Fluorescent Self-Assembled Monolayers of Umbelliferone: A

Jul 23, 2013 - Besides SAM structural characterizations by atomic force microscopy and X-ray reflectivity, we carried out contact angle measurements a...
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Fluorescent Self-Assembled Monolayers of Umbelliferone: A Relationship between Contact Angle and Fluorescence Luc Séro,†,‡ Lionel Sanguinet,*,† Séverine Derbré,‡ Frank Boury,§ Guillaume Brotons,∥ Sylvie Dabos-Seignon,† Pascal Richomme,‡ and Denis Séraphin‡ †

PRES L’UNAM, Laboratoire MOLTECH-Anjou, Université d’Angers, CNRS-UMR 6200, 2 bd Lavoisier, 49045 Angers Cedex, France ‡ PRES L’UNAM, Université d’Angers, EA 921 SONAS, 16 bd Daviers, 49045 Angers Cedex, France § PRES L’UNAM, Université d’Angers, INSERM U646 “Micro-Nanomédecine Biomimétiques”, IBS-CHU ANGERS, bâtiment IRIS, 4 rue Larrey, 49933 Angers Cedex, France ∥ PRES L’UNAM, Université du Maine, UMR-CNRS 6283, IMMM, 72085 Le Mans Cedex 9, France ABSTRACT: Self-assembled monolayers (SAMs) that contain fluorophore units are nowadays widely used to tune surface properties and design new chemical sensor chips. It is well-known that the nature of the substrate may strongly interfere with the emission properties of the grafted molecules, but the organization of the monolayer may also have an important role. To study the influence of the SAM organization on the luminescence properties, we prepared different coumarin-based derivatives endowed with tethered chains of different lengths and elaborated the corresponding SAMs on glass slides. Besides SAM structural characterizations by atomic force microscopy and X-ray reflectivity, we carried out contact angle measurements and applied the Van Oss−Chaudhury− Good theory, which was rarely used previously for self-assembled monolayers. As expected, by increasing the tethered chain length, a higher surface coverage, a higher degree of organization, and a stronger molecular packing were observed. However, it appears to facilitate the self-quenching process, and thus, this strongly affects the fluorescent properties of the SAMs.

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INTRODUCTION Since their first description in 1946 by Zysman et al.,1 selfassembled monolayers (SAMs) continue to be the focus of much attention. In particular, the elaboration of SAMs represents a simple, convenient, and flexible manner to chemically functionalize a metallic surface2 and also to obtain highly ordered molecular systems with well-defined dimensions at the nanoscale range.3 In this context, a wide variety of substrates (mainly coinage metals) from planar surfaces to patterned nanostructures3 have been used as supports for a large assortment of molecules and assemblies, including complex supramolecular dyads and triads.4−6 Such materials have found numerous applications,7,8 in particular in analytical chemistry for the development of chemical sensors,9 with chemically modified SAMs displaying recognition units.10,11 To monitor the corresponding host−guest complexation, fluorescence spectroscopy appears as one of the most promising techniques in terms of sensitivity, selectivity, speed, and localization of the interaction.12 However, the photophysical properties of a fluorophore can be drastically modified once bound to a surface.13 Indeed, the use of a metallic surface as the substrate may induce a fluorescence quenching through dipolar coupling between the chromophore and the substrate.14 Several © XXXX American Chemical Society

teams have studied the influence of the tethered chain length on the quenching efficiency.15,16 Among them, Whitesell’s works on 9-alkylfluorenethiols have shown that almost 65% of the fluorescence is still quenched by the metallic surface when the alkyl chains linking the fluorescent moieties and the substrate are as long as 12 carbon atoms.17 For all these reasons, silicon oxide substrates are generally preferred to metallic surfaces for building up fluorescent chemical sensors. Nevertheless, this choice does not allow for probing the organization of the SAM by common electrochemical techniques (cyclic voltammetry or impedance spectroscopy). In this context, it is interesting to evaluate the SAM organization on simple and cheap glass microscopy slides, and we studied the relationship between molecular organization and relevant parameters, such as surface density (molecular packing) and the photophysical properties of the integrated fluorophore. For this purpose, a series of monolayers on identical glass slides were prepared and characterized by contact Received: April 24, 2013 Revised: July 18, 2013

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angle, fluorescence, atomic force microscopy (AFM), and X-ray reflectivity (XR) measurements.

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(51 g, 170 mmol), and K2CO3 (2.48 g, 34 mmol) were dissolved in dry DMF (40 mL), and the resulting mixture was heated at 90 °C for 24 h. After being cooled to room temperature, the reaction mixture was poured into deionized water (200 mL) and extracted two times with CH2Cl2 (50 mL). The combined organic layers were then washed with brine (15 mL), dried over MgSO4, and concentrated under reduced pressure. The crude residue was purified by column chromatography (petroleum ether/CH2Cl2, 8/2) to afford the desired compound as a white powder (6.15 g, 91%). 1H NMR (CDCl3): δ 7.46 (d, 2H, J = 8.7 Hz, C8H), 3.82 (m, 2H), 6.09 (s, 1H), 3.98 (t, 2H, J = 6.3 Hz, CH2O), 3.38 (t, 2H, J = 6.9 Hz, CH2Br), 2.37 (s, 3H, CH3), 1.79 (m, 2H), 1.3 (m, 14H). 13C NMR (CDCl3): δ 162.2 (C2), 161.3 (C7), 155.2 (C8′), 152.5 (C4), 125.4 (C5), 113.3 (C4′), 112.6 (C3), 111.7 (C6), 101.3 (C8), 68.5 (CH2O), 33.9 (CH2), 32.7 (CH2), 29.25 (CH2), 29.19 (CH2), 28.89 (CH2), 28.64 (CH2), 28.06 (CH2), 25.85 (CH2), 18.58 (CH2). 7-[(10-Hydroxydecyl)oxy]-4-methylcoumarin (3). In a roundbottom flask, 7-[(10-bromodecyl)oxy]-4-methylcoumarin (4.61 g, 11.7 mmol) was dissolved in 25 mL of a H2O/HMPA mixture (15/85, v/ v), and the resulting mixture was stirred for 5 h at 130 °C. After being cooled to room temperature, the reaction mixture was poured into water and extracted three times with CH2Cl2. The combined organic layers were then washed with brine, dried over MgSO4, and concentrated under reduced pressure. The crude product was triturated in petroleum ether and then filtered to afford the desired compound as a white powder (3.06 g, 80%). 1H NMR (CDCl3): δ 7.48 (d, 2H, J = 8.7 Hz, C8H), 6.83 (m, 2H), 6.12 (d, 1H, J = 0.9 Hz, C3H), 4.00 (t, 2H, J = 6.6 Hz, CH2O), 3.64 (t, 2H, J = 6.6 Hz, CH2OH), 2.39 (d, 3H, J = 1.2 Hz, CH3), 1.78 (m, 2H), 1.26 (m, 14H). 13C NMR (CDCl3): δ 162.2 (C2), 161.4 (C7), 155.3 (C8′), 152.6 (C4), 125.4 (C5), 113.4 (C4′), 112.7 (C3), 111.8 (C6), 101.3 (C8), 68.6 (CH2O), 63.1 (CH2OH), 32.8 (CH2), 29.5 (CH2), 29.4 (CH2), 29.4 (CH2), 29.3 (CH2), 28.94 (CH2), 25.9 (CH2), 25.7 (CH2), 18.6 (CH3). N-[3-(Triethoxysilyl)propyl]carbamic Acid 10-[(4-Methyl-2oxo-2H-1-benzopyran-7-yl)oxy]dec-1-yl Ester (C10). To a solution of 7-[(10-hydroxydecyl)oxy]-4-methylcoumarin (0.60 g, 1.8 mmol) in dry THF (30 mL) were added 3-(triethoxysilyl)propyl isocyanate (0.669 g, 2.7 mmol) and triethylamine (0.183 g, 1.8 mmol) under a nitrogen atmosphere. The resulting mixture was refluxed and monitored by TLC (CH2Cl2). After 36 h, the reaction mixture was allowed to cool to room temperature, and the THF was removed under vacuum. The crude residue was purified by flash chromatography (CH2Cl2 to CH2Cl2/MeOH = 9/1 in 30 min, debit 20 mL/min, column Chromabond Flash RS80SiOH) to afford the desired compound as a white powder (650 mg, 62%). 1H NMR (CDCl3): δ 7.48 (d, 1H, J = 8.7 Hz, C8H), 6.84 (m, 2H), 6.12 (d, 1H, J = 0.9 Hz, C3H), 4.87 (s, 1H, NH), 4.00 (m, 4H), 3.81 (q, 6H, J = 6.9 Hz, CH3CH2OSi), 3.16 (m, 2H, SiCH2CH2CH2NH), 2.39 (d, 3H, J = 1.2 Hz, CH3), 1.83−1.28 (m, 18H), 1.22 (t, 9H, J = 6.9 Hz, CH3CH2OSi), 0.62 (t, 3H, J = 8.1 Hz, CH2Si). 13C NMR (CDCl3): δ162.2 (C2), 161.4 (C7), 156.8 (NHCO), 155.3 (C8′), 152.5 (C4), 125.4 (C5), 113.4 (C4′), 112.7 (C3), 111.8 (C6), 101.3 (C8), 68.6 (CH2OPh), 64.8 (CH2NH), 58.4 (CH2OSi), 29.4 (CH2), 29.3 (CH2), 29.3 (CH2), 29.1 (CH2), 29.0 (CH2), 29.0 (CH2); 25.9 (CH2), 25.8 (CH2), 23.3 (CH2), 18.7 (CH3), 18.3 (CH3CH2OSi), 7.6 (CH2Si). HRMS (m/z): calcd for (C30H49NO8Si + Na)+ 602.3125, found 602.3120. Sample Preparation. The microscopy glass slides were purchased from Chevallier Glass & Lux and cleaned by immersion in a piranha solution (7/3 (v/v) 98% H2SO4/30% H2O2) at 90 °C for 1 h. The substrates were then rinsed several times with copious amounts of ultrapure water, dried under high vacuum overnight, and then transferred into a glovebox filled with an argon atmosphere. The monolayers of the different alkylsiloxanes were prepared under a dry and oxygen-free atmosphere ([H2O] < 1 ppm, [O2] < 1 ppm) on fresh oxidized substrates from dilute solutions of alkyltriethoxysilanes (ca. 10 mmol) in anhydrous toluene for various immersion times. After the substrate was removed from the solution, it was rinsed with fresh toluene, sonicated for 5 min, and then rinsed one more time with toluene before being dried in a nitrogen stream.

MATERIALS AND METHODS

Materials. Unless otherwise stated, starting materials, reagents, and solvents were obtained from commercial suppliers and were used without further purification. 1H and 13C NMR spectra were recorded on a Bruker 300 or 500 MHz spectrometer. N-[3-(Triethoxysilyl)propyl]carbamic Acid 4-Methyl-2-oxo2H-1-benzopyran-7-yl Ester (C0).18 To a solution of 4-methylumbelliferone (3.00g, 17.05 mmol) in dry THF (20 mL) were added 3-(triethoxysilyl)propyl isocyanate (6.32g, 25.6 mmol) and triethylamine (1.72g, 17.05 mmol) under a nitrogen atmosphere. The resulting mixture was refluxed and monitored by TLC (CH2Cl2). After 24 h, the reaction mixture was allowed to cool to room temperature, and the THF was removed under vacuum. The crude product was triturated in petroleum ether and filtered to afford the desired compound as a white powder (7.10 g, 98%). 1H NMR (CDCl3): δ 7.54 (d, 1H, J = 9.3 Hz, C8H), 7.11 (m, 2H, C4H and C5H), 6.22 (s, 1H, C3H), 5.52 (br, 1H, NH), 3.83 (q, 6H, J = 7.2 Hz, CH3CH2OSi), 3.27 (m, 2H, CH2NH), 2.40 (s, 3H, CH3), 1.72 (m, 2H, CH2), 1.23 (t, 9H, J = 7.2 Hz, CH3CH2OSi), 0.68 (t, 2H, J = 8.1 Hz, CH2Si). 13C NMR (CDCl3): δ 160.8 (OCO), 154.1 (C8′), 153.7 (NHCO), 153.5 (C4), 152.1 (C7), 125.1 (C5), 118.0 (C6), 117.2 (C4′), 114.0 (C8), 110.07 (C3), 58.5 (CH2OSi), 43.6 (CH2NCO), 22.9 (CH2), 18.7 (CH3), 18.3 (CH3CH2OSi), 7.7 (CH2Si). HRMS (m/z): calcd for (C20H29NO7Si + Na)+ 446.1611, found 446.1606. 7-[(3-Hydroxypropyl)oxy]-4-methylcoumarin (1).19 In a round-bottom flask, 4-methylumbelliferone (2.0 g, 11.4 mmol), 3bromopropanol (2.36 g, 17 mmol), and K2CO3 (8.6 g, 62 mmol) were dissolved in 25 mL of acetone, and the resulting mixture was heated at 60 °C for 20 h. After being cooled to room temperature, the reaction mixture was poured into deionized water (100 mL) and then extracted three times with CH2Cl2. The combined organic layers were then washed with brine (15 mL), dried over MgSO4, and concentrated under reduced pressure. The corresponding residue was purified by column chromatography (CH2Cl2/AcOEt, 8/2) to afford the desired compound as a white powder (2 g, 74%). 1H NMR (CDCl3): δ 7.48 (d, 1H, J = 8.7 Hz, C8H), 6.85 (m, 2H, C5H and C6H), 6.12 (d, 1H, J = 1.2 Hz, C3H), 4.18 (t, 2H, J = 6 Hz, CH2OH), 3.87 (t, 2H, J = 6 Hz, CH2O), 2.39 (d, 3H, J = 1.2 Hz, CH3), 2.08 (q, 2H, J = 6 Hz, CH2), 1.6 (br, 1H, OH). 13C NMR (CDCl3): δ 161.9 (C8′), 161.3 (C4′), 155.2 (C2), 152.6 (C4), 125.5 (C7), 113.6 (C6), 112.5 (C5), 112.0 (C8), 101.4 (C3), 65.8 (CH2O), 59.7 (CH2OH), 31.8 (CH2), 18.7 (CH3). N-[3-(Triethoxysilyl)propyl]carbamic Acid 3-[(4-Methyl-2oxo-2H-1-benzopyran-7-yl)oxy]prop-1-yl Ester (C3). To a solution of 7-[(3-hydroxypropyl)oxy]-4-methylcoumarin (1.00 g, 4.27 mmol) in dry THF (20 mL) were added 3-(Triethoxysilyl)propyl isocyanate (6.32 g, 25.6 mmol) and triethylamine (1.72 g, 17.05 mmol) under a nitrogen atmosphere. The resulting mixture was refluxed and monitored by TLC (CH2Cl2/MeOH, 98/2). After 24 h, the reaction mixture was allowed to cool to room temperature, and the THF was removed under vacuum. The crude residue was purified by column chromatography to afford the desired compound as a white powder (1.78 g, 87%). 1H NMR (CDCl3): δ 7.47 (d, 1H, J = 8.7 Hz, C8H), 6.80 (m, 2H, C5H and C6H), 6.12 (d, 1H, J = 1.2 Hz, C3H), 4.96 (br, 1H, NH), 4.24 (t, 2H, J = 6 Hz, CH2), 4.09 (t, 2H, J = 6 Hz, CH2), 3.80 (q, 6H, J = 6.9 Hz, CH3CH2OSi), 3.16 (m, 2H, CH2), 2.38 (d, 3H, J = 1.2 Hz, CH3), 2.12 (m, 2H, CH2), 1.61 (m, 2H, CH2), 1.21 (t, 9H, J = 6.9 Hz, CH3CH2OSi), 0.62 (t, 2H, J = 8.4 Hz, CH2Si). 13C NMR (CDCl3): δ 161.9 (C2), 161.2 (C7), 156.4 (NCO), 155.2 (C8′), 152.5 (C4), 125.5 (C5), 113.6 (C4′), 112.7 (C3), 112.0 (C6), 101.3 (C8), 65.0 (CH2OPh), 61.1 (CH2OCO), 58.4 (CH3CH2OSi), 43.4 (CH2NCO), 28.8 (OCH2CH2), 23.2 (SiCH2CH2), 18.6 (CH3), 18.2 (CH3CH2OSi), 7.6 (CH2Si). HRMS (m/z): calcd for (C23H35NO8Si + Na)+ 504.2029, found 504.2024. 7-[(10-Bromodecyl)oxy]-4-methylcoumarin (2). In a roundbottom flask, 4-methylumbelliferone (3.0 g, 17 mmol), dibromodecane B

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Scheme 1. Synthesis of the Different Coumarin Derivatives Used To Elaborate the Corresponding Self-Assembled Monolayer

αin was varied from 0 to 2α. From the width of the specular peak measured from the rocking curves, we defined the incident angle offset for the longitudinal scans to Δαoffset = 0.1°. These scans were subtracted from the specular scans to obtain the so-called true specular reflectivity curves that were analyzed. Without this subtraction, specular reflectivities would be overestimated at large incidence angles and the film roughness would be underestimated by several angstroms. At small angles, when the beam footprint overspilled the film surface, a geometrical correction was used to compensate this effect by integrating the incident flux reaching the surface at each angle of incidence.

Characterization. Contact angles were measured at room temperature using the sessile drop technique on a commercial GBX Digidrop MCAT instrument (monitored by Digidrop+ software). Drops of 2 μL volume were formed at the tip of a capillary fitted to an automatic syringe and deposited on the substrate. All contact angles are an average of at least eight independent measurements performed under the same conditions in different places of the sample and are reported with their standard deviation. Fluorescence spectroscopy measurements and steady-state excitation and emission spectra were recorded on a Quantamaster spectrometer (from PTI) set to a 2 nm band pass for both the excitation and emission collection monochromators, and anthracene was used as the standard for the quantum yield determination. The emitted light was detected at an angle of 90° with respect to the excitation beam. The solid substrate was fixed in a special sample holder at constant distance from the source and detector, allowing reproducible fluorescence intensities recorded in front face mode. All surface fluorescence spectra were corrected for background emission from glass. AFM was performed using a PicoSPM microscope (Molecular Imaging Inc.). All images were recorded in the contact mode with a maximum scan size of 6.5 × 6.5 μm2. The images were processed with the WSxM program provided by Nanotec.20 The roughness parameter given in the following is an RMS calculated from the WSxM program. For each investigated sample, several scans were performed on different sites of the sample and at different scales. X-ray reflectivity measurements were carried out on a home-built reflectometer: The source is a Rigaku Ultra X18 rotating anode with a copper target set to 40 kV and 50 mA. An Osmic multilayer monochromator fixes the wavelength to 1.54 Å. After two cross sets of Huber collimating slits, the primary beam fwhm (full width at half-maximum) is 0.08° at the sample stage, 150 cm away from the source, with a peak intensity of 2 × 107 cps (counts per second after correction from automatic attenuators). A motorized Huber goniometer allows independent sample and detector rotations with a precision of 0.0005°, and the detector is a Cyberstar NaI(Ti) scintillator with a low background (0.1 cps) at 30 cm from the sample with two selection slits that define an acceptance angle smaller than the primary beam fwhm. Three types of in-plane incidence scans were collected on each sample. The specular reflection was measured by keeping the incident angle, αin, and the outcoming angle equal (αout = αin) by coupled (αin/2αin) reflectivity scans. Therefore, the momentum transfer vector, q, was kept perpendicular to the film interface (q = qz). To record nonspecular intensities, we performed standard longitudinal scans (offset scans) and rocking scans (transverse scans). The longitudinal scan was similar to the reflectivity scan except that the detection angle was intentionally offset by a fixed angle, Δαoffset. In the rocking scan, the detector did not move (i.e., the sum 2α = αin + αout was fixed) while the sample was rocked in the incident beam so that

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RESULTS AND DISCUSSION

The preparation of a self-assembled monolayer with a fluorophore unit can be conducted in two ways. The first one consists of a two-step procedure, which starts with the grafting of a molecule endowed with a terminal reactive group, such as an amino or isocyanate function, and finishes with the introduction of the fluorescent unit by surface reaction. The efficiency of this surface reaction strongly depends on the size of the reactant and the accessibility of the reactive group,21 which may be an issue in the context of SAMs displaying high surface coverage. By contrast, the second method of preparation requires the synthesis of fluorescent compounds functionalized with an anchoring group through a molecular spacer of different lengths. The modification of the tethered chain length allows one to generate different SAM organizations. A short chain gives less ordered films than a long one, because of the reduced van der Waals interactions between adjacent hydrocarbon chains.22,23 In this study, we prepared three different compounds based on the 4methylumbelliferone moiety. This fluorophore presents two main advantages: first, coumarin has been largely studied in the literature, and second, the hydroxyl group in position 7 allows one to introduce different spacer lengths by simple alkylation of this function or addition/elimination. The choice of the anchoring group was driven by the nature of the substrate. To elaborate SAMs on a glass substrate, the use of a silane coupling agent, generally trichloro- or trialkoxysilane, is the most widespread technique. However, it is important to note that in this case all molecules are not individually linked to the surface. The elaborated monolayer consists of a cross-polymerized network of molecules with only C

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Figure 1. Evolution of the emission spectra (with λex = 325 nm) for the different surfaces (a) S0, (b) S3, and (c) S10 after different immersion times. (d) Evolution of the fluorescence intensity at 390 nm with λex = 325 nm as a function of the immersion time. The solid lines represent the best fits obtained with a Langmuir adsorption model.

longer. As the fluorescence intensity was stable after a few hours of immersion, we suggest these compounds form single monolayers on the surface, in agreement with previous studies of SAMs incorporating similar coumarin derivatives.27,28 In fact, in the case of multilayer formation and/or polymers physisorbed on a substrate, the fluorescence intensity would be expected to grow again with the immersion time.29 As pointed out before, the spacer length has a strong influence on the adsorption kinetics. On the contrary, Hoffmann et al. obtained a constant growth rate for the formation of different alkylsiloxane monolayers on native silicon and concluded that the hydrocarbon chain length or the type of terminal substituent had a minor influence on the monolayer formation rates.22 To clarify this point, we fitted the evolution of the fluorescence intensity using a Langmuir adsorption curve (If(t) = If,max(1 − exp(−kct)) shown in Figure 1d and obtained similar constant rates for C0, C3, and C10 (respectively 1.5 ± 0.3, 1.9 ± 0.3, and 1.3 ± 0.3 L·mol−1·min−1) assuming that the absorption kinetics for all three compounds are similar and weakly influenced by the tethered chain length. Besides the evaluation of the kinetic constant, the fluorescence measurement provides information about the organization of the fluorophore inside the self-assembled monolayer. As is generally observed for the immobilization of a fluorophore on a surface, the emission spectra of all coumarin derivatives immobilized on glass substrates only display a slight red shift and an enlargement of the peak when compared to the solution spectra or to the condensed-phase spectra (see Figure

a few bonds to the surface. From this point, SAMs formed from a silane coupling agent can be considered very similar to Langmuir−Blodgett layers.24 If the use of dimethylalkoxysilane avoided the possibility of forming a multilayer, some studies have shown that it produces incomplete SAMs by nature.25,26 For all these reasons, a triethoxysilane function, as an anchoring group, was grafted to the coumarin unit by reaction between (3-isocyanatopropyl)triethoxysilane (3-ICPTS) and a hydroxyl function preliminarily placed at the end of the alkyl chain. The whole synthesis of the different compounds suitable for SAM elaboration is depicted on Scheme 1. Since it is essential to combine several characterization techniques to reach an accurate molecular representation of the SAMs, we used the incorporation of the coumarin moiety as a fluorophore to follow the growth of the monolayer by monitoring its fluorescence using steady-state fluorescence spectroscopy. To carry out this experiment, the three coumarin derivatives C0, C3, and C10 were immobilized by immersion of a glass slide preliminarily activated in the silinazing solution to form the corresponding self-assembled monolayers called respectively S0, S3, and S10. The ex situ measurement of the fluorescence intensity for different immersion times was used to evaluate the kinetics of the surface functionalization as plotted in Figure 1. As expected, for our three surfaces (S0, S3, and S10) the fluorescence intensity increases with the immersion time. However, the signal intensity did not significantly change after more than 2 h of immersion for S0 and S3. For S10, reaching the maximum of the fluorescence intensity took a bit D

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Figure 2. Comparaison of the normalized emission spectra of the different coumarin derivatives (a) C0, (b) C3, and (c) C10 recorded in ethanol solution (solid line) and immobilized on the surface (dashed line).

Since we are not able to extract the exact surface coverages of the SAMs from the fluorescence measurements, we characterized the S0, S3, and S10 samples to probe their organization. Among all available techniques, wettability measurements are certainly one of the most simple and robust methods to obtain the first insight into the structure and composition of a monolayer.35,36 For this reason, we first present these data and then additional surface characterizations using AFM and X-ray reflectivity techniques. Contact Angle Measurements. Contact angle measurements are often used in self-assembled monolayer research to determine the wettability of a functionalized surface. In this way, the surface free energy can be linked, to some extent, to the molecular composition and structure of the monolayer.36 These measurements consist of determining the so-called contact angle, θ, of a droplet of a given probe liquid with the functionalized surface to be studied. This angle depends on the surface free energies of the solid−liquid (γsl), solid−vapor (γsv), and liquid−vapor (γlv) interfaces following the Young equation:

2). However, one should note this variation is not equal for all our surfaces and is strongly affected by the tethered chain lengths. In fact, the bathochromic shift and the enlargement of the peak are higher with the longest linker. This was our first hint to presume a difference in fluorophore organization for the S0, S3, and S10 surfaces. As was already observed for different chromophores immobilized on a gold surface, the red shift of the emission spectra is due to intermolecular interactions within the monolayer.17,30 We can then presume that the packing of the SAM increases from S0 to S10. The different bathochromic shifts are not the only difference between these surface emission spectra. The fluorescence intensity also changed strongly from one surface to another. Figure 1d indicates that the fluorescence intensity obtained with coumarin C3 was 10 times higher than for C0 and C10 derivatives. This does not result from intrinsically different fluorescence quantum yields. Indeed, it has already been reported that the nature of the substituent at position 7 of the 4-methylcoumarin core strongly affects the luminescence properties. As expected,31,32 the determination of the quantum yield in solution (ethanol at 10−5 M) shows that, on one hand, the C0 compound presents the strongest fluorescence efficiency (ϕ = 28%) and, on the other hand, the quantum yields of C3 and C10 are quite similar (9% and 7%, respectively). It seems that the difference in the photophysical behavior in the condensed phase comes from different surface functionalization efficiencies and/or different SAM organizations that would affect the coumarin photophysical properties. We expect that a better organization would facilitate the dimerization of the coumarins under UV irradiation.33 The formation of such a dimer, which is known to present a very low quantum yield,34 would drastically decrease the surface fluorescence. Thus, the fluorescence differences between surfaces could be due to differences in the dimer/monomer ratio. Nevertheless, since the fluorescence intensity is stable over extended irradiation times, it seems unlikely that dimerization explains the fluctuation of fluorescence intensity among all three surface types. We rather propose that the different photophysical behaviors among S0, S3, and S10 arise from a modification of the self-assembled monolayer density with the variation of the tethered chain length. In fact, a difference in the monolayer density might bring two antagonist effects. On one hand, a better packing increases the density of fluorophore units grafted onto the surface, but on the other hand, it may also place the fluorophores in a specific environment which might facilitate fluorescence self-quenching.

−γsv + γsl + γlv cos θ = 0

(1)

In most studies, water is used as the probe liquid, and often, the hysteresis measurements, between advancing and receding contact angles, are reported. These measurements for all three surfaces are summarized in Table 1. As expected, the Table 1. Static (θs), Advancing (θa), and Receding (θr) Contact Angles (deg) with Their Standard Deviation of Water on Surfaces S0, S3, and S10 surface

θs

θa

θr

Δθ = θa − θr

S0 S3 S10

65.1 ± 0.9 68.0 ± 0.4 71.7 ± 1.2

70.4 ± 0.6 76.0 ± 2.0 73.6 ± 0.4

60.1 ± 1.7 62.6 ± 2.7 66.0 ± 1.3

10.3 ± 1.1 13.5 ± 2.3 7.6 ± 1.5

measurements indicate that the hydrophobicity of the surface increases with the tethered chain length (θwater varies from 65.1° to 71.7°). A contact angle hysteresis may be the sign of a poor degree of molecular ordering in the underlying monolayer, but one should keep in mind that such hysteresis could also be due to surface roughness and/or a particular SAM’s polarity.2 However, these measurements are useful in gauging the surface heterogeneity. In our case, the hysteresis measured is comparable to that obtained on an alkylsiloxane self-assembled monolayer.37−39 From these results, we presume that the S0, S3, and S10 functionalization of a glass slide E

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Table 2. Surface Tension Components (mJ·m−2) of Probe Liquids Used in Contact Angle Measurements41 and Apparent Contact Angles (deg) Measured on the Different Surfaces with Water, Formamide, and Diiodomethane as the Probing Liquid tested liquid

γTOT

γLW

γAB

γ+

γ−

S0

S3

S10

water formamide diiodomethane

72.8 58.0 50.8

21.8 39.0 50.8

51.0 19.0 0.0

25.5 2.3 0.0

25.5 39.6 0.0

65.1 ± 0.9 26.5 ± 0.3 35.2 ± 0.5

68.0 ± 0.4 40.1 ± 0.6 34.2 ± 0.5

71.7 ± 1.2 54.4 ± 0.9 35.7 ± 0.7

To interpret these results, we can presume that the organization of the self-assembled monolayer is strongly affected by the modification of the linker, as also supported by the fluorescence measurements. The strong increase in the basic term from S0 to S10 could be simply translated as an increase in the number of coumarin moieties available at the liquid−surface interface. It seems that the functionalization of the surface and SAM formation with derivatives of longer chains is more efficient. However, this higher functionalization could be interpreted not only as an increase of the number of immobilized molecules, but also as a modification of the selfassembled monolayer organization. The purely basic character of S10 could be explained by the SAM compacity of the monolayer exposing coumarin moieties toward the liquid and as a consequence the interaction between neighboring molecules that reduces interactions of the liquid with the glass substrate. To conclude, we can assume that the increase in the tethered chain length should lead to the formation of a selfassembled monolayer with a higher surface coverage, a higher degree of organization, and a stronger molecular packing. Consequently, the higher fluorescence intensity obtained with S3 than with S0 might come from a better surface coverage for S3 than for S0, which means that a higher number of fluorophores are present at the interface. In the same way, S10 should present a more important surface coverage than S3 and as a consequence a higher fluorescence intensity, but the increase of the fluorochrome surface density was accompanied by an increase in its compacity and organization, so that the interactions between neighboring molecules are more important in the case of S10, which facilitates the self-quenching of fluorescence, resulting in a decrease in the signal in comparison to that of S3. If the contact angle allows us to confirm that the modification of the SAM organization and compactness is induced by the modification of the tethered chain length, we can now assume that the larger red shift of the fluorescence emission observed in the case of compound C10 is due to the stronger interaction between neighboring molecules. Atomic Force Microscopy and X-ray Reflectivtity. We performed AFM and X-ray reflectivity experiments on the S0, S3, and S10 samples to investigate the structure of the SAMs. The AFM topographic images of the layers are presented with the same scales in Figure 3. The calculated roughness RRMS for each surface is reported with its standard deviation in Table 4; in addition, the roughness of the glass substrate used is also given. The three surfaces presented a different topography of their top level according to the change of the linkers’ length, even though the S0 and S10 surfaces were quite similar in terms of surface homogeneity and roughness, while S3 was obviously rougher with a higher surface coverage of large domains (submicrometer islands). These surface reliefs could reach a height of 6 nm for S0 and 15−30 nm for S10 with a lateral width ranging from 250 to 400 nm for both S0 and S10 layers. Due to the low surface coverage of these aggregates, we can reasonably assume that these aggregates might be due to some

corresponds to smooth and homogeneous layers where the silane coupling agent is chemisorbed to the surface.40 Several models have been developed for the contact angle and free energy dependence. The γsl term is often decomposed into several components, generally one for apolar interactions such as Van Der Waals interactions (γLW) and one for acid− base interactions (γAB), which is decomposed in the Van Oss− Chaudhury−Good model (eq 2) into an electron acceptor parameter (γ+) and an electron donor parameter (γ−). γl(1 + cos θ ) = 2( γsLWγlLW +

γs+γl− +

γs−γl+ )

(2)

In our case, this Van Oss−Chaudhury−Good model is remarkably well-suited because each parameter refers to the contribution of a different group of our molecules; γLW, γ+, and γ− refer respectively to the presence of the linker, the anchoring group at the glass surface in the best situations, and the coumarin moiety (with its ester function) (see Scheme 1). However, to determine all three parameters, the Van Oss− Chaudhury−Good model requires the measurement of the contact angle with three different probe liquids (of which two must be polar). For this study, we have chosen three liquids commonly used for contact angle measurements and wellcharacterized: water, formamide, and diiodomethane. Their surface energy parameters are summarized in Table 2 with the contact angle values measured on the different elaborated SAMs. It is quite surprising to observe that the values obtained with the diiodomethane are similar, which could indicate that van der Waals interactions did not strongly vary from one surface to another. Only the use of a polar solvent induces a modification of the contact angle. Moreover, this change is directly related to the value of the electron acceptor component (γ+). For water, a shift of only 7° is observed between S0 and S10, and when formamide is used, the contact angle almost doubles (from 26.5° to 54.4°). From these values we have calculated with eq 2 the van der Waals (γLW), electron acceptor (γ+), and electron donor (γ−) terms of the surface tension for each surface using the contact angles obtained with the three probe liquids. The results are summarized in Table 3 and show that both polar components are the most affected by a change of the linker length. Indeed, the basic component increases by almost a factor of 2, and the acidic term becomes null from surface S0 to surface S10. On the contrary, the van der Waals parameter remains constant (∼42 mJ/m2) for all surfaces. Table 3. Surface Tension Components (mJ·m−2) Derived from Table 2 for Each Self-Assembled Monolayer Obtained from Coumarin Derivatives C0, C3, and C10 surface

γLW

γ−

γ+

γAB

S0 S3 S10

41.94 ± 0.21 42.40 ± 0.22 41.70 ± 0.34

6.58 ± 0.71 8.50 ± 0.41 11.87 ± 1.37

2.86 ± 0.14 0.94 ± 0.09 0.00 ± 0.01

8.67 ± 0.68 5.67 ± 0.41 0.38 ± 0.64 F

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Figure 3. AFM topographic images of the different functionalized surfaces by C0, C3, and C10 named respectively S0, S3, and S10.

Table 4. AFM Measurements: Roughness, RRMS, and Average Height, Zava surface

calcd full molecule length, nm

measd Zav, nm (error)

measd RRMS, nm (error)

S0 S3 S10

1.4 1.8 2.8

1.7 (0.10) 3.2 (0.3) 2.2 (0.4)

0.95 (0.1) 1.52 (0.2) 0.74 (0.3)

a Error estimated from averaging measurements obtained on different places on the surface.

polymerization in solution or starting from the surface which occurs at sample preparation. The S0 and S10 surfaces are rather smooth with a very low roughness, lower for S0 than for S10, which is consistent with the fact that the SAMs become denser when the length of the tethered chain is increased, as discussed from the fluorescence and contact angle measurements. For S3, the roughness is much higher and does not follow the trend observed from measurements of the contact angle and fluorescence. We observed a larger population of islands for S3, and some reached 15 nm or more in height (see a profile for the S3 sample in Figure 3). The roughness was calculated from the height distribution histogram. From these histograms, we can also determine an estimate of the mean thickness of the deposited layer assuming that the lowest level reached in the data corresponds to the bare substrate level. Each histogram was fitted to a Gaussian shape, the center of the Gaussian gave the average height of the layer assimilated to its mean thickness (Zav given in Table 4) and the fwhm gave an estimate of the layer homogeneity (Figure 4 shows the resulting fits for the three different surfaces studied). The Zav is coherent with a monolayer for the S0 and S10 compounds. Since S3 came with a higher roughness partially due to the large island domains, the fitted Gaussian center of the histogram was shifted to higher values and is not compatible with a single molecular layer anymore. The fwhm was also wider (as shown in Figure 4) for the same reasons. We also carried out XR experiments on these samples. The X-ray true specular reflectivity curves with the best fits to the data are plotted in Figure 5, and the corresponding fitting parameters are given in Table 5. We obtained good fits to the data in the frame of Fresnel optics assuming a single-layer box model to represent the SAM grafted onto the bare glass substrate but with a large roughness of the air/film interface for all samples. A single broad oscillation (first “Kiessig” fringe) appears on the specular reflectivity curve before the specular intensity reaches the longitudinal intensity level. Such a pronounced oscillation, particularly for S0 and S10, was only expected for a welldefined film thickness over the whole surface, but the loss of

Figure 4. Height histogram of the S0, S3, and S10 layers corresponding to the images shown in Figure 3.

Figure 5. True specular reflectivity curves of the bare glass substrate and S0, S3, and S10 samples plotted with the best fits to the data (solid lines).

signal at larger angles shows the presence of rough interfaces. For each sample, an index of refraction profile was built from a single homogeneous layer separated by rough interfaces. The profile was adjusted with the help of a finite set of parameters per layer: the thickness (d), the real and imaginary parts of the index of refraction expressed in the form of a complex scattering length density (SLD, Re(ρb) + i Im(ρb)), and the RMS roughness (σ). The reflectivity was then calculated using Parratt’s recursive algorithm42 based on the dynamical formalism originally due to Airy43 for light and exact when including the instrumental X-ray resolution effects. The best fit corresponds to the smaller distance between experimental and calculated data, measured in the form of the classical χ2 function and refining the set of parameters with a Monte G

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Table 5. Parameters Obtained for the Best Fits of the X-ray True Specular Reflectivity Data Shown in Figure 5a layer on top of substrate sample bare glass substrate S0 S3 S10 a

d, Å 17.3 ± 5 34.9 ± 4 23.2 ± 6

Re(ρb), 10−6 Å−2 14.15 ± 2.4 13.16 ± 5.6 10.9 ± 3.8

substrate

Im(ρb), 10−6 Å−2 0.05 ± 0.04 0.1 ± 0.05 0.05 ± 0.04

σ, Å

Re(ρb), 10−6 Å−2

Im(ρb), 10−6 Å−2

σ, Å

11.9 ± 2.6 12.6 ± 3.5 11.9 ± 4.1

22 ± 1 22* 22* 22*

0.228 ± 0.1 0.228* 0.228* 0.228*

10.3 ± 1.5 10.3* 10.3* 10.3*

Parameters marked with an asterisk were fixed during the fitting procedure.

Carlo routine. A minimum value of χ2 does not ensure that this minimum is absolute, and several sets of parameters can give acceptable fits. Thus, we reduced the number of fitting parameters in the following way: we determined the glass refraction index by fitting the bare glass curve, and we obtained the same glass parameters in all cases: Re(ρbSub) = 22 × 10−6 Å−2 and Im(ρbSub) = 2.28 × 10−7 Å−2. The real part corresponds to an electron density of 0.78 Å−3 (calculated from Re(ρbSub)/r0, with r0 = 2.818 × 10−5 Å, the classical electron radius). The bare substrate parameters could be fixed for fitting the SAM curves, and we ensured that they kept similar values when floated. The best XR fit corresponded to a SAM thickness of 17.3 Å for S0, 34.9 Å for S3, and 23.2 Å for S10, which are in excellent agreement with the AFM Zav values obtained on the same samples but probed on a local scale while X-rays probed square millimeters over the same samples. We obtained SLDs of 14.2 × 10−6, 13.2 × 10−6, and 10.9 × 10−6 Å−2 (i.e., electronic densities of 0.504, 0.468, and 0.387 Å−3, respectively, for S0, S3, and S10), in agreement with the calculated values expected for a dense SAM of these molecules from molecular modeling (i.e., calculated electronic densities of 0.404, 0.335, and 0.311 Å−3, respectively, for C0, C3, and C10). The top SAM roughness corresponding to the monolayer roughness in our case was comparable for all samples (∼12 Å). We conclude that the X-ray data are in good agreement with the AFM data and a flat SAM grafted onto the glass substrate but with a nonconformal roughness and amplitude (RMS) that show a nonperfectly flat top interface. Note that the clusters that cover S3 do not drastically affect the XR measurements due to their low surface coverage and their particularly irregular aspects. They increased the S3 roughness and the off-specular scattering when probed at the smallest angles, but this effect is partially subtracted when we build the true specular reflectivity curve where longitudinal scans were subtracted from the corresponding specular ones.

Good theory. In addition, the AFM and XR measurements show that the tethered chain compounds with the smallest and the biggest chain lengths, respectively C0 and C10, gave smooth monolayers, while the compound with an intermediate chain length, C3, surprisingly gave a bilayer formation, since it was prepared with the same protocol. This result was confirmed several times and reveals that the formation of bilayer islands on top of the monolayer is difficult to predict for such molecules.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +33 0-241 735 374. Fax: +33 0-241 735 405. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the Pays de la Loire region (PROVASC) and the University of Angers. We thank B. Siegler and Dr. I. Freuze for their assistance with NMR spectroscopy and MS, respectively, and Dr. E. Levillain for helping with contact angle measurement error calculations.

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REFERENCES

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CONCLUSIONS As expected, the functionalization of a simple glass slide by the coumarin derivatives C0, C3, and C10 resulted in fluorescent SAMs. However, the intensity and the spectral characteristics of the emitted light are strongly affected by the chain length linking the fluorophore unit to the anchoring group. To demonstrate the organization is mainly responsible for these fluorescence differences, we used a cheap and simple technique of contact angle measurements and applied the Van Oss− Chaudhury−Good theory, which is rarely used for selfassembled monolayers. The results show that the layer compacity increases with the length of the tethered chain and induces a bathochromic shift of the luminescence. The AFM and X-ray reflectivity data obtained on the different surfaces are in perfect agreement with the conclusions obtained from contact angle measurements using the Van Oss−Chaudhury− H

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