Steady-State Fluorescence-Based Investigation of the Interaction

Langmuir 2002, 18, 2413-2420

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Steady-State Fluorescence-Based Investigation of the Interaction between Protected Thiols and Gold Nanoparticles Michael Ming Yu Chen and Alexander Katz* Department of Chemical Engineering, University of California at Berkeley, Berkeley, California 94720-1462 Received December 12, 2001 The first demonstration of thioester and thiocarbonate binding to gold is provided via steady-state fluorescence spectroscopy of probe molecule adsorption onto a nanoparticle surface. Both thioester 1 and thiocarbonate 2 bind noncovalently to gold from aqueous solution, with 2 possessing a greater affinity for the gold surface relative to 1. The surface-bound molecules assemble into high packing densities comprising molecular footprints of 24.5 ( 1.0 Å2 per molecule. Both 1 and 2 deprotect catalytically on the gold surface to yield hydrolysis products and surface-bound thiolate. This process is thermally activated and can be described by an Arrhenius-type expression with activation energies of 14.0 and 16.7 kcal/mol for 1 and 2, respectively. The results presented herein demonstrate the role of gold in catalyzing thioester and thiocarbonate hydrolysis and diversify the synthetic repertoire of organosulfur functional groups that can be used for binding to gold.

Introduction The strong binding of thiolates to gold surfaces has been investigated extensively owing to fundamental interest in monolayer formation and the widespread use of this system in various aspects of technology, including the synthesis of materials based on colloidal templates,1 immunogical probes,2 biosensors,3 and molecular electronics devices.4 Despite the ubiquity of the thiolate-gold system for surface modification,5 there are few approaches (1) (a) Storhoff, J. J.; Mirkin, C. A. Chem. Rev. 1999, 99, 1849-1862. (b) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609. (c) Martin, B. R.; Dermody, D. J.; Reiss, B. D.; Fang, M.; Lyon, L. A.; Natan, M. J.; Mallouk, T. E. Adv. Mater. 1999, 11, 1021-1025. (d) Whetten, R. L.; Shafigullin, M. N.; Khoury, J. T.; Schaaff, T. G.; Vezmar, I.; Alvarez, M. M.; Wilkinson, A. Acc. Chem. Res. 1999, 32, 397-406. (e) Loweth, C. J.; Caldwell, W. B.; Peng, X.; Alivisatos, A. P.; Schultz, P. G. Angew. Chem., Int. Ed. Engl. 1999, 38, 1808-1812. (f) Alivisatos, A. P.; Johnsson, K. P.; Peng, X.; Wilson, T. E.; Loweth, C. J.; Bruchez, M. P., Jr.; Schultz, P. G. Nature 1996, 382, 609-611. (g) Sarathy, K. V.; Raina, G.; Yadav, R. T.; Kulkarni, G. U.; Rao, C. N. R. J. Phys. Chem. B 1997, 101, 9876-9880. (h) Giersig, M.; Mulvaney, P. Langmuir 1993, 9, 3408-3413. (i) Brust, M.; Fink, J.; Bethell, D.; Schiffrin, D. J.; Kiely, C. J. Chem. Soc., Chem. Commun. 1995, 1655-6. (j) Brust, M.; Bethell, D.; Schiffrin, D. J.; Kiely, C. J. Adv. Mater. 1995, 7, 795-797. (k) Brust, M.; Walker, M.; Bethell, D.; Schriffrin, D. J.; Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 801-2. (l) Liz-Marza´n, L. M.; Giersig, M.; Mulvaney, P. Langmuir 1996, 12, 4329-4335. (m) Liz-Marza´n, L. M.; Giersig, M.; Mulvaney, P. Chem. Commun.1996, 731-732. (n) Fan, H.; Zhou, Y.; Lope´z, G. P. Adv. Mater. 1997, 9, 728-731. (o) Bethell, D.; Brust, M.; Schiffrin, D. J.; Kiely, C. J. Electroanal. Chem. 1996, 409, 137-143. (2) (a) Roth, J. Histochem. Cell. Biol. 1996, 106, 1-8. (b) Bhatnagar, R. Microsc. Res. Tech. 1998, 42, 1. (c) Hermann, P.; Walther, P.; Mu¨ller, M. Histochem. Cell. Biol. 1996, 106, 31-39. (d) Powell, R. D.; Halsey, C. M. R.; Hainfeld, J. F. Microsc. Res. Tech. 1998, 42, 2-12. (e) Robinson, J. M.; Vandre´, D. D. J. Histochem. Cytochem. 1997, 45, 631-642. (f) Robinson, J. M.; Takizawa, T.; Vandre´, D. D.; Burry, R. W. Microsc. Res. Tech. 1998, 42, 13-23. (3) (a) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. J. Am. Chem. Soc. 2000, 122, 7837-7838. (b) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078-1081. (4) (a) Fan, F.-R. F.; Yang, J.; Dirk, S. M.; Price, D. W.; Kosynkin, D.; Tour, J. M.; Bard, A. J. J. Am. Chem. Soc. 2001, 123, 2454-2455. (b) Jones, L., II; Schumm, J. S.; Tour, J. M. J. Org. Chem. 1997, 62, 1388-1410. (c) Pearson, D. L.; Tour, J. M. J. Org. Chem. 1997, 62, 1376-1387. (d) Cygan, M. T.; Dunbar, T. D.; Arnold, J. J.; Bumm, L. A.; Shedlock, N. F.; Burgin, T. P.; Jones, L., II; Allara, D. L.; Tour, J. M.; Weiss, P. S. J. Am. Chem. Soc. 1998, 120, 2721-2732. (e) Reese, S.; Fox, M. A. J. Phys. Chem. B 1998, 102, 9820-9824.

for the synthesis-by-design of organosulfur molecules to have a specific degree of affinity for a gold surface. This capability could be useful for, among other applications, controlling kinetics6 and electrostatic charge on the gold surface during monolayer formation,7-9 which may be particularly important in systems involving nanoparticles, since the time scale of these events relative to colloidal phenomena, such as coagulation, can be critical in maintaining sol stability. We postulated that a protected (oxidatively stable) thiol may be a good starting point for the design of organosulfur molecules for adsorption onto gold, since the electronic environment surrounding the sulfur atom can, in principle, be controlled via synthesis to accommodate different amounts of electron density. Investigators have previously used thioesters as precursors to adsorbed thiols on gold surfaces,10 and the binding of thioesters to gold has been studied using reflectionabsorption infrared spectroscopy and X-ray photoelectron spectroscopy.11 However, elucidating the mechanism of adsorption in these instances has proven rather difficult. One of the complicating factors has been characterizing whether it is the protected thiol that actually adsorbs or whether it is the deprotected form, which may be present (5) (a) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 448183. (b) Strong, L.; Whitesides, G. M. Langmuir 1988, 4, 546-558. (c) Bain, C. D.; Evall, J.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7155-7164. (d) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7164-7175. (e) Sabatani, E.; Cohen-Boulakia, J.; Bruening, M.; Rubinstein, I. Langmuir 1993, 9, 2974-2981. (f) Frostman, L. M.; Bader, M. M.; Ward, M. D. Langmuir 1994, 10, 576-582. (g) Yang, D. B.; Wakamatsu, T. Surf. Int. Anal. 1996, 24, 803-810. (h) Ulman, A. Chem. Rev. 1996, 96, 1533-1554. (6) (a) Peterlinz, K. A.; Georgiadis, R. Langmuir 1996, 12, 47314740. (b) Bain, C. D.; Troughton, E. B.; Tao, Y.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321-335. (7) Lindberg, B. J.; Hamrin, K.; Johansson, G.; Gelius, U.; Fahlman, A.; Nordling, C.; Siegbahn, K. Phys. Scr. 1970, 1, 286-298. (8) Widrig, C. A.; Chung, C.; Porter, M. D. J. Electroanal. Chem. 1991, 310, 335-359. (9) Biebuyck, H. A.; Bain, C. D.; Whitesides, G. M. Langmuir 1994, 10, 1825-1831. (10) Tour, J. M.; Jones, L., II; Pearson, D. L.; Lamba, J. J. S.; Burgin, T. P.; Whitesides, G. M.; Allara, D. L.; Parikh, A. N.; Atre, S. V. J. Am. Chem. Soc. 1995, 117, 9529-9534. (11) Skulason, H.; Frisbie, C. D. J. Am. Chem. Soc. 2000, 122, 97509760.

10.1021/la015729f CCC: $22.00 © 2002 American Chemical Society Published on Web 02/22/2002

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in small amounts as an impurity and can dominate adsorption on the metal surface.12 Previous results are consistent with the possibility that thioesters may be able to adsorb on the gold surface;10,11 however, definitive proof for their adsorption has yet to be provided. Using fluorophores 1 and 2 as probe molecules, we wished to investigate whether protected thiols in the form of thioesters and thiocarbonates, respectively, can bind to a gold surface. These molecules were synthesized with a

pyrene fluorescence tag that is sensitive to the degree of isolation via its monomer and excimer emission signatures,13 which can be analyzed using steady-state fluorescence spectroscopy. The butyl tether attached to the sulfur of 1 and 2 maintains a certain degree of hydrophobicity within the layer of bound molecules, so as to promote organization into hydrophobic domains within the monolayer, while simultaneously facilitating a limited degree of water solubility. Although previous studies of pyrene-based fluorophores incorporated within monolayer matrixes have been performed,14-16 in the current investigation, the fluorescence tag is covalently attached to the molecule being bound to the surface and is present as the major component in the monolayer (as opposed to an additive that is present in small proportions relative to other nonfluorescent components). The results of this investigation unequivocally demonstrate, for the first time, that thioesters and thiocarbonates bind noncovalently to a gold surface from aqueous solution. In addition, the gold surface is shown to have an unexpected catalytic role in hydrolyzing the protected thiol derivatives, and the kinetic parameters (Arrhenius pre-exponential factors and activation energies) associated with these reactions are measured for 1 and 2. The steady-state fluorescence-based approach described herein is put forth as a general method for investigating functional group binding to nanoparticle surfaces. Experimental Section General. 1H and 13C NMR were performed on Bruker AMX 300 and 400 MHz machines at University of Califiornia at Berkeley (UCB). FAB mass spectra were recorded at the UCB Mass Spectrometry Facility. UV/vis spectroscopy was performed on a Varian Cary 400 Bio UV/vis spectrophotometer. Materials. 1-Pyrenebutanol, 1-butanethiol, sodium citrate, sodium ethoxide, and carbonyldiimidazole were purchased at the highest possible level of purity from Aldrich and were used as received. Tetrachloroauric acid was purchased from Acros and was used as received. Absolute ethanol was distilled in a glass apparatus under nitrogen prior to use. 1-Pyrenebutyric (12) Zhong, C.; Brush, R. C.; Anderegg, J.; Porter, M. D. Langmuir 1999, 15, 518-525. (13) (a) Birks, J. B. Rep. Prog. Phys. 1975, 38, 903-974. (b) Barashkov, N. N.; Sakhno, T. V.; Nurmukhametov, R. N.; Khakhel, O. A. Usp. Khim. 1993, 62, 579-593. (14) Flink, S.; van Veggel, F. C. J. M.; Reinhoudt, D. N. J. Phys. Org. Chem. 2001, 14, 407-415. (15) Karpovich, D. S.; Blanchard, G. J. Langmuir 1996, 12, 55225524. (16) Chen, S. H.; Frank, C. W. In Surfactant Adsorption and Surface Solubilization; Sharma, R., Ed.; ACS Symposium Series 615; American Chemical Society: Washington, DC, 1995.

Chen and Katz acid was purchased from Aldrich at the highest available purity and subsequently recrystallized three times from benzene prior to use. Unless stated otherwise, water that was used was distilled, purified with a Barnstead Nanopure Infinity system to possess at least 18 MΩ purity, and subsequently passed through a 0.2 µm filter. For colloidal gold syntheses, this water was further distilled three times in a glass distillation apparatus. 1-Pyrenebutanethioic Acid S-Butyl Ester (1). Carbonyldiimidazole (0.44 g, 2.72 mmol) was dissolved in 6 mL of anhydrous THF in a dry airless flask. 1-Pyrenebutyric acid (0.75 g, 2.60 mmol) was dissolved in 2 mL of anhydrous THF, and this solution was added dropwise to the reaction mixture, which was subsequently stirred overnight at room temperature. In a 250 mL two-neck flask, sodium ethoxide (0.4 g, 5.88 mmol) was dissolved in 50 mL of THF, and 1-butanethiol (0.26 g, 0.288 mmol) was added to this mixture. The contents of the airless flask were then mixed into the two-neck flask. The reaction mixture was carefully heated with an oil bath maintained at 40 °C for approximately 70 min and was monitored using thin-layer chromatography. Subsequently, the reaction mixture was cooled to room temperature and 450 mL of ether and 6 g of silica gel were added. The mixture was filtered, concentrated to a dark yellow oil, and purified by silica chromatography (Silica Gel 60 and 10.0/1.0 v/v hexane/ethyl acetate) to yield an oil (0.17 g, 0.468 mmol, yield 18%). 1H NMR (CDCl3): 0.925 (3H, t, J ) 7.4 Hz, CH3); 1.385-1.598 (4H, m, CH2); 2.210-2.261 (2H, m, CH2); 2.684-2.733 (2H, t, J ) 7.4 Hz, CH2); 2.892-2.940 (2H, t, J ) 7.2 Hz, CH2); 3.363-3.414 (2H, t, J ) 7.7 Hz, CH2); 7.852-8.295 (9H, m, Ar-H). 13C{1H} NMR (CDCl3): 13.64 (CH2); 21.99 (CH2); 27.33 (CH2); 28.61 (CH2); 31.68 (CH2); 32.36 (CH2); 32.56 (CH2); 43.57 (CH2); 123.15 (Ar-C); 124.69 (Ar-C); 124.81 (Ar-C); 125.72 (Ar-C); 126.59 (Ar-C); 126.71 (Ar-C); 127.34 (Ar-C); 127.46 (ArC); 128.67 (Ar-C); 129.97 (Ar-C); 130.86 (Ar-C); 131.38 (Ar-C); 135.52 (Ar-C); 199.46 (CdO). Mass spectrum (FAB): m/z 360.1555 (C24H24OS, 360.1548). Thiocarbonic Acid O-(4-Pyren-1-ylbutyl) S-Butyl Ester (2). Carbonyldiimidazole (0.62 g, 3.82 mmol) and a catalytic amount of sodium ethoxide were dissolved in 6 mL of anhydrous THF in a dry airless flask. 1-Pyrenebutanol (1.0 g, 3.64 mmol) was dissolved in 2 mL of anhydrous THF, and this solution was added dropwise to the reaction mixture, which was subsequently stirred overnight at room temperature. The solution was cooled in an ice bath, and the precipitated white powder (the imidazolide condensation product of 1-pyrenebutanol and carbonyldiimidazole) was collected in a glass frit (0.88 g, 2.39 mmol), mixed with a molar excess of sodium ethoxide, and dissolved in 150 mL of THF. Subsequently, 1-butanethiol (0.37 g, 4.12 mmol) was added to the reaction mixture, which was brought to reflux for a period of 20-24 h and monitored by thin-layer chromatography. The reaction was then cooled to room temperature, and 450 mL of ether and 6 g of silica gel were added. The mixture was filtered, concentrated to a dark yellow oil, and purified by silica chromatography (Silica Gel 60 and 10.0/1.0 v/v hexane/ethyl acetate) to yield a brilliant white powder (0.55 g, 1.41 mmol, yield 38%). 1H NMR (CDCl ): 0.911 (3H, t, J ) 7.4 Hz, CH ); 1.371-1.445 3 2 (2H, m, CH2); 1.545-1.651 (3H, m, CH2); 1.832-1.973 (4H, m, CH2); 2.832-2.869 (2H, t, J ) 7.4 Hz, CH2); 3.363-3.401 (2H, t, J ) 7.6 Hz, CH2); 4.274-4.306 (2H, t, J ) 6.4 Hz, CH2); 7.8548.274 (9H, m, Ar-H). 13C{1H} NMR (CDCl3): 13.63 (CH3); 21.88 (CH2); 27.97 (CH2); 28.71 (CH2); 30.75 (CH2); 31.91 (CH2); 32.99 (CH2); 67.16 (CH2); 123.32 (Ar-C); 124.77 (Ar-C); 124.85 (Ar-C); 124.94 (Ar-C); 125.05 (Ar-C); 125.13 (Ar-C); 125.87 (Ar-C); 126.69 (Ar-C); 127.26 (Ar-C); 127.35 (Ar-C); 127.55 (Ar-C); 128.65 (ArC); 129.92 (Ar-C); 130.94 (Ar-C); 131.47 (Ar-C); 136.23 (Ar-C); 171.47 (CdO). Mass spectrum (FAB): m/z 390.1656 (C25H26O2S, 390.1654). Steady-State Fluorescence. Steady-state fluorescence was performed on a Hitachi F-4500 fluorimeter equipped with a 150 W Xe-lamp source. The signal-to-noise ratio was greater than 100/1 for the Raman band of water. All spectra were corrected using a Rhodamine B standard, unless noted otherwise. Wavelength accuracy was checked using the xenon line at a wavelength of 450.1 nm with a diffuser cell and was within (2.0 nm. A thermostated extended temperature compartment with the temperature range 5-60 °C was used to control the cell temperature. The F-4500 was interfaced to a computer through

Interaction between Protected Thiols and Gold a National Instrument PCI-GPIB interface card, and data acquisition and analysis software was provided by the Hitachi F-4500 Fluorescence Spectroscopy FL solutions software. Emission experiments were conducted with an excitation wavelength of 278.0 nm, which is located at a maximum ultraviolet absorbance for all of the compounds investigated. The entry beam was passed through a 320 nm filter (Hoya UV-32) to eliminate second-order Rayleigh scatter from the excitation beam. A PMT voltage of 950 V and an excitation slit width of 5.0 nm were employed in all fluorescence experiments, unless specified otherwise. All emission scans were collected from 360 to 600 nm. The fluorescence cuvettes used were purchased from Hitachi Instruments (Hitachi AN0-1804) and comprised 1 cm2 ultrahigh-quality quartz cells with four sides polished and no background fluorescence. Background fluorescence measured from the colloidal gold was insignificant compared to the signal resulting from fluorophore emission for the conditions used in this investigation. Approximately 30 s of argon purging was performed on all samples prior to measurement. Preparation of Gold Sol. On the basis of a review of previous gold sol syntheses,17 a gold sol procedure was chosen that maintained a relatively large concentration of colloidal gold in solution, to increase the signal-to-noise ratio of the bulk fluorescence measurements used in this study for the investigation of surface-related phenomena.18 All glassware used in the gold sol preparation was first treated with base bath (890 mL of absolute ethanol, 107 mL of purified water, and 107 g of KOH) overnight, rinsed with large amounts of water, soaked in aqua regia (3/1 v/v HCl/HNO3) overnight, and finally rinsed with excess amounts of water. The colloid synthesis was performed according to a previously published procedure.18 Transmission electron microscopy (TEM) was performed on a random sampling of particles and verified the presence of spherical 12.5 nm colloidal gold particles having a standard deviation of 1.5 nm in the diameter and a concentration of 17 nM.18 Following synthesis, colloidal gold was transferred to a brown bottle and stored at 5 °C. The characteristics of the colloidal gold were verified via UV/vis spectroscopy. The gold sol diluted with four parts of water per part of sol had a measured λmax of 519 nm (516-520 nm literature specification18), an absorbance of 0.7 (0.7-0.9 nm literature specification18), and a full peak width at half maximum of 86 nm (80-90 nm literature specification18). Concentration-Dependence Studies. Samples for titrating colloidal gold at a fixed fluorophore concentration were prepared as follows. Fluorophore stock solutions of 1 and 2 in absolute ethanol (0.246 mM) were freshly prepared and stored in the dark at 5 °C. Water (4 mL) was added to clean scintillation vials, and a set of solutions containing 0, 3, 7, 10, 15, 20, and 29 µL of gold sol was added to these vials. Fluorophore stock solution (2 µL) was finally added to these vials immediately prior to measurement. Separate sets of solutions for cases using 5 and 8 µL of the fluorophore stock solution were also prepared. For each of these cases corresponding to greater concentrations of fluorophore in solution, the amount of added gold sol was scaled in proportion to the fluorophore concentration. The cuvette was then placed in a thermostated cell holder, which was maintained at a fixed temperature of 7 °C. The fluorescence emission spectra were measured for each solution, and the emission slit width was adjusted to maximize the signal-to-noise ratio (set to 2.5 nm for solutions containing 8 µL of 1 and 2 stock solution and 5 µL of 2 stock solution and to 5.0 nm for solutions containing 2 µL of 1 and 2 stock solution and 5 µL of 1 stock solution). For evaluating the equilibrium binding constant, solutions of varying amounts of dilution were made that had a fixed ratio of 7.25/1 v/v gold sol solution/fluorophore stock solution. The emission slit width was 2.5 nm for solutions of 1, as well as solutions of 2 above 0.3 µM of 2. For solutions of 2 below 0.3 µM of 2, an emission slit width of 5.0 nm was used (for all data in Figure 3b). Kinetics Studies. Fluorophore stock solutions of 1 and 2 in absolute ethanol (0.246 mM) were freshly prepared and stored in the dark at 5 °C. Water (3.12 mL), gold sol (0.88 mL), and (17) Turkevich, J.; Stevenson, P. C.; Hillier, J. Discuss. Faraday Soc. 1951, 11, 55-75. (18) Keating, C. D.; Musick, M. D.; Keefe, M. H.; Natan, M. J. J. Chem. Educ. 1999, 76, 949-955.

Langmuir, Vol. 18, No. 6, 2002 2415 fluorophore stock solution (2 µL; corresponding to approximately 1.65% of saturation coverage) were mixed in a scintillation vial. The pH of the final solution was measured to be 6.34. The solution was transferred from the vial to a fluorescence cuvette via a glass pipet. Emission scans were recorded at 3-min intervals using the time-delay feature of the fluorescence software and with an emission slit width of 5 nm. To quantify the extent of reaction from measurements of the fluorescence emission band area versus time (fluorescence emission in these studies comprised entirely the excimer band for both 1 and 2 under the conditions of excess colloidal gold used in the investigation), a linear correlation was established between fluorophore concentration and fluorescence emission band area under the conditions of the kinetics studies (calibration curves are included in the Supporting Information). Using this information, the amount of deprotection could be investigated at various temperatures by calculating the relative amounts of bound fluorophore on the basis of the measured decrease in the fluorescence emission area with time. The experiment was performed at the temperatures 7, 15, 25, and 35 °C. Background rates for fluorophore deprotection in aqueous solution were measured by preparing a 0.1234 µM fluorophore solution in water at a pH of 6.34 and monitoring the initial rate of fluorophore deprotection via decreases in the fluorescence excimer band intensity at various temperatures. The emission and excitation slit widths were both 10 nm for these fluorescence measurements of the background deprotection rate. Topographic (Two-Dimensional) Fluorescence Emission Studies. Fluorophore stock solutions of 1, 2, 1-pyrenebutyric acid, and 1-pyrenebutanol in absolute ethanol were freshly prepared and stored in the dark at 5 °C. Solutions of various fluorophores in water were freshly prepared by adding 2 µL of the corresponding stock solution to 4 mL of water in a scintillation vial. Samples containing colloidal gold were prepared by adding 3.42 mL of water, 0.58 mL of colloidal solution, and 2 µL of the particular fluorophore stock solution. To avoid fluorescence quenching by excess colloidal gold present in samples containing fluorophore after deprotection, the cuvette was gently heated for a brief period of time to facilitate colloid precipitation from solution. The emission-excitation scans were measured using the emission and excitation wavelength ranges 200-600 nm and the emission slit width 5.0 nm.

Results Steady-State Fluorescence Spectroscopy of Fluorophore Binding. The fluorescence emission spectra of 1 and 2 in aqueous solution are shown in Figure 1. Upon addition of colloidal gold (at a concentration corresponding to approximately 70% of saturation coverage of fluorophore on the colloid surface), a significant amplification in the fluorophore excimer emission is observed (26-fold increase for 1 and 40-fold increase for 2), as well as a corresponding increase in the excimer to monomer band areas, which are summarized in Table 1. Also shown in Figure 1 are the corresponding fluorescence emission spectra of fluorophores comprising 1-pyrenebutyric acid and 1-pyrenebutanol, both in aqueous solution and in the presence of colloidal gold. The concentration dependence of the binding of 1 and 2 to gold was investigated by titrating standard solutions of fixed fluorophore concentration with varying amounts of colloidal gold. The total area of the emission spectrum (which comprises mostly the excimer band for the bound protected thiols, as shown in Figure 1) was calculated for each of these solutions to generate a titration curve, two of which are represented in parts a and b of Figure 2 for 1 and 2, respectively, corresponding to a fixed fluorophore concentration of 123.4 nM. Equilibrium limitations of fluorophore binding to gold were investigated by successively diluting samples comprising a fixed relative amount of fluorophore to colloidal gold corresponding to saturation coverage and measuring

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Figure 1. Fluorescence emission spectra of aqueous solutions containing (a) 1 without colloidal gold, (b) 1 with colloidal gold, (c) 2 without colloidal gold, (d) 2 with colloidal gold, (e) 1-pyrenebutyric acid without colloidal gold, (f) 1-pyrenebutyric acid with colloidal gold, (g) 1-pyrenebutanol without colloidal gold, and (h) 1-pyrenebutanol with colloidal gold. The fluorophore solution concentration in the solutions is 0.1234 µM, and for spectra containing colloidal gold, the colloid concentration is 0.085 nM. The emission slit width in parts a-d is 5.0 nm, and it is 2.5 nm in parts e-h. Table 1. Area of Excimer Emission Band and Monomer Emission Band of 1 and 2 in the Presence of and without Colloidal Gold in Aqueous Solution

fluorophore/colloid

excimer band area (nm counts)

monomer band area (nm counts)

excimer area/monomer area

1 without colloid 1 with colloid 2 without colloid 2 with colloid

14969 370106 10927 381738

4087 14716 3402 8440

3.7 25 3.2 45

the fluorescence emission spectrum. Figure 3 represents the fluorescence emission band area as the concentration is varied for these samples at a fixed colloid-to-fluorophore ratio corresponding to saturation coverage, i.e. 2000 molecules of fluorophore per colloid in aqueous solution, for 1 and 2. Steady-State Fluorescence Spectroscopy of Fluorophore Hydrolysis. The insets in Figure 4 show the fluorescence emission spectra of surfacebound 1 and 2 during the course of hydrolysis. The deprotection kinetics of thioester 1 and thiocarbonate 2 were investigated under pseudo-first-order conditions in fluorophore (the amount of colloidal gold was in large excess relative to saturation coverage). Pseudo-first-order rate coefficients that are based on the measured initial rate of surface-bound fluorophore consumption are represented in Figure 4. Further identification of reactants and products involved in the hydrolysis reaction was performed using topographic fluorescence emission plots that are shown in Figures 5 and 6. These are two-dimensional representations of the fluorescence emission intensity landscape versus excitation and emission wavelength. The lines in these figures represent regions of constant emission intensity for 1 and 2, respectively, in proceeding from (a)

fluorophore in aqueous solution to (b) initial state of surface-bound fluorophore in the presence of excess colloidal gold, at a similar surface coverage to that studied in the kinetics experiments described above to (c) final state of surface-bound fluorophore in (b) to (d) either pyrenebutyric acid (Figure 5d) or 1-pyrenebutanol in aqueous solution (Figure 6d). Discussion Fluorophore Binding to Colloidal Gold. The significant amplification in the excimer emission intensity upon addition of colloidal gold to aqueous solutions of 1 and 2 (Figure 1 and Table 1) is consistent with an increase in the degree of fluorophore aggregation upon adsorption onto the gold surface. Surface-bound fluorophores of 1 and 2 are expected to organize into hydrophobic aggregates in which adjacent pyrenes can form an excimer species (van der Waals contact distance is required between pyrenes for excimer formation),13 which minimizes exposure to aqueous solvent. The observed amplification in excimer emission in this case is similar to that reported by other investigators studying naphthalene fluorescence on the periphery of third generation dendrimers in an aqueous solvent environment.19 A similar amplification in excimer relative to monomer emission has also been observed upon self-assembled monolayer formation of a pyrene-based fluorophore from solution.14 Control experiments with fluorophores that are unable to displace the adsorbed citrate anion and subsequently bind to the colloidal gold surface, such as 1-pyrenebutyric acid and 1-pyrenebutanol, show an insignificant decrease in the monomer emission intensity and no measurable change (19) Ghaddar, T. H.; Whitesell, J. K.; Fox, M. A. J. Phys. Chem. B 2001, 105, 8729-8731.

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Figure 2. Normalized fluorescence emission band area (total area from 360 to 600 nm corresponding to both monomer and excimer bands) versus amount of gold nanoparticles per molecule of (a) 1 and (b) 2. A smooth curve was fit to the data for visualization purposes. A fixed fluorophore concentration of 123.4 nM was used in the titration experiment. Inset: Relationship between the highest colloidal gold concentration corresponding to saturation (maximum in emission area versus colloids per fluorophore in the titration experiment of Figure 2) and the concentration of (a) 1 and (b) 2. Each data point represents a separate titration experiment, and the result of a linear regression of the inset data is drawn as a solid line.

Figure 3. Fluorescence emission band area (total area from 360 to 600 nm corresponding to both monomer and excimer bands) versus fluorophore concentration of (a) 1 and (b) 2. The ratio of fluorophore molecules to colloidal gold nanoparticles was held fixed at 2000 for all data points investigated, which is near the saturation coverage value. The line labeled K ) ∞ represents the behavior expected due to dilution alone, without a finite equilibrium constant for binding.

Figure 4. Plot of the natural logarithm of the first-order rate constant versus the inverse of temperature for (a) deprotection of 1 in the presence of excess colloidal gold and (b) deprotection of 2 in the presence of excess colloidal gold. Lines represent linear regression of the experimentally measured data to the Arrhenius equation. Inset: Fluorescence emission spectra of (a) 1 in the presence of colloidal gold (50% of saturation coverage of 1 on the colloid surface) and (b) 2 in the presence of colloidal gold (36% of saturation coverage of 2 on the colloid surface).

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Figure 5. Topographic (two-dimensional) fluorescence emission spectra of aqueous solutions containing (a) 1, (b) 1 immediately following the addition of colloidal gold, (c) the solution in part b after thioester deprotection to 1-pyrenebutyric acid and surface-bound thiol, and (d) 1-pyrenebutyric acid at the same fluorophore concentration as that in parts a-c.

Figure 6. Topographic (two-dimensional) fluorescence emission spectra of aqueous solutions containing (a) 2, (b) 2 immediately following the addition of colloidal gold, (c) the solution in part b after thiocarbonate deprotection to 1-pyrenebutanol and surface-bound thiol, and (d) 1-pyrenebutanol at the same fluorophore concentration as that in parts a-c.

in the excimer emission band upon addition of the same amount of gold colloid (Figure 1e and f for 1 and g and h for 2). The minor quenching observed upon addition of colloidal gold in these cases is similar to that reported in previous studies of fluorescence in the presence of colloidal gold surfaces.20

Chen and Katz

The concentration dependence of fluorophore binding to the gold surface is studied in Figure 2. The fluorescence emission area is observed to follow a plateau for small amounts of gold relative to protected thiol. Excess fluorophore in this regime has a quenching effect on the fluorescence emission, which is not well-understood at this time. One possibility is a bilayer-type fluorophore structure on the nanoparticle surface, with the pyrenes forming the hydrophobic tail, which predominates at relative amounts of fluorophore to colloidal gold that are more than twice that required for saturation of the nanoparticle surface (vide infra). Subsequent addition of colloidal gold beyond the plateau regime (the end of the plateau regime is at 0.00025 colloids per fluorophore in Figure 2) leads to a sharp increase in the fluorescence emission signal, up to a maximum at approximately twice the colloidal gold concentration corresponding to the onset of the increase in the fluorescence emission and the concomitant end of the plateau regime. The colloid concentration at the location of this maximum represents the highest colloid concentration corresponding to saturation of the nanoparticle surface, at which the surface coverage of fluorophore is maximized. Further addition of colloid beyond this maximum results in a slight decrease in the emission intensity, which is likely due to the same fluorescence quenching phenomenon observed in the case of excess colloid (vide supra) and signifies the presence of an unpassivated gold surface in the system.20 The concentration of colloidal gold necessary to achieve this maximum in fluorescence emission area within a titration experiment is illustrated within the inset of Figure 2 for a series of fluorophore concentrations (corresponding to several different titration experiments). The relationship between this colloid concentration and the fluorophore concentration is linear and passes through the origin, with the slope representing the number of fluorophore molecules per saturated gold nanoparticle. This number is calculated to be 2006 ( 70 molecules for 1 and 2023 ( 60 molecules for 2 per gold nanoparticle using the inset data, which requires a footprint size of approximately 24.5 ( 1.0 Å2 for 1 and 2 on the gold surface, with the pyrenes pointing outward and away from the surface. Comparison of this value with those reported in the literature for the adsorption of alkanethiols onto gold nanoparticles suggests a high packing density at saturation coverage of 1 and 2 on the nanoparticle surface. The footprint area is likely limited by van der Waals contact between adjacent adsorbed fluorophore molecules, although comparisons of the absolute footprint size require a more detailed knowledge of the nanoparticle surface roughness.21-24 The results above provide the first demonstration of thioester and thiocarbonate binding to gold and show that the binding of these functional groups to a gold surface from an aqueous environment does not necessitate immediate deprotection to a surface-bound thiolate species. The equilibrium limitations of fluorophore binding onto the surface of gold were investigated by studying the concentration dependence of the fluorescence emission of solutions with a fixed colloid-to-fluorophore ratio corresponding to saturation coverage (i.e. 2000 molecules of (20) Wang, C. Y.; Liu, C. Y.; Yan, X. B.; He, J. J.; Zhang, M. H.; Shen, T. J. Photochem. Photobiol., A 1997, 104, 159-163. (21) Badia, A.; Singh, S.; Demers, L.; Cuccia, L.; Brown, G. R.; Lennox, R. B. Chem. Eur. J. 1996, 2, 359-363. (22) Camillone, N., III; Chidsey, C. E. D.; Liu, G.; Putvinski, T. M.; Scoles, G. J. Chem. Phys. 1991, 94, 8493-8500. (23) Mohri, N.; Inoue, M.; Arai, Y. Langmuir 1995, 11, 1612-1616. (24) Molecular models show a minimum footprint size of 2.8 Å × 7.1 Å for 1 and 2.

Interaction between Protected Thiols and Gold

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Scheme 1. Representation of Fluorophore Hydrolysis Reactants and Products

Table 2. Summary of Activation Energies and Pre-exponential Factors for the Hydrolysis of 1 and 2 on the Surface of Colloidal Gold, Which Were Calculated from Linear Regression Analysis of the Data in Figure 4 fluorophore system

activation energy

pre-exponential factor

1 with colloidal gold 2 with colloidal gold

14.0 kcal/mol 16.7 kcal/mol

3.88 × 107 (1/s) 8.73 × 108 (1/s)

fluorophore per colloid in aqueous solution). The expected intermediate concentration range behavior is observed in the top-right portion of the plots in Figure 3 for both fluorophores. The linear relationship between amount of surface-bound fluorophore and system concentration represents the expected effect of dilution as the concentration is varied (line passes through the origin). The extent of dilution controls the amount of surface-bound fluorophore in the system as the concentration decreases along the line labeled K ) ∞, until a point where fluorophore binding becomes equilibrium limited, which is in part representative of, among other effects, the competition between enthalpy of adsorption and translational entropy in this system. That is to say, beyond a certain limiting fluorophore concentration in Figure 3, the magnitude of the fluorescence emission band area is seen to decline more than would be predicted from dilution, suggesting the presence of unbound fluorophore at these low concentrations, which is indicative of an equilibrium limitation of fluorophore binding to the gold surface. A quantitative binding constant that is representative of the concentration scale of departure from the K ) ∞ line can be estimated using a rather simple equilibrium model based on a Langmuir isotherm. Since the ratio of the fluorescence emission area at the concentration of departure from the K ) ∞ line to its value on the K ) ∞ line represents the fractional surface coverage of fluorophore at a particular concentration (assuming small perturbations from the K ) ∞ line), this ratio can be used directly in the expression for a Langmuir isotherm to calculate a binding constant using the data in Figure 3, which results in binding constants of approximately 7.2 × 107 ( 2.5 × 107 M-1 for 1 and 1.7 × 108 ( 5.9 × 107 M-1 for 2 to colloidal gold. The larger binding constant of 2 relative to 1, which is evident in the smaller concentration of departure from the K ) ∞ line for 2 relative to 1 in Figure 3, can be rationalized using molecular mechanics calculations based on energy-minimized conformations of fluorophore. The calculated partial charges on the sulfur atoms within these molecules show a significantly higher negative charge on

the sulfur atom in 1 relative to 2 (charges of -0.44 and -0.38 for 1 and 2, respectively, were calculated for the sulfur atom in these molecules).25 Since the binding of organosulfur compounds to gold involves the transfer of electron density from gold to the sulfur atom, as reported previously for the adsorption of thiols and sulfides to gold,7-9,12 the stronger binding constant for 2 relative to 1 in this case is consistent with the more electronegative sulfur in 2, which can act as a better electron acceptor from the gold surface during fluorophore adsorption. Hydrolysis Kinetics of Bound Fluorophore. Previous investigations of thioesters and gold surfaces have indicated that formation of a surface-bound thiolate species can occur.10,11 The hydrolysis reactions are represented above for 1 and 2, and result in the formation of the thermodynamically stable thiolate species, which are known to adsorb on the gold surface.5 The disappearance of the excimer band in the insets of Figure 4 corresponds to depletion in the amount of surface-bound fluorophore during the course of hydrolysis, and the concomitant appearance of a new band in the monomer region of pyrene emission is consistent with fluorescent hydrolysis products that are unable to bind to gold, comprising either 1-pyrenebutyric acid for 1 or 1-pyrenebutanol for 2 (vide supra). The information in Figures 5 and 6 can be used as the equivalent of a fingerprint to further identify the species involved during the course of fluorophore binding and hydrolysis, although the underlying photophysical phenomena governing the detailed features within these curves are quite complex. The significant changes seen during binding in going from part a to part b are representative of the different environments of 1 and 2 in going from a free translational state in aqueous solution to a surface-bound state on the gold surface. During the course of hydrolysis of the bound species, the curves in part b change to those in part c, which represent a new environment indicative of predominantly monomer pyrene emission. Qualitative identification of hydrolysis products can be performed by comparing the similarity of the features between parts c and d, which represent reference spectra of hydrolysis products. Although gold is known to be catalytic for certain classes of reactions that do not currently include thioester and thiocarbonate deprotection,26 we wished to quantitatively (25) Molecular mechanics force field calculations were performed on energy optimized conformations of 1 and 2 using a Dreiding 2.21 force field with Cerius2 version 4.0 software (MSI). (26) Haruta, M. Catal. Today 1997, 36, 153-166.

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investigate its role in catalyzing the hydrolysis of 1 and 2. The temperature dependence of the rate coefficient data shown in Figure 4 can be described by an Arrhenius equation (linear relationship between the logarithm of the first-order rate coefficient and reciprocal temperature), indicating that the gold-catalyzed hydrolysis reactions in this case are thermally activated processes. The corresponding Arrhenius pre-exponential factors and activation energies calculated from Figure 4 are presented in Table 2 and are consistent with values from other catalyzed hydrolysis reactions.27 The significantly higher preexponential factor for 2 relative to 1 is consistent with stronger noncovalent binding (larger sticking coefficient) of 2 to the gold surface, which is in agreement with the equilibrium binding constant results for 1 and 2 (vide supra).28 Background rates for the hydrolysis reaction in aqueous solution in the absence of colloidal gold and at the same conditions of pH and temperature as investigated in Figure 4 were too small to be measurable on the time scale of the kinetics experiment (activation energies for the background hydrolysis reactions in aqueous solution at the same pH were measured to be 32 and 52 kcal/mol for 1 and 2, respectively). In conclusion, the binding of (27) (a) Aksnes, G.; Libnau, F. O. Acta Chem. Scand. 1991, 45, 463468. (b) Li, J.; Brill, T. B. J. Phys. Chem. A 2001, 105, 6171-6175. (28) Somorjai, G. A. Introduction to Surface Chemistry and Catalysis; John Wiley & Sons: New York, 1994.

Chen and Katz

model thioester and thiocarbonate probe molecules to the surface of colloidal gold was investigated via steady-state fluorescence measurements. The results of this study show that 1 and 2 bind noncovalently to the surface of gold nanoparticles from aqueous solution and display a saturation coverage behavior on the nanoparticle surface that is likely limited by van der Waals contact distances. Gold catalyzes the hydrolysis of the bound protected thiols via a thermally activated process that can be described by an Arrhenius equation. The binding of other organofunctional groups on the surface of gold nanoparticles is currently being investigated in our laboratory, and the use of such organized assemblies on nanoparticles as templates for the synthesis of materials will be reported in due course. Acknowledgment. The authors wish to acknowledge the UC Berkeley Department of Chemical Engineering for start-up funding. M.M.Y.C. is grateful to the Undergraduate Research Apprenticeship Program (URAP) at UC Berkeley for a summer fellowship. Supporting Information Available: Description of equilibrium binding constant calculation using Langmuir isotherm model and four figures (gold-catalyzed hydrolysis kinetics data and background hydrolysis kinetics data). This material is available free of charge via the Internet at http://pubs.acs.org. LA015729F