Optical Method for Predicting the Composition of Self-Assembled

Jan 23, 2012 - the two pyrrole rings), falling in a spectral range distant from the LSPR of Ag .... striking.7e However, the 112° value refers again ...
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Optical Method for Predicting the Composition of Self-Assembled Monolayers of Mixed Thiols on Surfaces Coated with Silver Nanoparticles Piersandro Pallavicini,*,† Claire Bernhard,§ Giacomo Dacarro,‡ Franck Denat,§ Yuri A. Diaz-Fernandez,† Christine Goze,§ Luca Pasotti,† and Angelo Taglietti† †

InLAB, Dipartimento di Chimica and ‡Dipartimento di Fisica “A. Volta”, Università di Pavia, 27100 Pavia, Italy § Institut de Chimie Moléculaire de l’Université de Bourgogne, UMR CNRS 6302, Université de Bourgogne, 21078 Dijon, France S Supporting Information *

ABSTRACT: With a simple optical method, based on UV− vis absorption spectra on glass slides, it is possible to predict the composition of self-assembled monolayers of mixed thiols, grafted on monolayers of silver nanoparticles. Glass slides are modified with the layer-by-layer technique, first forming a monolayer of mercaptopropyltrimethoxysilane, then grafting a monolayer of silver nanoparticles on it. These surfaces are further coated by single or mixed thiol monolayers, by dipping the slides in toluene solutions of the chosen thiols. Exchange constants are calculated for the competitive deposition between the colorless 1-dodecanethiol or PEG5000 thiol and BDP-SH, with the latter being a thiol-bearing molecule containing the strongly absorbing BODIPY (4,4-difluoro-4bora-3a,4a-diaza-s-indacene) moiety, synthesized on purpose. The constants are calculated by determining the fraction of BDPSH deposited on the surface from a solution with a given molar fraction, directly measuring the absorption spectra of BDP-SH on the slides. Then, the exchange constant for the competitive deposition between 1-dodecanethiol and PEG5000 thiol is calculated by combining their exchange constants with BDP-SH. This allows to predict the fraction of the two colorless thiols coating the silver nanoparticles slides obtained from a toluene solution with a given molar fraction, for example, of PEG5000 thiol. The correctness of the calculated surface fraction is verified by studying the coating competition between 1-dodecanethiol and a PEG5000 thiol remotely modified with a strongly absorbing fluorescein fragment.

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INTRODUCTION The deposition of a stable monolayer of metal nanoparticles (NPs) on flat surfaces has become a common practice. In particular, gold NPs can be easily grafted on surfaces like mica or glass by means of silanization of the surface with a monolayer of functional silanes (typically mercaptopropyltrimethoxy silane, MPTS, or aminopropyltrimethoxy silane APTS), whose terminal group allows easy and stable binding of gold NPs.1 This approach, see Scheme 1A, B, has often been called LbL, that is, layer-by-layer,2 and has been used mainly for the assembling of materials with enhanced sensing and optical properties. Beside gold, the same approach has been used, although much less frequently, to prepare monolayers of silver NPs.3 In this case, in addition to the optical and sensing properties, the well-known antibacterial activity of Ag can be exploited to prepare surfaces with a significant disinfectant capability, as we have recently shown.4 Another interesting feature of this approach is that if the used bulk material is transparent (glass, quartz), the peculiar absorption spectrum of the Ag or Au NPs monolayer, due to the localized surface plasmon resonance (LSPR) phenomenon, can be easily studied © 2012 American Chemical Society

by means of conventional UV−vis spectrophotometers, as the absorption of the NPs monolayer is sufficiently intense to observe well-defined bands in the visible. These are centered around 390 and 520 nm for Ag and Au, respectively.4 A huge chapter of the development of the nanochemistry of inorganic surfaces was dedicated to the study of molecular selfassembled monolayers (SAMs) of thiols on films of gold and silver.5 These films, often prepared on mica or glass, are considered flat, and SAMs are obtained by dipping the bulk surface in a solution of the chosen thiol. This simple but powerful technique is capable of producing ordered, densepacked SAMs of linear thiols, whose properties (e.g., contact angles) depend essentially on the terminal X group of the chosen HS−(CH2)n−X molecule, with X being what the surface exclusively exposes to the external environment.6 A flat, ordered, metal surface is an essential prerequisite to obtain such ordered, and dense-packed SAMs. This kind of metal surface is Received: August 1, 2011 Revised: January 23, 2012 Published: January 23, 2012 3558

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Scheme 1. Pictorial Scheme of the Approach Used to Prepare the Multilayer Glass Slides

commonly prepared by physical vapor deposition of gold or silver on pretreated flat silicon wafers, mica, or glass slides. One intriguing subchapter regards the formation of mixed monolayers on flat gold or silver, by competitive deposition from mixed solutions of thiols, X−(CH2)n−SH and Y− (CH2)m−SH. As demonstrated by the seminal papers of Whitesides, dating back to 1988, given a certain molar fraction of the two thiols in solution, the molar fraction on the surface may be different, depending mainly on the nature of X, Y, on the solvent, and on m and n values.7 Mixed coating has been studied also on the surface of metal nanoparticles, dispersed (i.e., dissolved) in a solvent. In this field, the series of papers of Murray, in the late 1990s, demonstrated that thiol-coated gold NPs undergo placeexchange reactions of the coating thiol with solutions containing mixtures of competing X−(CH2)n−SH and Y− (CH2)m−SH.8 Also in this case, the ratio of the two thiols on the NP surface is a complex function of X, Y, solvent, and chain lengths. Formation of a SAM of a chosen X−(CH2)n−SH on a monolayer of gold or silver NPs grafted on a flat surface is also a described procedure, obtained by simply dipping the surfaces bearing the NPs monolayer in a solution of the chosen thiol (as in Scheme 1C). Electrochemical1a,d,9 or optical sensors have been made by this approach, in the latter case by exploiting the changes in the LSPR of the Au or Ag NPs monolayer due to the changes in the local refractive index brought by interaction of external species with the terminal X group.10 Moreover, a number of papers was published regarding the coating of the gold (and to a lesser extent silver) NPs monolayer with α,ωdithiols, for example, HS−(CH2)n−SH, then exploiting the free, upper −SH layer to deposit a second gold NPs monolayer. The operation may be repeated for several steps, allowing one to obtain materials with new optical, conductive and Raman (SERS) properties.11

To the best of our knowledge, no attempt has been done to coat gold or silver NPs monolayers grafted on a surface with a mixed SAM of different thiols. This paper represents the first attempt to prepare, characterize, rationalize, and control the method to obtain such a new type of hybrid surfaces, that may be of great importance in the field of materials for medicine and of antibacterial ones in particular. The possibility of coating the exposed free portion of a monolayer of firmly grafted silver NPs with two (or more) different molecules is appealing, as this could be a straightforward approach to prepare materials capable of carrying on (and releasing from) their surface a cocktail of molecular drugs (e.g., antibiotic, antimicrobial, antimicotic, anti-inflammatory, etc.). This, in addition to silver strong antibacterial action, would result in poly active materials. However, by merging the literature on competitive SAM formation on flat surfaces and nanoparticles, it is expected that also in this case the surface composition is not the same of the solutions used for the coating process. A method allowing the prediction of surface composition knowing that of the coating solution appears thus necessary. In this paper, we prepare monolayers of silver NPs grafted on MPTS-coated glass slides, then we use the free, exposed portion of the grafted NPs to append a thiol-functionalized BODIPY molecule (BDP-SH) from its solutions in toluene. BDP-SH has been prepared on purpose in our laboratories and is a stable, intensely colored thiolated dye, whose concentration on the surface can be easily calculated by UV−vis absorption spectrometry, thanks to the typical BODIPY band12 that falls in the visible range but well shifted from the LSPR of silver NPs. Then formation of mixed monolayers of BDP-SH and R-SH is studied by deposition from toluene solutions containing mixtures of the two competing molecules; see Scheme 1D and E. The chosen R-SH species are C12-SH (1-dodecanethiol) and PEG5000-SH, that is, one lipophilic and one hydrophilic molecule. Both molecules are colorless, as they do not absorb in the 300−850 nm range, but the composition of the mixed 3559

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the solvent was evaporated to give a yellow solid. 1H NMR (300 MHz, CDCl3): δ = 1.45 (t, 1H, SH), 2.75−2.68 (m, 2H, CH2CH2SH), 3.14− 3.08 (m, 2H, NCH2), 3.65 (m, 440 H, OCH2), 6.79−6.62 (m, 6H), 7.13 (d, 1H), 8.00 (d, 1H), 8.17 (s, 1H). 3. Methods. Glass Slides and Cuvettes Bearing Ag NPs Monolayer. First, glass slides were coated with a monolayer of MPTS, according to a published procedure.14 Then, the thiolmodified glass slides were immersed into a colloidal suspension of silver nanoparticles (d = 7 ± 4 nm), prepared as described elsewehere,4a,15 and kept at 30 °C for 18 h, as described in a previous work.4a The same procedure was applied to optical glass cuvettes, that in the last step were filled with the Ag NPs colloidal suspension. Coating Procedure with Pure Thiols (BDP-SH, PEG5000-SH, FLPEG5000-SH, C12-SH) on Slides and Cuvettes Bearing the Ag NPs Monolayer. In each preparation, eight glass slides bearing an Ag NPs monolayer were kept in a vertical position into a eight-place slides holder, filled with 40 mL of toluene solution of the chosen thiol. Solutions were 10−4 M. The 40 mL volume was sufficient to completely cover the slides. The cuvettes bearing the Ag NPs monolayer were simply filled with the same solutions. After 2 h, the coating solution was removed, and the obtained glasses were placed in toluene (while the cuvettes were filled with the same solvent) and sonicated for 5 min. This procedure was repeated twice, then glasses or cuvettes were dried under a gentle N2 stream and stored in air. For each slide four contact angles (two for each side) were measured within 1 h. On the slides coated with BDP-SH, UV−vis spectra were also recorded. The spectra were corrected for background absorption and the corrected absorbance used to calculate ns,max by means of eq 1b (vide infra, Results and Discussion). Coating Procedure with Thiols Mixtures. A 10−4 M stock solution of each thiol was prepared in toluene. Sixteen new solutions were obtained by blending different amounts of the stock solutions. By this, the 16 different solutions have a total thiol concentration of 10−4 M and the molar fraction of one of the two competing thiol is 0.000; 0.125; 0.200; 0.250; 0.333; 0.375; 0.460; 0.470; 0.500; 0.570; 0.622; 0.666; 0.750; 0.800; 0.900; 1.000. Two samples were prepared for each molar ratio, by dipping two slides bearing Ag NPs in the toluene solutions. Slides were kept in a vertical position into a five-place slides holder and were completely immersed in the coating solution. After 2 h the coating solution was removed, and the slides were washed twice with pure toluene. This procedure was repeated three times for each pair of competitors. For each slide four contact angles (two for each side) were measured within one hour from preparation. In case that one of the two competing thiols was BDP-SH, UV−vis absorption spectra were also recorded, background corrected, and the corrected absorbance used to calculate ns. Verification of BDP-SH Concentration on Surface by Its Release in Solution. The molar extinction coefficient of BDP-SH in ethanol containing 10−4 M of PEG5000-SH was calculated by recording UV− vis spectra at different BDP-SH concentrations, in the 3 × 10−6−1 × 10−5 M range. The absorbance versus concentration plot is linear, and ε526 = 76 007 M × cm−1 is calculated from the slope. Five glass slides coated with BDP-SH were kept in a vertical position into a five-place slide holder that contains 17 mL of PEG5000-SH 10−4 M in ethanol. Every 15 min, a 3 mL portion of the solution was rapidly transferred to a quartz cuvette, the absorption spectrum measured, and the 3 mL portion reintegrated in the bulk solution. After the first hour, spectra were taken at 1 h intervals. Release was complete in less than 2 h. The total BDP-SH released was calculated taking a spectrum on the solution after 24 h (the entire experiments were repeated with toluene as solvent with identical final total released BDP-SH; in some cases, C12-SH was used instead of PEG5000-SH, obtaining identical results). Determination of FL-PEG5000-SH on Surface by Its Release in Solution. The molar extinction coefficient of FL-PEG-SH in a 10−4 M ethanol solution of PEG5000-SH was calculated as described above, obtaining ε501 = 60 706 M × cm−1. Five glass slides coated with FLPEG5000-SH were kept in a vertical position into a five-place slide holder that contained 17 mL of PEG5000-SH 10−4 M in ethanol. After 1, 2, 16, and 24 h, a 3 mL portion of the solution was transferred to a

SAMs obtained on the Ag NPs can be determined observing the absorption of the BDP-SH molecule. As expected, the dependence of the surface composition on that of the solution is not linear. Making the toluene solution composition to vary and calculating by means of the BDPSH absorption the surface composition, exchange constants are obtained for the BDP-SH/C12-SH and BDP-SH/ PEG5000-SH competitions. Finally, the combination of the two exchange constants allows one to calculate the constant for the deposition from solutions of the two competing colorless species C12-SH and PEG5000-SH, so to predict the surface composition from a given solution mixture. We confirmed the correctness of this approach by studying the surface composition after deposition from solution mixtures of C12-SH competing with a PEG thiol that was remotely functionalized with a dye molecule, this allowing direct evaluation of its surface concentration.

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MATERIALS AND METHODS

1. Reagents. PEG5000-SH (CH3O−(CH2CH2O)nCH2CH2SH, MW = 5000) and NH2−PEG5000−SH hydrochloride (H2N− (CH2CH2O)nCH2CH2SH, MW = 5000) were purchased from Rapp Polymere and used as received; fluorescein isothiocyanate (>90%) and 1-dodecanethiol (>98%) were purchased from Sigma-Aldrich and used as received; 1,1-carbonyldiimidazole (CDI) and diisopropylamine (DIPEA) were purchased from Acros and used as received. Organic solvents were purchased from Carlo Erba. Water was distilled three times. Microscopy cover glass slides (2.4 × 2.4 cm2) were purchased from Forlab (Carlo Erba), and glass and quartz spectroscopy cuvettes from Sigma-Aldrich. 2. Syntheses. BDP-SH. 1,1-Carbonyldiimidazole (CDI; 1.37 g, 8.5 mmol) was added to a solution of 4-carboxyphenyl1,3,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacene13 (3.0 g, 7.0 mmol) in dimethylformamide (DMF; 200 mL) at 0 °C. After stirring for 1 h, the solution was allowed to return to room temperature. Diisopropylethylamine (DIPEA; 1.8 g, 14 mmol) and 2-aminoethanethiol hydrochloride (0.795 g, 7 mmol) were successively added. After total conversion of the starting material (12 h, followed by TLC), the solvent was removed on a rotary evaporator. The solid obtained was washed with water (2 × 60 mL) and extracted with dichloromethane (200 mL). The organic phases were dried over MgSO4, and the solvent was evaporated to give a red oil. The crude product was purified by column chromatography on silica gel (AcOEt/hexane 4:6). Recrystallization in CH2Cl2/ hexane/EtOH gave pure BDP-SH as red solid (1.75 g, 52%). 1 H NMR (300 MHz, CDCl3, 300 K) δ (ppm): 0.95 (t, 6H, J = 7.6 Hz, CH3), 1.23 (s, 6H, CH3), 1.43 (t, 1H, J = 8.5 Hz, SH), 2.26 (q, 4H, J = 7.6 Hz, CH2), 2.51 (s, 6H, CH3), 2.82 (dt, 2H, J = 6.2 Hz, J = 8.4 Hz, CH2CH2SH), 3.67 (dt, 2H, J = 6.0 Hz, J = 6.2 Hz, CH2CH2SH), 6.70 (t, 1H, J = 6.0 Hz, NH), 7.36 (d, 2H, J = 8.4 Hz, Ar), 7.92 (d, 2H, J = 8.4 Hz, Ar). 13C{1H} NMR (75 MHz, CDCl3, 300 K) δ (ppm): 12.1, 12.7, 14.8, 17.2, 24.9, 43.1, 127.9, 129.1, 130.6, 133.3, 134.7, 138.3, 138.8, 139.7, 154.5, 166.9. ESI-MS: m/z = 506.23 [M+Na]+, 989.46 [2M +Na]2+. HRMS: Calculated for C26H32BF2N3O2Na m/z = 506.2241. Obtained: 506.2273. Elemental analysis: calculated for C26H32BF2N3O2 + 2 EtOH: C (62.20%), N (7.30%), H (7.71%). Obtained: C (62.69%), N (7.09%), H (7.73%). FL-PEG5000-SH. Fluorescein isothiocyanate (6.0 mg, 0.015 mmol) and NH2−PEG5000−SH hydrochloride (78 mg, 0.015 mmol) were dissolved in 50 mL of acetonitrile. Then 30 μL of triethylamine was added under magnetic stirring. After total conversion of the reagents (12 h) checked by TLC (methanol/ethyl acetate 1/1 + 1% acetic acid), the solvent was removed with a rotary evaporator. The solid obtained was washed with 2 mL water and extracted with dichloromethane. The organic phases were dried over MgSO4, and 3560

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Figure 1. (A) absorption spectrum of BDP-SH in toluene solution. (B) Blue line: absorption spectrum on an air-exposed, dry glass slide bearing an Ag NP monolayer coated with a BDP-SH monolayer. Red line: corrected spectrum, after background subtraction. quartz cuvette, to measure the absorption spectrum, after which it was reintegrated in the bulk solution. Release was complete in less than 2 h. The total FL-PEG5000-SH released was calculated recording a spectrum on the solution after 24 h Determination of 1-Dodecanethiol Concentration on Surface by Its Release in Solution. Five glass slides coated with 1-dodecanethiol were crushed in a 10 mL vial, covered with a PEG5000-SH 10−4 M solution in ethanol and gently shaken in Heidolph Promax 1020 reciprocating platform shaker for 24 h. Three samples were prepared in the same manner. The quantities of released 1-dodecanethiol were measured with GC/MS ThermoScientific DSQII instrument by comparison of the area peak of the chromatogram with that of different standard solutions. See the Supporting Information for GC/ MS conditions. Determination of the Local Refractive Index As a Function of the Solvent Refractive Index for Ag NPs Monolayers Coated with Thiols. Dry cuvettes bearing in the internal walls an Ag NPs monolayer coated with the chosen thiol (BDP-SH, C12-SH, PEG5000-SH) were filled with the chosen solvent and allowed to equilibrate for 1 h at 25 °C, after which time an UV−vis absorption spectrum was recorded and corrected for background. After each spectrum the solvent was discarded, the cuvette was gently dried in a N2 flux and then washed three times with the next solvent before refilling. In order to achieve the total removal of the previous solvent, the following sequence was used: water, acetonitrile, DMF, n-butanol, ethyl acetate, toluene, and n-heptane. The Ag NPs LSPR absorption data were treated as already described4b to obtain the local refractive index. In brief, the corrected spectra were interpreted applying Mie’s formalism16 combined with the dielectric function data for silver.17 This approach allows calculation of the effective refractive index (ηeff) felt by the NPs anchored to the thiol-functionalized surface in contact with different solvents. A linear relationship is experimentally observed between the square effective refractive index, calculated from our LSPR data (ηeff 2), and the square refractive index of the solvent (ηs2) reported in literature,18 allowing one to calculate the fraction of silver NP surface exposed to the solvent (φ): ηeff2 = φηs2 + κ . Full details are available in the Supporting Information. 3. Instrumentation. Atomic force microscope images were obtained with a Thermo Microscopes CPII AFM, operated in tapping mode with NT-MDT silicon tips NSG03. Data analysis (manual width and height calculation on line profiles) was carried out with Image Processing and Data Analysis software, version 2.1.15 by TM Microscopes. UV−vis absorption spectra on solutions were taken with a Varian Cary 100 spectrophotometer in the 200−1000 nm range. Spectra on functionalized surfaces were obtained placing the glass slides on the same apparatus equipped with a dedicated Varian solid sample holder, or directly using the modified cuvettes. Contact angle determinations were made with a KSV CAM200 instrument.

Molecular modeling (MM+ level) was carried out using the Hyperchem 7.5 platform.

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RESULTS AND DISCUSSION 1. The Ag NP Monolayer and Its Coating with BDP-SH. To prepare glass surfaces coated with a silver NPs monolayer, we adopted a procedure that we have already described.4 Briefly, an MPTS monolayer is obtained on precleaned glass by dipping slides in a MPTS solution in toluene (Scheme 1A). Then, the −SH terminated glass is immersed in a citratestabilized Ag NPs colloidal solution (average diameter of the NPs is 7 ± 4 nm). This leads to the self-assembly of a NP monolayer firmly attached to the modified glass (Scheme 1B). We had shown that by dipping the NPs-coated slides in a series of solvents and by plotting the variation of the effective refraction index felt by the grafted NPs versus the refraction index of the solvent it is possible to calculate that 66% of the NPs surface is exposed to the environment.4a At least, in principle, this surface fraction is available for further coating with different thiols. The Ag NPs-coated glasses prepared by our method are stable for weeks (as proven by the invariability of UV−vis absorption spectra) when exposed to air or toluene. It has also to be stressed that the Ag NPs monolayer is not compact, as the NPs are separated one from the other, with an average distance of 17 ± 8 nm.4a For this work, we prepared monolayers of Ag NPs both on 24 mm × 24 mm glass slides and on the internal walls of glass cuvettes. The latter are used to measure variations of the absorption spectrum connected to the exposure to solvents, as when absorption spectra are recorded on the slides, the surfaces must be dry (N2 fluxed). The LSPR of Ag NPs on dry glass surfaces has λmax = 394 nm, while for the same surfaces dipped in toluene λmax is at 427 nm. BDP-SH is a molecule prepared on purpose in our laboratories. We have chosen it as the bodipy moiety (bodipy = borondipyrromethene, i.e. 4,4-difluoro-4-bora-3a,4a-diaza-sindacene) is widely used,12 stable, with an intense absorption centered at 500−530 nm (depending on the substituents on the two pyrrole rings), falling in a spectral range distant from the LSPR of Ag NPs. BDP-SH is soluble in toluene, where it displays the absorption spectrum shown in Figure 1A, with λmax = 528 nm and ε528 = 64883 M−1 cm−1. The coating of the Ag NPs monolayer with BDP-SH (Scheme 1C) is obtained by dipping the NPs-coated surfaces in a toluene solution of BDPSH (10−4 M). The coating process is complete in 20 min, as 3561

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and released in solution (see also the Supporting Information). No residual BDP-SH absorption is found on the slides after 2 h, and UV−vis determination of the concentration of BDP-SH in the measured volume of toluene allows to determine the effective number of released molecules. This, divided by the surface of the slides, gives ns = 1.2 × 1014 cm−2 (σ = 1.6 × 1013 cm−2). This indicates a slight underestimation of ns,max by using the spectra on the surface and ε533 in solution. A correction factor is calculated, c = 1.2 × 1014/9.7 × 1013 = 1.24. This value is used to obtain a corrected relation 1a, that allows one to calculate directly ns from A533 on glasses

indicated by UV−vis spectra carried out on a series of slides, taken off from the BDP-SH solution at different times (see the Supporting Information). At any rate, to rule out the possibility of partial functionalization, all coating procedures were carried out with a 2 h exposure to thiol solutions.19 Figure 1b displays a typical spectrum in air of a dry slide (blue line), with Ag NPs coated with BDP-SH. Both LSPR and BDP-SH absorptions are present and well separated. The LSPR shifts from 394 to 414 nm (average on eight measurements, σ = 1 nm) due to the functionalization with thiol groups of the available Ag NPs surface. In toluene-dipped surfaces, LSPR shifts from 427 to 433 nm (average on eight measurements, σ = 1). The absorption band of BDP-SH is slightly influenced by the formation of a monolayer on Ag NPs, as λmax moves to 533 nm (both in toluene-wet and in air-exposed surfaces). The surface concentration of BDP-SH molecules grafted on the Ag NPs monolayer can be calculated by the relation 1a ns(cm−2) = 6 × 1020A /2ε

ns(cm−2) = c × 6 × 1020A /2ε

(1b)

The value ns,max = 1.2 × 1014 cm−2 has been used as the maximum surface concentration for Ag NPs slides coated with BPD-SH in all the following calculations. By measuring the LSPR position of Ag NPs coated with the BDP-SH layer when the surface is immersed in solvents of different refractive index, one can calculate the effective refractive index (ηeff) felt by the NPs anchored to the thiolfunctionalized surface in contact with different solvents. A linear correlation is found between the square of ηeff and of the solvent bulk refractive index, whose slope gives the fraction of the NPs surface that is exposed to the solvent (see Figure S2 and Supporting Information). We found a 23% exposed fraction, with a considerable decrease with respect to the 66% exposed fraction found for grafted and uncoated Ag NPs.4b However, the exposed fraction is still significantly different from zero. This is not surprising considering that the NP monolayer is not a regular, flat surface, but it is made of small spherical objects with an average radius only ∼2 times larger than the length of BDP-SH (∼1.5 nm, evaluated by molecular modeling, MM+ level). Solvent penetration near the NP surface may take place between BDP-SH molecules, that are significantly divergent if arranged perpendicularly to the NPs surface. Moreover, solvent penetration is expected to be promoted also at the lower border of the NPs, where steric repulsion between BDP-SH and the underlying MPTS monolayer may result in less efficient coating. The advancing water contact angle (CA) on the BDP-SH coated Ag NPs surfaces is 65° ± 3, with a significant increase with respect to the uncoated Ag NPs layer (42° ± 5) and a value similar to what measured on the underlying MPTS monolayer (62° ± 3). It must be pointed out that all the observed CA values result from the contact of a macroscopic water droplet on a surface that is heterogeneous on the nanoscale, as the underlying MPTS layer remains exposed in the area between the Ag NPs. The measured CAs are thus averaged for the contact of water with the various types of surface components. Finally, both the LSPR and BDP-SH bands do not change position and intensity with time (72 h checking), when exposed to air at room temperature, indicating good stability. 2. Coating with 1-Dodecanethiol (C12-SH) or PEG5000-SH. We have chosen one linear hydrophobic n-alkyl thiol (C12-SH) and one thiol-terminated linear hydrophilic polyethylene glycol (PEG5000-SH) as competitors for BDP-SH in the coating process. These two thiols have been used in a number of papers as thiolated coating agents for the surface modification of gold and silver nanoparticles or bulk surfaces.5 They impart a sharp hydrophobic or hydrophilic character, respectively. As an example, a C12-SH coating makes gold nanoparticles stable and soluble in nonaqueous solvents,21

(1a)

where ns is the number of chromophores in the unit area, that is, 1 cm2, A is the absorbance at the chosen wavelength, and ε is the molar extinction coefficient of the chromophore. The factor 2 is used as our samples are coated on both sides. Spectra taken on surfaces may be strongly influenced by the absorption of the bulk material. In particular, untreated glass slides or glass slides with a MPTS monolayer display a weak unstructured absorption, that is, they may be not perfectly transparent in the 300−800 range, as it is has been reported in the literature.4,14,20a This unstructured absorption may have different intensities in different preparations (see also Supporting Information, S10), even if using identical materials and reactions conditions, so it is problematic to define a unique sample for background subtraction. Accordingly, the absorption spectra were analyzed using a homemade software4a (see also ref 20b), the background was interpolated and subtracted with a cubic-spline, and the parameters of the peaks (λmax, absorbance) were determined. A representative corrected spectrum is included in Figure1b (red line). Minor spectral features may be lost in the subtraction process (e.g., the shoulder at lower wavelength of the 533 nm peak, as it can be seen on comparing blue and red lines in Figure 1b). Moreover, the slight shift in the absorption maximum of BDP-SH from solution (λmax = 528 nm) to its SAM on dry Ag NPs surfaces (λmax = 533 nm) rises the problem of what ε value is to be used on calculating ns with relation 1a, as either the ε value of the maximum of absorption of BDP-SH in solution (ε528 = 64883 M−1cm−1) or the ε value at 533 nm in solution (ε533 = 55879 M−1cm−1) can be considered. Opting for the ε533 value, we obtain ns,max = 9.7 × 1013 cm−2 (σ = 1.7 × 1013). Finally, it may be argued that a molecular monolayer grafted on a NPs monolayer is a different environment with respect to a toluene solution, and that the extinction coefficient may thus be significantly different. Due to all this, the reliability of the background subtraction and ns,max calculation was validated with an alternative method. BDP-SH is fully released from the Ag NPs monolayer into accurately measured volumes of toluene. This is obtained by dipping the BDP-SH coated slides for 24 h in a 10−4 M toluene solution of RSH (RSH = PEG5000-SH or C12-SH). Competition with the concentrated solutions of the thiols makes BDP-SH to be displaced by RSH on the Ag NPs surface 3562

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Figure 2. AFM imaging on the 500 nm × 500 nm scale for Ag NP slides coated with PEG5000-SH (A) and for uncoated Ag NP slides (B).

linear and flexible molecules, while the bodipy moiety of BDPSH is a large, flat, rigid fragment. It is thus not reasonable to assign the higher percent of exposed NP surface to a number of C12-SH or PEG5000-SH molecules per surface unit lower than that of BDP-SH. Sterical demand and intermolecular stacking interactions, that are possible only for BDP-SH (vide infra), should instead be considered. Moreover, direct counting of ns with alternate methods (vide infra) shows that it is identical within standard deviation for the three thiols. Atomic force microscopy (AFM) imaging carried out on the uncoated Ag NPs surfaces and on surfaces coated with BDPSH, C12-SH, and PEG5000-SH are displayed in Figure 2 and Supporting Information Figure S7. Images on the 500 nm × 500 nm scale show that the surfaces are dominated by the morphology of the Ag NPs monolayer, with no difference between coated and uncoated slides (Figure 2A: the representative case of PEG5000-SH coating; identical images are obtained for C12-SH and BDP-SH coating, Supporting Information Figure S7; Figure 2B: uncoated slide). The average height, calculated on 500 nm × 500 nm images, is 4.9 nm (standard deviation = 1.3 nm) in the case of the uncoated Ag NPs layer. For coated Ag NPs, lower values are found: 3.3 nm (1.0) for BDP-SH, 3.8 nm (0.9) for PEG5000SH, and 4.5 nm (1.7) for C12-SH. This indicates a smoothing effect exerted by the coating agents on the surface morphology, that is still dictated by the Ag NPs layer. As C12-SH and PEG5000-SH molecules have no absorption bands in the visible, the straightforward spectroscopical method described for BDP-SH is not available to calculate ns with eq 1b. To have a direct information on ns for PEG5000-SH, a bifunctional H2N-PEG5000-SH molecule is used. We modified H2N-PEG5000-SH at the −NH2 group with fluorescein isothiocyanate to obtain the FL-PEG5000-SH molecule. The fluorescein fragment has an intense absorption band in water or other protic, polar solvents (e.g., ethanol)24 (see Supporting Information for spectra in ethanol). However, when it is dissolved in aprotic, poorly polar solvents like toluene, it undergoes an intramolecular esterification yielding a weakly absorbing form.24 The same happens on dry slides, as they appear colorless. Direct molecule/cm2 counting by UV−vis spectroscopy on glass slides is thus not possible, but we were able to measured the quantity of FL-PEG5000-SH grafted to Ag NP by displacing it from the slides using a 10−4 M PEG5000-SH solution in ethanol. By measuring the absorbance at 501 nm and using the ε501 value determined with a calibration procedure (see Materials and Methods section), from the

while a PEG5000-SH coating makes gold nanorods stable and soluble in in-vivo-like aqueous environment.22 Beside opposite solvophilicity, they also have considerably different dimensions (length = ∼1.5 nm for C12-SH and ∼27 nm for PEG5000-SH, obtained by molecular modeling at the MM+ level for the fully extended molecule). Both are nonabsorbing in the 300− 820 nm range. The sum of these properties makes them perfect candidates to demonstrate the validity of our approach. Slides bearing Ag NPs monolayers coated with C12-SH and PEG5000-SH are prepared with the same experimental procedure used for BDP-SH. The LSPR shifts consistently with what observed with BDP-SH. We obtain λmax = 413 and 412 (dry slides) and 436 and 447 nm (toluene) for C12-SH and PEG5000-SH coating, respectively. This confirms that the LSPR change is related to the functionalization of the Ag NPs on their available surfaces. Advancing water contact angles are 74° (±2) for C12-SH and 33° (±3) for PEG5000-SH, with a variation of +32° and −11° with respect to the uncoated monolayer of Ag NPs. The CA found with PEG5000-SH is comparable to that reported in the literature for monolayers of similar molecules, even if CAs with slightly lower values are found in the case of ordinate SAMs on flat gold surfaces (e.g., water advancing CAs for SAMs of HS-(CH2)15(CO)NH−(CH2−CH2−O)n−H are 28° and 30° for n = 4 and 6, respectively23). In our case, despite the heterogeneous nature of the surface, the CA is low, most probably because the long PEG5000-SH molecules extend from each Ag NPs, hiding also the underlying MPTS exposed areas. On the other hand, in the case of C12-SH, the difference found for the CA of a SAM of the same molecule on flat Ag, 112°, is striking.7e However, the 112° value refers again to a compact, ordered 2D-crystalline molecular monolayer. Comparable compactness is to be excluded with C12-SH on Ag NPs, due in part to the curved nature of each Ag NPs (r = 3.5 nm, C12SH length =1.5 nm by molecular modeling) and mainly to the intrinsically heterogeneous nature of the surface, exposing a significant area of the underlying MPTS monolayer. The plot of ηeff 2 versus the square of the solvent bulk refractive index, measured observing the Ag NPs LSPR variations on dipping surfaces in different solvents (Supporting Information, Figure S2b), allows to calculate that 35% of the NPs surface is exposed to the solvent in the case of C12-SH coating and 34% in the case of PEG5000-SH (Supporting Information, Figure S2c). The percent of exposed surface of the Ag NPs is higher than that found for BPD-SH. Both C12-SH and PEG5000-SH are 3563

dx.doi.org/10.1021/la202995w | Langmuir 2012, 28, 3558−3568

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Article

Figure 3. Experimental BDP-SH fraction (θBDP‑SH, blue symbols, determined by UV−vis spectroscopy) versus molar fraction of BDP-SH in the coating solution (χsol,BDP‑SH), in the case of BDP-SH/C12-SH competition (A) and BDP-SH/PEG5000-SH (B) competition. Dashed red curves are obtained by fitting experimental data with expression 3.

volume of the solution and the area of the slides ns is safely calculated, obtaining 1.1 × 1014 cm−2 (σ = 0.15 × 1014 cm−2), a value remarkably identical within experimental error to the number of BDP-SH molecules per surface units (ns,max = 1.2 × 1014 cm−2). The structural difference between PEG5000-SH and FL-PEG5000-SH regards the remote end of the molecules with respect to the −SH function. The PEG5000-SH and FLPEG5000-SH molecules can be reasonably considered identical in the coating processes. Also in the case of C12-SH, its surface concentration was checked by means of an alternative, nonspectroscopic method. Glass slides coated with C12-SH were crushed inside a vial and then covered with a measured volume of ethanol containing 10−4 M PEG5000-SH for prolonged time (24 h) in order to fully displace C12-SH from the NP surface. Its concentration in solution was then determined by GC-MS (mass spectroscopy gas chromatography), allowing to obtain ns = 1.3 × 1014 cm−2 (σ = 0.2 × 1014 cm−2) surface concentration, superimposable within standard deviation to the ns,max value of 1.2 × 1014 cm−2 (σ = 1.6 × 1013 cm−2) found for BDP-SH. 3. Competitive Coating between BDP-SH and C12-SH or PEG5000-SH. Freshly prepared glass slides coated with Ag NPs are dipped for 2 h in toluene solutions containing BDP-SH and RSH (RSH = C12-SH or PEG5000-SH). In the solutions, the total thiol concentration (BDP-SH + RSH) is constant and equal to 10−4 M, and the molar fraction of BDP-SH, χsol,BDP‑SH, (eq 2), is varied between 0 and 1. χsol,BDP‐SH =

[BDP‐SH] [BDP‐SH] + [RSH]

As expected, the observed trend is not linear, but it is fitted by expression (3) (fitting curves: dashed red profiles in Figure 3): θBDP ‐ SH =

1 ⎛ [R‐SH] ⎞α ⎟ 1 + KS⎜ ⎝ [BDP‐SH] ⎠

(3)

This equation corresponds to a modified Langmuir isotherm, and it can be rearranged to obtain an explicit expression for the thermodynamic exchange constant Ks for the case of the competition between BDP-SH and C12-SH KS,BDP/C12 =

[BDP‐SH]α (1 − θBDP‐SH) [C12‐SH]α θBDP‐SH

(4a)

and in the case of competition of BDP-SH with PEG5000SH KS,BDP/PEG =

[BDP‐SH]α (1 − θBDP‐SH) [PEG5000‐SH]α θBDP‐SH

(4b)

By linearization and fitting experimental data to eqs 4a and 4b, we obtained the concentration exponent α and the exchange constants KS for the two competitions: log KS,BDP/C12 = −0.28 and log KS,BDP/PEG = −0.54, with αBDPH/C12 = 0.45 (r2 = 0.96) The concentration exponents on eqs 4a and 4b can be associated to the molecularity of the single adsorption and desorption kinetic steps (see the Supporting Information for further discussion). In our case, the fitted values of α are smaller than 1, indicating an island-type adsorption on the surface (or phase-segregation).26 AFM images obtained on glass slides bearing Ag NPs coated with mixtures BDP-SH/C12-SH and BDP-SH/PEG5000-SH obtained from 0.5 molar fraction solutions (Supporting Information, Figure S7) do not display remarkable differences with respect to what observed for the slides coated with single thiols. Although α values in eqs 3 and 4 indicate the formation of islandlike superstructures in the coating layer, the invariance of the AFM images is not surprising, considering that the differences in the average height between the slides with Ag NPs covered with each single-thiol are smaller than the standard deviation affecting the height value (the high standard deviation originates from the large dimensional distribution of

(2)

Quantities in square parentheses are molar concentrations in solution. [BDP-SH] + [RSH] = 10−4 M. The slides are then washed with toulene and gently dried with a N2 stream before measuring their UV−vis spectrum. From the BDP-SH absorption, its surface concentration (ns) is calculated with eq 1a, and dividing by the maximum ns value determined for pure BPD-SH coating (ns,max = 1.2 × 1014cm−2) we calculate the fraction of BDP-SH deposited on surfaces, θBDP‑SH. Figure 3, blue points displays the variation of θBDP‑SH versus χsol,BDP‑SH.25 3564

dx.doi.org/10.1021/la202995w | Langmuir 2012, 28, 3558−3568

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the synthetized Ag NP, as d = 7 nm ±4 nm). It is thus unlikely that islands of any of the three employed RSH may be distinguishable by AFM on surfaces whose morphology is dominated by the Ag NPs layer. Moreover, the discrete nature of the Ag NP SAM must be again considered. As we have pointed out, the centers of two adjacent NP are positioned at distance larger than the sum of their radii (center−center distance =17 ± 8 nm, r = 3.5 nm4a). At least for short molecules like C12-SH and BDP-SH (length