiol reaction with gold is not thermally or optically activated. The SERS signal ratio between NCs diameters observed in this experiment is thus only due to the effect of the LSPR position compared to the excitation wavelength 30 and does not reflect the modification in the coverage rate of the thiophenol on the NCs with different diameters. As expected for experiment C3, the ratio between SERS signal on 110 nm NCs is 2.5 larger than the one measured on the 200 nm due to plasmonic effects on the SERS signal. In the case of the thiol-ene reaction, this ratio is much higher, close to 6. Such a large ratio can only be explained by the higher efficiency of the thiol-ene reaction in thiophenol grafting. Thus, when the LSPR is shifted relative to the excitation wavelength, the thiol-ene reaction is less efficient whereas it is enhanced when the LSPR is close to the excitation wavelength. This is clear evidence that radical reaction is induced and can be tuned by the plasmonic properties of the NCs.

Plasmonic mechanisms

Thanks to NMR experiments (see supplementary information), we have demonstrated that the thiol-ene reaction monitored herein on the plasmonic substrates cannot be initiated at 660 nm wavelength without the plasmonic substrates. In a conventional thiol-ene experiment either a UV or a thermal activation is necessary to initiate the reaction, even if this latter effect seems to be less efficient (see supplementary information). Thus, we propose two processes related to the plasmonic properties of the NCs to interpret our results. The first one, which we call the photonic process, is related to the optical excitation of the radical initiator. It is known that when a molecule is in the close vicinity of a metallic surface its electronic levels are broadened as illustrated in figure 5a 31,32 . Electronic transitions can then occur at wavelengths outside the UV range whereas they are forbidden for the same molecules in solution and without metal. Through this process, the radical can be formed under initiation with a 660 nm wavelength. As the electromagnetic near-field is enhanced when the LSPR is close to the excitation wavelength, the formation of the radical should be favored as well. As a consequence, the reaction rate is highly improved on resonant NCs.

The second contributing mechanism is related to a process. Discrete Dipole Approximation (DDA) calculations were performed to determine the near-field at the vicinity of the NCs surface (see supplementary information) and the increase in temperature around the NCs was then derived (figure 5b) 33 . Though an increase in temperature was obtained for all diameters it was found to be strongly dependent on the LSPR shift relative to the excitation wavelength. Nearly the same increase in temperature was calculated for 110 nm and 140 nm NCs, close to 50-55°C, whereas it was close to 15°C for 200 nm NCs. The attained temperatures were then high enough to induce the formation of the radical (70-75°C for the 110 and 140 nm NCs and 35°C for the 200 nm NCs). he LSPR dependency on the kinetics (figure 3b) can be explained by such LSPR dependency on the temperature.

Comparing plots in figures 3b and 5b, one can notice that the slope of the temperature curve is opposite to the one of the reaction time constant,  More precisely, the temperature increase induced around the 110 and 140 nm NCs accelerates the speed of the thiol-ene coupling reaction, while a lower temperature increase found in the 200 nm NCs results in a rather slow reaction rate. We assume that both processes, thermal and photonic, are taking place since they are directly related to the plasmonic properties of the NCs. Both processes contribute simultaneously to the enhancement of the thiol-ene reaction efficiency.

To confirm this conclusion and to further improve thiol-ene reaction localisation, we set up one last experiment. To do this we performed an experiment contrary to the one carried out previously (Figure 2c). In this case, the thiol groups (butanedithiol) were grafted onto the 110 nm NCs surface whereas the molecules consisting in alkene function (pentenoyl-nitroaniline) and AAPH were dispersed in solution (figure 6a). As demonstrated in figure 6c, the reaction measured on three different points was working by means of a few minutes ( measured from 2.1 to 7.9 minutes). Similar behaviour was observed for all recorded spots and the reaction was reproducible from one point to another. These results confirm that in this configuration the radical formed on the thiol group is only present at the focal point. In previous experiments (allyl mercaptan grafted on the surface), the radicals were formed on the thiophenol in the solution and then the initiation of the thiol-ene reaction outside the laser spot could not be excluded. In the present experiment, the thiol-ene reaction could only occur locally inside the laser spot where the thiol groups were grafted. Accordingly, the reaction was limited to the laser spot and thus was highly localised. This paves the way to a quick and easy method to localise surface functionalisation via the "click" process at the nanometer scale through plasmonic effects.

Conclusions

We have demonstrated that the thiol-ene reaction can be induced at the surface of gold NCs even if the experimental conditions are not favourable, through the exploitation of the plasmonic properties of the nanostructures. These experiments demonstrate that the thiol-ene reaction can be performed in a few minutes and that by tuning the LSPR position we are able to control the kinetics of the reaction as well as the reaction localisation. This effect interpreted as the combination of both photonic and thermal effects can be used to enhance and control chemical reactions at the nanoscale level. To investigate the reaction mechanism and to have a better understanding on the nanoplasmonic effects, further experiments can be done involving different thiols and alkenes compounds as well as different radical initiators. Moreover, the careful selection of nanoparticles pursuing different plasmonic properties could also be meaningful to determine which process, thermal or photonic ones, is the dominant effect causing the initiation of thio-lene reaction.

Figure 1 :

 1 Figure 1: Schematic illustration of the free-radical thiol-ene coupling mechanism.

Figure 2 :

 2 Figure 2: a) Schema of the nanocylinders (NCs) used in the experiments. b) Schema of the experimental configuration. c) Schema of the thiol-ene reaction configuration. d) Scanning electron microscopy image of an array of NCs with a diameter of 200 nm. e) Extinction spectroscopy measurements on 110 nm (black curve), 140 nm (red curve) and 200 nm (blue curve) NCs arrays. The grey line indicates the laser wavelength used for thiol-ene reaction initiation and SERS investigation. f) SERS spectra of grafted thiophenol onto gold NCs of 110 nm (black spectra), 140 nm (red spectra) and 200 nm (blue spectra) diameter.

Figure 3 :

 3 Figure 3: a) Relative SERS intensities of the integrated peak at 1572 cm -1 versus reaction time for the 110 nm (black dot), 140 nm (red dot) and 200 nm (blue dot) NCs. Solid lines correspond to the fitting of the experimental points using the equation (1) (110 nm: black line, 140 nm: red line and 200 nm: blue line). b) Absolute SERS intensity at saturation and reaction time plotted versus the mismatch between the LSPR position of NCs and the laser excitation wavelength. Hollow dot: reaction time for the 110 nm, 140 nm and 200 nm NCs. Hollow square: absolute SERS intensities of the thiophenol measured on the 110 nm, 140 nm and 200 nm NCs for the thiol-ene reaction after surface saturation. Plain square: absolute SERS intensities of the thiophenol measured on the 110 nm, 140 nm and 200 nm NCs for the experiment C3 and after surface saturation.

Figure 5 :

 5 Figure 5: a) Schema of the photonic mechanism. On the left, the electronic configuration of an individual molecule with two levels (ea and eb) is represented. On the right, the same molecule is shown but in interaction with a metallic surface. The upper electronic level of the molecule is then broadened (eb' in the middle schema) due to the interaction with the metallic electronic states (right schema with EF the Fermi level). The dotted arrow in the middle schema represents the excitation of the molecule through the 660 nm illumination. b) Temperature increases plotted versus the mismatch between LSPR position of NCs and the laser excitation wavelength on the 110 nm, 140 nm and 200 nm NCs. The temperature increases were calculated using Discrete Dipole Approximation method.

Figure 6 :

 6 Figure 6: a) Schema of the thiol-ene reaction configuration. b) SERS spectra of grafted pentenoyl-nitroaniline to 110 nm NCs. c) Relative SERS intensities of the integrated peak at 1332 cm -1 versus reaction time for three different points on the SERS substrate. The solid lines correspond to the fitting of the experimental points using the equation (1). The reaction time constant, , in minutes is given in insert for each point.

Acknowledgements

The authors want to acknowledge the REMANTAS ANR project (ANR-11-ECOT-0010) for financial support.