Surface Modification of Colloidal Gold by Chemisorption of

All solutions having colloidal gold were stored in polypropylene centrifuge tubes in the dark to prevent light-induced flocculation of the colloids an...
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Langmuir 2002, 18, 6059-6065

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Surface Modification of Colloidal Gold by Chemisorption of Alkanethiols in the Presence of a Nonionic Surfactant Kadir Aslan and Vı´ctor H. Pe´rez-Luna* Illinois Institute of Technology, Department of Chemical and Environmental Engineering, 10 West 33rd Street, Chicago, Illinois 60616 Received April 1, 2002. In Final Form: May 25, 2002 Surface modification of colloidal gold with 11-mercaptoundecanoic acid or 16-mercaptohexadecanoic acid was performed in the absence or in the presence of the nonionic surfactant polyoxyethylene (20) sorbitan monolaurate (Tween 20). The stability of the colloidal systems was assessed with optical absorption spectroscopy. The surface-modified nanoparticles were stable only within a narrow range of intermediate pH values when chemisorption of alkanethiols was performed in the absence of Tween 20. This was explained in terms of partial ionization of the surface carboxylic groups and charge neutralization at high pH values by counterions present in the buffer solutions. Formation of a physisorbed monolayer of Tween 20 onto the nanoparticles prior to chemisorption of alkanethiols resulted in surface-modified colloidal gold that was stable over a broader range of pH values. Parallel experiments demonstrated that self-assembled monolayers could form on flat substrates in the presence of Tween 20. Therefore, possible incorporation of alkanethiols within micelles or the presence of a physisorbed layer of Tween 20 on gold surfaces did not prevent their chemisorption. The chemisorption of alkanethiols on gold surfaces with a physisorbed layer of Tween 20 is slow and may be due to a decreased sticking coefficient of the alkanethiols on gold with a physisorbed layer of surfactant. Nanoparticles whose surface was modified in the presence of Tween 20 do not appear to undergo irreversible aggregation. They can be frozen or dried and resuspended again with mild sonication.

Introduction Colloidal gold is extensively used in immunocytochemistry and cell biology,1 and it has been in existence for a considerable amount of time dating back to its use in stained glass. Despite its long history, there is currently a considerable amount of research being conducted on metallic nanoparticles for a variety of applications. With the discovery and characterization of self-assembled monolayers (SAMs) of alkanethiols chemisorbed on coinage metals during the past two decades,2,3 there has been a resurgence of interest in colloidal gold because of the possibility it offers in terms of modification of the surface of these nanoparticles with a variety of functional groups using thiol chemistry. Some recently reported uses of metallic nanoparticles are in the development of colorimetric assays for detection of DNA hybridization,4-6 biosensors,7,8 catalysis,9 and the assembly of quantum dots into ordered arrays or nanocrystal superlattices.10 The optical and electronic properties of these materials offer an enormous potential in nanoscale science and engineering applications.10-12 Therefore, surface functionalization of metallic nanoparticles will greatly expand the range of * Corresponding author. E-mail: [email protected]. Phone: 312567-3963. Fax: 312-567-8874. (1) Hayat, M. A. Colloidal Gold: Principles, Methods, and Applications; Academic Press: San Diego, CA, 1989. (2) Ulman, A. An Introduction to Ultrathin Organic Films: from Langmuir-Blodgett to Self-Assembly; Academic Press: Boston, MA, 1991. (3) Whitesides, G. M.; Laibinis, P. E. Langmuir 1990, 6, 87-96. (4) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078-1081. (5) Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959-1964. (6) Reynolds, R. A.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 2000, 122, 3795-3796. (7) Kim, J. H.; Cho, J. H.; Cha, G. S.; Lee, C. W.; Kim, H. B.; Paek, C. W. Biosens. Bioelectron. 2000, 14, 907-915. (8) Velev, O. D.; Kaler, E. W. Langmuir 1999, 15, 3693-3698. (9) Li, H.; Luk, Y. Y.; Mrksich, M. Langmuir 1999, 15, 4957-4959. (10) Collier, C. P.; Vossmeyer, T.; Heath, J. R. Annu. Rev. Phys. Chem. 1998, 49, 371-404.

applications of these materials. Two basic approaches can be taken toward the creation of functionalized gold nanoparticles. One is performing the surface modification of already available colloidal gold preparations by chemisorbing thiols, and the other implies the synthesis of colloidal metals with an organic monolayer in a one-step procedure. The latter is most commonly based on modifications to the synthetic procedure originally reported by Brust to create metallic nanoparticles.13,14 Brust’s synthesis consists of reducing a metallic salt (e.g., AuCl4H) in the presence of an alkanethiol and results in nanoparticles protected by an organic monolayer which are called monolayer-protected clusters (MPCs). Such a monolayer confers these systems with new surface properties and also with extraordinary stability such that they can be isolated and manipulated with a variety of chemical and physical procedures to the extent that they can be dried and resuspended in solution again without suffering irreversible aggregation or decomposition.15-22 Several groups in the world are now actively involved in the study and synthesis of these nanoparticles.15-23 Noteworthy are (11) Wang, Y.; Herron, N. J. J. Phys. Chem. 1991, 95, 525. (12) Bawendi, M.; Stierderwald, M. L.; Brus, L. E. Annu. Rev. Phys. Chem. 1990, 4, 477. (13) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 801-802. (14) Brust, M.; Fink, J.; Bethell, D.; Schiffrin, D. J.; Kiely, C. J. Chem. Soc., Chem. Commun. 1995, 1655-1656. (15) Templeton, A. C.; Wuelfing, W. P.; Murray, R. W. Acc. Chem. Res. 2000, 33, 27-36. (16) Shon, Y. S.; Mazzitelli, C.; Murray, R. W. Langmuir 2001, 17, 7735-7741. (17) Cliffel, D. E.; Zamborini, F. P.; Gross, S. M.; Murray, R. W. Langmuir 2000, 16, 9699-9702. (18) Aguila, A.; Murray, R. W. Langmuir 2000, 16, 5949-5954. (19) Templeton, A. C.; Chen, S.; Gross, S. M.; Murray, R. W. J. Phys. Chem. B 2000, 104, 564-570. (20) Kometani, N.; Tsubonishi, M.; Fujita, T.; Asami, K.; Yonezawa, Y. Langmuir 2001, 17, 578-580. (21) Yee, C.; Scotti, M.; Ulman, A.; White, H.; Rafailovich, M.; Sokolov, J. Langmuir 1999, 15, 4314-4316. (22) Chen, S.; Kimura, K. Langmuir 1999, 15, 1075-1082.

10.1021/la025795x CCC: $22.00 © 2002 American Chemical Society Published on Web 07/30/2002

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Scheme 1. Chemisorption of Alkanethiols to Colloidal Gold Particles Can Eliminate the Negative Charges that Normally Stabilize Them against Aggregationa

a Additionally, in a partially formed self-assembled monolayer, the exposed methylene groups of alkanethiol molecules confer these particles with a hydrophobic character, thereby promoting their aggregation. On colloidal gold particles having a physisorbed layer of Tween 20, the presence of oligo(ethylene glycol) headgroups prevents their aggregation by means of steric interactions. This steric repulsion continues to exist during chemisorption of alkanethiols, thereby allowing full monolayer coverage of the nanoparticles without inducing their aggregation.

presence of the nonionic surfactant polyoxyethylene (20) sorbitan monolaurate (Tween 20). The hypothesis behind this approach was that physical adsorption of Tween 20 onto the gold nanoparticles prior to chemisorption of alkanethiols would stabilize them against aggregation because the oligo(ethylene glycol) moieties of the surfactant provide steric stabilization. Since the interaction of gold surfaces with Tween 20 would be weak compared with their interaction with alkanethiols (physisorption versus chemisorption), the weakly adsorbed surfactant could be subsequently displaced by alkanethiols chemisorbing onto the gold surface. One of the possible mechanisms leading to irreversible aggregation of colloidal gold is the fusion of the metallic cores of aggregating particles. Therefore, chemisorption of alkanethiol in the presence of Tween 20 would ensure that irreversible particle aggregation does not occur because of the intermediate layer of adsorbed surfactant and/or alkanethiol. That is, a protective layer of oligo(ethylene glycol) moieties on the surface of the particles will exist (preventing fusion of the metallic core of these particles) until complete monolayer coverage of alkanethiols is achieved around the colloids. Once a fully formed SAM was present on the colloidal gold particles, the surfactant Tween 20 could be removed and irreversible aggregation of the metallic nanoparticles would not occur. These ideas are depicted schematically in Scheme 1.

the efforts during the past decade of Murray et al. to produce and characterize MPCs following the initial report by Brust et al.13,14 on the synthesis of gold clusters stabilized by monolayers of alkanethiolate ligands.15-19 In general, the synthesis of MPCs yields polydisperse size distributions of nanoparticles of sizes under 10 nm.12,16-22 For some applications, it may be desirable to obtain functionalized gold nanoparticles of sizes larger than these MPCs and with monodisperse sizes. For example, quantum dot (QD) superlattices can be better assembled from single-sized nanoparticles or from colloids with narrow size distributions.12 Superlattice formation from broad particle size distributions has been achieved;24 however, it is generally recognized that single- or narrowsized nanocrystals are easier to assemble into ordered arrays of QDs.12 This and the fact that larger colloids may offer a broader range of properties and applications have motivated us to study the stability of commercial preparations of colloidal gold upon surface modification by chemisorption of alkanethiols. Colloidal gold is commercially available in monodisperse sizes on the order of tens of nanometers, whereas MPCs have typical polydisperse size distributions on the order of 1-10 nm. Also, the synthesis and characterization of MPCs may become expensive and time consuming. Thus, functionalization of commercial monodisperse preparations of colloidal gold would provide these systems with different surface properties and may offer advantages similar to those of MPCs. However, colloidal gold can undergo irreversible aggregation upon chemisorption of alkanethiols.15,25,26 Our approach to obtain stable surfacemodified colloidal gold dispersions was to perform the chemisorption of carboxyl-terminated alkanethiols in the

Materials. Colloidal gold (monodisperse, 20 nm average particle size), hydrogen peroxide, sulfuric acid, phosphoric acid, acetic acid, sodium acetate, sodium phosphate monobasic, sodium phosphate dibasic, and sodium carbonate were obtained from Sigma. Absolute ethanol, Tween 20, 16-mercaptohexadecanoic

(23) Imahori, H.; Arimura, M.; Hanada, T.; Nishimura, Y.; Yamazaki, I.; Sakata, Y.; Fukuzumi, S. J. Am. Chem. Soc. 2001, 123, 335-336. (24) Ohara, P. C.; Leff, D. V.; Heath, J. R.; Gelbart, W. M. Phys. Rev. Lett. 1995, 75, 3466-3469.

(25) Weisbecker, C. S.; Merritt, M. V.; Whitesides, G. M. Langmuir 1996, 12, 3763-3772. (26) Mayya, K. S.; Patil, V.; Sastry, M. Langmuir 1997, 13, 39443947.

Experimental Section

Surface Modification of Colloidal Gold acid (16-MHDA), and 11-mercaptoundecanoic acid (11-MUDA) were obtained from Aldrich. All chemicals were used as received. Premium quality glass slides were purchased from Fisher Scientific (75 × 25 mm); gold evaporation material (99.999% pure) and titanium (99.995% pure) were supplied by Plasmaterials, Inc. Buffers and Solutions. All buffer solutions were prepared to 0.010 M concentration as follows: pH 3, phosphoric acid/sodium phosphate monobasic; pH 5, acetic acid/sodium acetate; pH 7, sodium phosphate monobasic/sodium phosphate dibasic; pH 8, sodium phosphate dibasic; pH 9, sodium phosphate dibasic; pH 11, sodium carbonate. Exact pH values for buffer solutions were obtained using an Accumet AB15 pH meter. Deionized (DI) water (electric resistivity > 18 MΩ cm) was obtained from a Barnstead Nanopure Infinity UV/UF water purification system. All glassware used was treated with “piranha solution” (3:7 30% hydrogen peroxide/concentrated sulfuric acid; CAUTION: piranha solution reacts violently with most organic materials and should be handled with extreme care). Solutions of 0.5 mM 16-MHDA and 0.5 mM 11-MUDA were prepared in degassed ethanol. Tween 20 solutions were prepared in the corresponding buffers. Surface Modification of Colloidal Gold by Chemisorption of Alkanethiols. Gold sols with a concentration of 0.8 nM were degassed with nitrogen before use. Equal volumes of colloidal gold and Tween 20 in buffer were gently mixed and allowed to stand for a minimum of 20 min for physisorption of Tween 20 to the colloidal gold to occur. Then, the thiol solution was added and the final mixtures (final concentrations: [colloidal gold] ) 0.27 nM, [Tween 20] ) 0.12-3.65 mg/mL, [alkanethiols] ) 0.17 mM) were allowed to stand up to 4 h. Experiments were also done in the absence of Tween 20 in order to compare the effect of added surfactant on the stability of the system. The optical absorption spectra of these systems were acquired with a Shimadzu UV-2401PC dual-beam spectrophotometer using 1 cm path length quartz cuvettes. The optical absorption spectra were collected for systems containing thiols, surfactant, and colloidal gold in situ (to monitor their stability during surface modification) and also following removal of excess thiols and surfactant from the system (in order to determine if the colloidal suspension would remain stable once the surfactant was removed). Excess thiols and Tween 20 were removed from the surface-modified colloids by centrifugation for 10 min at 15700g followed by decantation of supernatants and resuspension in buffer. The absorbance of the supernatants was also measured in order to check for the presence of gold. All solutions having colloidal gold were stored in polypropylene centrifuge tubes in the dark to prevent lightinduced flocculation of the colloids and oxidation of the alkanethiolates.25 Determination of Flocculation. Mie’s scattering theory shows that metallic nanoparticles with a radius much smaller than the wavelength of light and dispersed in a dielectric medium absorb strongly at certain wavelengths due to resonance excitation of surface plasmons.27,28 For colloidal gold in an aqueous medium, this occurs around 520 nm. However, if the colloidal particles aggregate such that the distance between aggregating spheres becomes small compared to their radius, additional resonances will occur at wavelengths longer than those of the individual particles.29 This results in red-shifting and broadening of the absorption spectra. Based on this observation, a “flocculation parameter” was defined by Weisbecker25 and later modified by Mayya26 as the integral of the absorption spectra between 600 and 800 nm, as a semiquantitative measure of aggregation. Since the absorbance of colloidal solutions increases at longer wavelengths upon particle aggregation, the flocculation parameter increases with the extent of particle aggregation.25,26 In this paper, we adopted this convention with the optical absorption spectra normalized to the absorption intensity of the surface plasmon peak.26 Formation of SAMs on Flat Substrates. Glass slides were cleaned in piranha solution, rinsed with DI water and absolute grade ethanol, and blown dry with a stream of nitrogen before placing them in a metal evaporator system. An adhesion layer (27) Mie, G. Ann. Phys. 1908, 25, 377-445. (28) Mulvaney, P. Langmuir 1996, 12, 788-800. (29) Quinten, M.; Kreibig, U. Surf. Sci. 1986, 172, 557-577.

Langmuir, Vol. 18, No. 16, 2002 6061 of 20 Å of titanium was deposited on the glass slides before depositing 500 Å of gold by evaporation of metals at 10-7 Torr using an e-beam-gun metal evaporator system. The substrates were used immediately after evaporation. One set of gold-coated slides was immediately immersed in a 0.5 mM thiol solution to allow for SAM formation to occur overnight. A second set of freshly deposited gold films was immersed in solutions of Tween 20 for 2 h. Then, the films were immersed in thiol solutions containing Tween 20 and buffer and incubated overnight to allow for SAM formation. The samples were pulled out of solution, rinsed with ethanol, DI water, 0.1 M HCl, water, and ethanol again, and finally blown dry with a stream of nitrogen and used for contact angle measurements. A minimum of five advancing contact angle measurements were done using a Rame-Hart model 100 goniometer.

Results and Discussion Physisorption of Tween 20 on Colloidal Gold. Our hypothesis was that the presence of a physisorbed layer of Tween 20 on the surface of colloidal gold could prevent irreversible aggregation of gold nanoparticles during and after chemisorption of alkanethiols. In view of this, the kinetics of adsorption of Tween 20 onto these nanoparticles was investigated first. This was done by monitoring the change in absorbance at 550 nm of the colloidal solutions with time upon addition of Tween 20. The selection of this wavelength is based upon theoretical predictions27,28,30,31 and experimental observations30-32 that the extinction coefficient and the position of the surface plasmon resonance peak of colloidal gold are sensitive to changes in the dielectric constant, m, around the particles. In general, the formation of a dielectric layer around a metallic nanoparticle results in a shift of the surface plasmon resonance peak to lower energies (longer wavelengths) and an increase in the extinction coefficient (absorbance) if the dielectric constant of this layer is larger than that of the medium in which the particle is embedded.28,30-32 Figure 1A shows the optical absorption spectra of colloidal gold before and after adsorption of Tween 20. The shift in the position of the surface plasmon peak is consistent with the formation of a layer of adsorbed surfactant around the nanoparticles. This shift correlates directly with the dielectric constant of the medium around the particles. Such a correlation can be considered, to a first approximation, as linear over small ranges of m. Nath and Chilkoti demonstrated that the absorbance at wavelengths longer than the surface plasmon peak could be used to monitor changes in the dielectric constant around the colloids and that these changes could be approximated by a linear relationship provided that the appropriate wavelength is selected.32 We used the latter approach because single-wavelength measurements can provide real-time information on fast adsorption kinetics and because they are more sensitive than monitoring the position of the peak maximum. Figure 1B shows the adsorption kinetics of Tween 20 as measured through the changes in the absorbance at 550 nm. At this wavelength, the extinction coefficient follows an almost linear relationship with wavelength and thus changes in absorbance are linearly correlated to shifts in the surface plasmon peak. The physical adsorption of Tween 20 onto colloidal gold proceeded at similar rates regardless of the surfactant concentration used in these experiments. This is because, above the critical micellar concentration, the concentration (30) Schmitt, J.; Ma¨chtle, P.; Eck, D.; Mo¨hwald, H.; Helm, C. A. Langmuir 1999, 15, 3256-3266. (31) Eck, D.; Helm, C. A.; Wagner, N. J.; Vaynberg, K. A. Langmuir 2001, 17, 957-960. (32) Nath, N.; Chilkoti, A. Anal. Chem. 2002, 74, 504-509.

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Figure 2. Adsorption kinetics at pH ) 5 of 11-MUDA (0.167 mM) on colloidal gold after a physisorbed layer of the nonionic surfactant Tween 20 was allowed to adsorb for 20 min previous to chemisorption of the alkanethiol.

Figure 1. (A) Optical absorption spectra of colloidal gold (solid line) and colloidal gold after 4 h of adsorption of Tween 20 at 0.61 mg/mL (dotted line). (B) Adsorption kinetics of Tween 20 onto colloidal gold at pH 7 as measured by the increase in absorbance at 550 nm.

of free surfactant remains nearly constant and only the size and number of micellar aggregates change. We also note that a plot of the change in absorbance versus xt shows a linear trend, indicating diffusion-limited kinetics (not shown). Therefore, monolayer formation of Tween 20 around gold nanoparticles proceeds by diffusion of free surfactant in solution rather than from micellar aggregates. The adsorption of Tween 20 on colloidal gold reached 95% saturation coverage in 20 min. Thus, we chose to perform the physisorption of Tween 20 onto the gold nanoparticles for a minimum of 20 min before adding alkanethiols. This ensured that the colloids were covered by an adsorbed surfactant layer before SAMs were formed on their surfaces by chemisorption of alkanethiols. In fact, when chemisorption of alkanethiols was performed before the adsorption of Tween 20 reached saturation levels the resulting nanoparticles were less stable and the results were less reproducible. We ascribe this to the fact that incomplete monolayer coverage did not effectively stabilize the nanoparticles against aggregation. We also note that the presence of Tween 20 prevented aggregation at high ionic strengths since colloidal gold underwent irreversible aggregation upon incubation with Dulbecco’s phosphate buffer solution (the reddish color disappeared in a couple of minutes) whereas it remained stable in this buffer when Tween 20 was present in the system. Chemisorption of Alkanethiols on Colloidal Gold. Figure 2 shows the kinetics of chemisorption of 11-MUDA on colloidal gold. The chemisorption process in this case is slower than previous reports on the kinetics of SAM formation on flat gold surfaces by chemisorption of alkanethiols.33,34 We speculate that the presence of physisorbed surfactant on the colloids would decrease the sticking coefficient35,36 of alkanethiols reaching the surface because the adsorbed surfactant would have to be displaced by alkanethiol molecules in order to chemisorb.

Figure 3. Normalized optical absorption spectra of the selfassembled monolayers of 16-MHDA on colloidal gold in the presence of Tween 20, at pH 7 after 4 h. Symbols have been put to differentiate between the lines: (b) gold; (3) gold + 0.61 mg/mL Tween; (9) gold + 16-MHDA; (]) gold + 16-MHDA + 0.12 mg/mL Tween; (2) gold + 16-MHDA + 0.61 mg/mL Tween; (O) gold + 16-MHDA + 3.65 mg/mL Tween.

Figure 3 shows the optical absorption spectra normalized to the surface plasmon resonance peak for colloidal gold before and after addition of Tween 20 and for colloidal gold after 4 h of chemisorption of 16-MHDA in the absence and in the presence of varying amounts of Tween 20 at pH ) 7. The surface plasmon resonance peak of colloidal gold occurs at 520 nm as expected. A shift to 523 nm is observed upon addition of Tween 20. This results from physical adsorption of the surfactant on the nanoparticles and is consistent with models predicting small shifts of the surface plasmon absorption band upon formation of dielectric layers around colloidal metals.28,30,31 Upon chemisorption of 16-MHDA, the surface plasmon peak shifts to 524-525 nm, indicating a thicker monolayer forming around the metallic nanoparticles. We note that upon adsorption of Tween 20, and 16-MHDA when Tween 20 was previously physisorbed, the spectral shift is not accompanied by broadening of the spectrum. The fact that the absorption spectrum does not broaden indicates lack of particle aggregation. In contrast, the shift in the surface plasmon resonance peak to 550 nm for colloidal gold exposed to 16-MHDA in the absence of Tween 20 is accompanied by further broadening of the spectra. This clearly indicates aggregation of gold particles as 16-MHDA (33) Peterlinz, K. A.; Georgiadis, R. Langmuir 1996, 12, 4731-4740. (34) Himmelhaus, M.; Eisert, F.; Buck, M.; Grunze, M. J. Phys. Chem. B 1999, 104, 576-584. (35) Weaver, D. R.; Pitt, W. G. Biomaterials 1992, 13, 577-584. (36) Jung, L. S.; Nelson, K. E.; Stayton, P. S.; Campbell, C. T. Langmuir 2000, 16, 9421-9432.

Surface Modification of Colloidal Gold

Figure 4. Flocculation parameter versus pH of colloidal gold with SAMs of (A) 11-MUDA and (B) 16-MHDA after chemisorption proceeded for 4 h in various surfactant concentrations. Tween 20 and alkanethiols were not removed from the system.

chemisorbs on the gold particles. It also illustrates the basis for defining the flocculation parameter as the area under the curve (between 600 and 800 nm) since this value would increase significantly when broadening of the spectrum occurs. We note, however, that small changes in the flocculation parameter may result from adsorption of a dielectric layer on the nanoparticle’s surface even when this is not accompanied by particle aggregation. The above-mentioned observations further motivated the approach of physisorbing a nonionic surfactant prior to surface modification with alkanethiols in order to achieve stable surface-modified colloidal gold. Flocculation of Colloidal Gold in the Presence of Surfactant and Thiols. Electrostatic forces are the most important factor stabilizing colloidal gold preparations. These result from anions adsorbed on the surface of colloidal gold particles, which confer them with a net negative charge. Chemisorption of alkanethiols onto colloidal gold results in a net loss of this negative charge, and it has been speculated that nanoparticle aggregation occurs upon SAM formation because of decreased electrostatic repulsion when this charge is decreased or lost.25 However, if a ω-functionalized alkanethiol with an ionizable group (e.g., carboxyl) is chemisorbed, the net charge on the functionalized nanoparticles should depend strongly on pH. Thus, we expect the stability to depend critically on the pH of the solution. To determine the effect of pH and alkanethiol molecule on the aggregation of colloidal gold, experiments were conducted using 16-MHDA and 11-MUDA at pH values ranging from 3 to 11. Figure 4 shows the flocculation parameter versus pH upon chemisorption of alkanethiols on colloidal gold in the presence of Tween 20 after 4 h of exposure to 11-MUDA (Figure 4A) and 16-MHDA (Figure 4B). These data represent measurements in situ; that is, the surfactant and thiols were not separated from the system. The flocculation parameters for colloidal gold with SAMs of 11-MUDA and 16-MHDA in the absence of Tween 20 reached a minimum at pH 5. However, colloidal gold with SAMs of 16-MHDA had slightly lower values of the flocculation parameter, indicating more stability. Surface

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modification with the longer alkyl chains (16-MHDA) may result in more stable colloids because the packing density and order of self-assembled monolayers increases with the number of methylene groups.25,37 Also, repulsive electrostatic forces between two ionized surfaces increase with increasing thickness of the monolayer surrounding the colloidal particles because the charge is located at the surface of the SAMs.25 This is consistent with a decrease in the flocculation parameter with chain length as reported before.25 Our choice of 11-MUDA and 16-MHDA resulted thus from their ability to confer surface-modified colloidal gold with more stability. As evident from Figure 3 (solid squares) and Figure 4 (inverted empty triangles), surfacemodified colloidal gold was stable only within a narrow range of pH. To increase the stability of colloidal gold during chemisorption of the thiols over a broader range of pH, the surface modification was performed using three different concentrations of the nonionic surfactant Tween 20; these were 0.12, 0.61, and 3.65 mg/mL. As shown in Figure 4, the use of Tween 20 dramatically lowered the flocculation parameter and extended the range of stability at pH values higher than 7. That is, the colloidal system became stable at higher pH values than in the absence of surfactant. Stabilization by Tween 20 might be explained by the formation of a physisorbed layer of surfactant chains that repel other colloidal particles due to the presence of ethylene oxide moieties in Tween 20. Partial ionization of carboxylic acid groups at larger pH values also contributes to this increased stability. In the absence of Tween 20, colloidal gold modified with carboxylic groups is more stable at intermediate pH values. This may seem counterintuitive since it is expected that the degree of ionization of the carboxylic groups would increase with pH. These data suggest that the ionization of carboxylic groups at the surface of the nanoparticles is maximum at intermediate pH. Maximum ionization of surface carboxylic groups at intermediate pH values was observed by White et al. for SAMs of 11-MUDA on Ag(111) electrodes38 and by Mulvaney et al. for SAMs of 11-MUDA on gold.39 Stability of Surface-Modified Colloidal Gold Upon Separation of Excess Thiols and Surfactant. It was found that gold colloids with SAMs of carboxyl-terminated thiols were stable over a wider range of pH when the chemisorption of thiols was performed in the presence of Tween 20 (Figure 4). However, it was not clear if they would aggregate or remain stable upon removal of Tween 20 (which helped stabilize the dispersions). To answer this question, unadsorbed thiols and Tween 20 were removed after chemisorption of alkanethiols took place for 4 h. This was done by washing the particles using repeated centrifugation and resuspension in buffer. This procedure was repeated twice, and the final colloidal dispersion was examined by optical absorption spectroscopy. Chemical analysis of the “washed” colloids to test for complete removal of Tween 20 was not attempted. Thus, we cannot rule out the possibility of residual surfactant remaining on the particles. However, contact angle measurements on flat substrates provided indirect evidence that the amount of residual surfactant was small (vide infra). During this procedure, the optical absorption of the supernatant was also measured in order to determine the efficiency of this procedure to retrieve the colloidal particles. It was found that less than 5% of the colloids were discarded on the supernatant after the (37) Camillone, N., III.; Chidsey, C. E. D.; Liu, G. Y.; Putvinsky, T. M.; Scoles, G. J. Chem. Phys. 1991, 94, 8493-8502. (38) White, H. S.; Peterson, J. D.; Cui, Q.; Stevenson, K. J. J. Phys. Chem. B 1998, 102, 2930-2934. (39) Kane, V.; Mulvaney, P. Langmuir 1998, 14, 3303-3311.

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Figure 5. The flocculation parameter versus the pH of the colloidal gold with SAMs of (A) 11-MUDA and (B) 16-MHDA after 4 h in various surfactant concentrations. Tween 20 and thiols were separated from the medium.

repeated centrifugation and resuspension procedure. That is, more than 95% of the surface-modified gold nanoparticles were recovered during the removal of unadsorbed thiols and surfactant. Figure 5A,B shows the flocculation parameter versus pH of colloidal gold whose surface was modified by performing the chemisorption of 11-MUDA and 16-MHDA for 4 h in the presence of Tween 20 at various concentrations followed by removal of excess thiols and Tween 20 using the centrifugation and resuspension procedure described above. When the centrifugation and resuspension procedure was applied to colloidal gold that was exposed to 11-MUDA and 16-MHDA in the absence of Tween 20 at the lowest pH, this resulted in irreversible aggregation of the particles. This was evident from visual examination of the samples and from the fact that very small absorbances (within noise level) were measured for these samples. In these two cases, all colloidal particles underwent irreversible aggregation and settled down on the bottom of the cuvettes. Neither agitation nor sonication could redisperse them. We have arbitrarily assigned the highest possible value of the flocculation parameter, which is 200, to these two points due to the irreversible aggregation of the colloids. The flocculation parameter for colloidal gold with SAMs of 11-MUDA in the absence of Tween 20 followed the same trend as in Figure 2, exhibiting a minimum around pH 5 and then increasing and leveling off at pH 8. However, when Tween 20 was used, the flocculation parameter minimum shifted to pH 7 where it continued to increase thereafter. Colloidal gold with SAMs of 16-MHDA in the absence of Tween 20 was stable within a broader range of pH. The flocculation parameter had a minimum around pH 5-7 and then increased as the pH increased. Clearly, the dependence of the flocculation parameter with pH for surface-modified colloidal gold was different in the presence of excess surfactant and unreacted thiols than when the thiols and Tween 20 were removed from the system. It is possible that further aggregation of surface-modified colloidal gold was induced by centrifugation because of the observed increase in the flocculation parameter. In fact, samples prepared at pHs of 3 and 5

Aslan and Pe´ rez-Luna

showed a significant extent of irreversible aggregation of the colloidal particles as evident by the presence of a precipitate. However, samples prepared between pH values of 7 and 9 remained in suspension. The dependence of the flocculation parameter with pH (exhibiting a minimum) appears counterintuitive. It would be expected that at larger pH values, the ionization of carboxylic groups on the surface of these particles would increase. A larger number of ionized groups on the surface of gold nanoparticles should translate in stronger electrostatic repulsion forces and thus in more stability. Yet, the system shows maximum stability at intermediate pH values. This observation is also evident in the work of Weisbecker, where the flocculation parameter of surfacemodified colloidal gold is minimum at intermediate pH values.25 White et al.38 observed a similar dependence with pH of the surface charge density of a Ag(111) electrode coated with mixed monolayer of 11-MUDA and 1-decanethiol. By the use of cyclic voltammetry, a maximum surface charge density was observed at a pH of 8.5 in all electrolyte solutions used. A second observation was that the number of ionized groups was smaller than the total number of carboxylic groups on the surface of the electrode. That is, the number of ionized groups did not increase continuously with pH but a maximum was obtained at pH ) 8.5 and only a fraction of the carboxylic groups were ionized to carboxylate groups.38 These observations are consistent with our results since maximum ionization at intermediate values of pH translates into maximum electrostatic repulsion at intermediate pH. An explanation for the maximum ionization of carboxylic groups on self-assembled monolayers was provided by Kane and Mulvaney after measuring the surface potential of 11-MUDA SAMs on gold with the atomic force microscope (AFM).39 They observed that the surface potential was maximum at intermediate values of pH and that this potential was smaller than what would be expected for fully ionized surface groups. The low degree of ionization of the carboxyl groups prompted them to interpret their observations using a model based on the Gouy-Chapman theory of the electrical diffuse double layer that included sodium binding as a “potential determining ion” that reduced the surface potential by neutralizing a large number of ionized carboxyl groups. To model the counterion binding, they used a mass-action approach for both metal ions and protons as follows:

COOH T COO- + H + (surf)

with Ka )

aCOO-aH+ aCOOH (1)

COONa T COO- + Na + (surf) with KNa )

aCOO-aNa+ (2) aCOONa

Thus, the surface charge density (σ0) would be

σ0 ) -e[COO-] ) [COO-] (3) -eNs {[COOH] + [COONa] + [COO-]} σ0 ) -

eNs +

{1 + [H ]/Ka + [Na+]/KNa}

(4)

where e is the charge of the electron, Ns is the site density

Surface Modification of Colloidal Gold

of surface groups, Ka and KNa are reaction equilibrium constants, and the quantities within brackets represent surface concentrations (we assume here that concentrations accurately represent activity). From eq 4, it is evident that for large enough values of pH (low [H+]), the denominator becomes dominated by the term 1 + [Na+]/ KNa. Thus, at large pH values surface charge density becomes independent of pH and depends on the concentration of Na+ (the potential determining ion), and it is smaller than the maximum surface charge density that would be attained at 100% ionization, σ0max ) -eNs. Kane and Mulvaney also estimated KNa ) 4 × 106, which explains why even small concentrations of sodium ions can dramatically reduce the number of ionized groups by neutralizing charged carboxylate groups (eq 2).39 Surface-modified colloidal gold that was prepared at pH 11 in the presence of Tween 20 and further resuspended at pH 7 shows a decreased flocculation parameter to levels similar to those of nanoparticles whose surface was modified at pH 7. This appears to indicate that, at least for pH values above 7, the observed aggregation may be reversible. That may be a result of a fully formed SAM on the gold nanoparticles that prevents fusion of the metallic cores. Additionally, the surface-modified colloidal gold particles obtained using this procedure can be frozen or dried and then resuspended again with mild sonication yielding reddish, stable colloidal suspensions that show a definite surface plasmon resonance peak at 527 nm in their optical absorption spectrum (data not shown). Although these observations may hold true only for the carboxyl-functionalized alkanethiols employed in this work, the surface modification procedure using nonionic surfactants as stabilizing agents may open the possibility of the creation of colloidal gold with a broader range of surface properties. SAMs on Planar Gold in the Presence of Tween 20. Stable systems were obtained when exposing colloidal gold to 11-MUDA and 16-MHDA in the presence of a nonionic surfactant. However, the possibility exists that stability could have been achieved if the physisorbed surfactant layer prevented chemisorption of alkanethiols or if the micelles sequestered the alkanethiol molecules within their cores, thus preventing them from reaching the surface of the nanoparticles. To prove the formation of alkanethiol SAMs on gold in the presence of Tween 20, we measured the advancing contact angles of water on planar gold films. Initially, Tween 20 was allowed to physisorb on the surface and advancing contact angles of 56-59° were obtained (the contact angle of freshly evaporated gold film was 85°). Then, the films were immersed into thiol solutions and contact angles smaller than 10° were obtained for both 11-MUDA and 16-MHDA, in agreement with the literature.40 This indicated that carboxylic acid terminated thiols displaced physisorbed Tween 20 and that SAMs were actually formed because (40) Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321-335.

Langmuir, Vol. 18, No. 16, 2002 6065

the chemisorption process of the alkanethiols on gold was energetically favored over physisorption of the surfactant. Furthermore, the fact that water contact angles on SAMs of 11-MUDA and 16-MHDA formed in the presence of Tween 20 are smaller than 10° appears to indicate that Tween 20 does not remain bound to the surface to a large extent. We have further reacted the carboxylic groups on gold nanoparticles to produce ligand-bearing colloidal gold that binds proteins through specific molecular recognition. These results will be presented in a future publication. Conclusions Surface modification of colloidal gold promises to expand the number of applications it already encounters in biology, microscopy, electronics, biosensors, and detection assays among others. Although the formation of self-assembled monolayers by chemisorption of alkanethiols on gold surfaces has been exploited for more than a decade, the extension of this technique to modify the surface of colloidal gold is not as straightforward because of the inherent destabilization of the latter upon alkanethiol chemisorption. In this work, the capability to modify the surface of commercial, well-characterized preparations of colloidal gold without compromising their stability through the chemisorption of 11-MUDA and 16-MHDA was demonstrated. Although direct surface modification using thiol chemistry results in particle aggregation, this can now be circumvented by forming a physisorbed monolayer of the nonionic surfactant Tween 20 prior to chemisorption of the alkanethiols. We hypothesize that the adsorbed layer of Tween 20 prevents aggregation of the nanoparticles by means of steric interactions due to the presence of oligo(ethylene glycol) moieties on the headgroup of this surfactant. Thus, the steric interactions prevent the nanoparticles from aggregating even after repulsive electrostatic interactions on colloidal gold are weakened or eliminated by the chemisorption of alkanethiols. The adsorbed layer of Tween 20 prevents aggregation during surface modification but also retards the chemisorption process, most likely by decreasing the sticking coefficient of the alkanethiols on gold. We hypothesize that once a fully formed SAM is present on the particles, removal of excess surfactant from the system does not result in irreversible aggregation because the fully formed SAM further prevents fusion of the metallic cores. Nanoparticle stability was achieved over a broader range of pH and ionic strength and under conditions as extreme as drying and resuspension. Thus, this method may offer an alternative to monolayer-protected clusters in a limited number of applications. Acknowledgment. We gratefully acknowledge the financial support for this project provided by the Armour College of Engineering and the Department of Chemical and Environmental Engineering at the Illinois Institute of Technology. LA025795X