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Monofunctional Group-Modified Gold Nanoparticles from Solid Phase Synthesis Approach: Solid Support and Experimental Condition Effect James G. Worden, Qiu Dai, Andrew W. Shaffer, and Qun Huo* Department of Polymers and Coatings, North Dakota State University, 1735 NDSU Research Park Drive, Fargo, North Dakota 58105 Received July 6, 2004. Revised Manuscript Received July 20, 2004
We herein report a systematic study of solid-phase place exchange reactions for the synthesis of gold nanoparticles with a single surface functional group. This approach is based on a “catch and release” mechanism to control the number of functional groups attached to the nanoparticle surface. Bifunctional thiol ligands with a carboxylic end group were first immobilized on a solid polymer support in a controlled density. The density was low enough that neighboring thiol ligands were far apart from each other. When the modified polymer support was incubated in a butanethiol-protected nanoparticle solution, a one-to-one place exchange reaction took place between the polymer-bound thiol ligands and the nanoparticles. After cleaving off from the solid support, nanoparticles with a single carboxylic group were obtained as the major product. By varying the solid supports and reaction conditions, we succeeded to obtain monofunctional gold nanoparticles with enhanced yield and high purity.
Introduction In the bottom-up approach toward nanomaterials and nanodevice development, two important aspects must be investigated. The first is the synthesis of the nanobuilding block itself and the second is how to assemble these nanobuilding blocks together into predefined structures with desired properties. While there has been much success with the synthesis of nanobuilding blocks, such as nanoparticles, nanorods, nanotubes,1-6 the assembling of these materials remains a significant challenge. Among the various approaches, the supramolecular chemistry-based self-assembling technique appears to be most promising in the design and development of a variety of nanomaterial structures, including one-dimensional wires, two-dimensional arrays, threedimensional crystals, and nanocomposites.7-13 To prepare even more complex nanomaterial-based structures and devices, one must first be able to ac* To whom the correspondence should be addressed. Tel: 701-2318438. Fax: 701-231-8439. E-mail:
[email protected]. (1) Nalwa, H. S., Ed. Nanostructured Materials and Nanotechnology; Academic Press: London, 2002. (2) Schmid, G. Clusters and Colloids: From Theory to Applications. VCH: New York, 2004. (3) Zhou, O.; Shimoda, H.; Gao, B.; Oh, S.; Fleming, L.; Yue, G. Acc. Chem. Res. 2002, 35, 1045-1053. (4) Ouyang, M.; Huang, J.-L.; Lieber, C. M. Acc. Chem. Res. 2002, 35, 1018-1025. (5) Kim, F.; Song, J. H.; Yang, P. J. Am. Chem. Soc. 2002, 124, 14316-14317. (6) Duan, X.; Huang, Y.; Agarwal, R.; Lieber, C. M. Nature 2003, 421, 241-245. (7) Daniel, M.-C.; Astruc, D. Chem. Rev. 2004, 104, 293-346. (8) Templeton, A. C.; Wuelfing, M. P.; Murray, R. W. Acc. Chem. Res. 2000, 33, 27-36. (9) Shenhar, R.; Rotello, V. M. Acc. Chem. Res. 2003, 36, 549-561. (10) Hamley, I. W. Angew. Chem., Int. Ed. 2003, 42, 1692-1712. (11) Feldheim, D. L.; Keating, C. D. Chem. Soc. Rev. 1998, 27, 1-12. (12) Schmid, G.; Baumle, M.; Geerkens, M.; Heim, I.; Osemann, C.; Sawitowski, T. Chem. Soc. Rev. 1999, 28, 179-185. (13) Fendler, J. H. Chem. Mater. 2001, 13, 3196-3210.
curately control the chemical structure and functionality of the nanobuilding blocks at the molecular level. Unfortunately, most nanobuilding blocks developed so far lack such a control. Given the example of gold nanoparticles, synthetic methods commonly used for monolayer-protected gold nanoparticle synthesis, including the Shiffrin reaction and place exchange reaction, can only lead to nanoparticles with either no functional groups or with multiple unknown numbers of functional groups.14-19 To address this challenge, our group recently reported a preliminary study on a solidphase place exchange reaction to synthesize gold nanoparticles with monofunctional group attached to the surface.20 This approach is based on a “catch and release” mechanism as outlined in Scheme 1. Bifunctional thiol ligands with a carboxylic end group were first immobilized on a solid support such as a polymer resin with a controlled density. The density was low enough that neighboring thiol ligands were far apart from each other. When the modified polymer support was incubated in a butanethiol-protected gold nanoparticle solution, a one-to-one place exchange reaction took place between the polymer-bound thiol ligands and the (14) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 801-802. (15) Hostetler, M. J.; Wingate, J. E.; Zhong, C.-J.; Harris, J. E.; Vachet, R. W.; Clark, M. R.; Londono, J. D.; Green, S. J.; Stokes, J. J.; Wignall, G. D.; Glish, G. L.; Porter, M. D.; Evans, N. D.; Murray, R. W. Langmuir 1998, 14, 17-30. (16) Hostetler, M. J.; Templeton, A. C.; Murray, R. W. Langmuir 1999, 15, 3782-3789. (17) Templeton, A. C.; Hostetler, M. J.; Warmoth, E. K.; Chen, S.; Hartshorn, C. M.; Krishnamurthy, V. M.; Forbes, M. D. E.; Murray, R. W. J. Am. Chem. Soc. 1998, 120, 4845-4849. (18) Templeton, A. C.; Chen, S.; Gross, S. M.; Murray, R. W. Langmuir 1999, 15, 66-76. (19) Templeton, A. C.; Cliffel, D. E.; Murray, R. W. J. Am. Chem. Soc. 1999, 121, 7081-7089. (20) Worden, J. G.; Shaffer, A. W.; Huo, Q. Chem. Commun. 2004, 518-519.
10.1021/cm048907+ CCC: $27.50 © 2004 American Chemical Society Published on Web 08/26/2004
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Scheme 1. Solid-Phase Synthesis of Gold Nanoparticles with a Single Carboxylic Group and Its Coupling Reaction with Alkyldiamine
nanoparticles. After cleaving off from the solid support, nanoparticles with a single carboxylic group were obtained as the major product. Following our pioneer work, Jacobson et al. published an almost identical approach toward the synthesis of gold nanoparticles with a single amino acid moiety.21 These nanoparticles with a single functional group attached can be treated as giant “molecules” and linked together into very sophisticated structures through traditional chemical reactions, just like the total synthesis of complicated natural product from small molecular units! Prior to our published work, there were two examples associated with the concept of nanoparticles with a single surface functional group. The first example is based on triphenylphosphine-protected gold nanoclusters reported by Hainfeld et al.22 This method is limited to gold nanoclusters with a diameter smaller than or around 1.4 nm. The functional ligand is attached to the nanoparticle with other nonfunctionalized ligands by controlling the stoichiometric ratio of these two different ligands during nanoparticle synthesis. However, this method is unlikely to be successful when applied to larger nanoparticles due to technical challenges. Furthermore, triphenylphosphine-protected gold nanoparticles have poor environmental and thermal stability, which limits the number of applications for which they may be used. In a second example, Alivisatos et al. reported the separation of gold nanoparticle-DNA conjugates with discrete numbers of DNA molecules by electrophoresis.23 However, this approach is not a general synthetic method to prepare gold nanoparticles with different organic functional groups in large quantities.
It has been argued and hypothesized that by strict stoichiometric control of incoming ligand ratio versus the nanoparticle-bound ligands, one may be able to attach a single functional group to the nanoparticle surface using the typical solution-phase place exchange reaction.16 We recently have conducted a comparison study on the solution-phase versus solid-phase place exchange reaction.24 It was found from solution-phase place exchange reaction that an even distribution of gold nanoparticles with one, two, three, and other discrete numbers of functional groups was obtained. The efficiency of solid-phase place exchange reaction is clearly much higher than the solution-phase place exchange reaction. However, as revealed in our preliminary study, there are still a significant number of obstacles that must be overcome before the solid-phase synthesis approach can become a versatile synthetic strategy for the controlled chemical functionalization of nanoparticle materials. Due to the large size, high molecular weight, and many other unique properties associated with nanoparticles compared to small organic and inorganic compounds, extensive modification of the solid-phase reaction conditions needs to be made to accommodate these properties. In this study, we explored systematically different aspects of solid-phase reactions, including the solid supports, bifunctional ligand loading levels, reaction temperatures, and solvents on the product yield and purity. Seven samples, AuNP1 to AuNP7, were prepared under different experimental conditions as summarized in Table 1. This study revealed the most appropriate conditions to obtain monofunctionalized gold nanoparticles with optimum yield and high purity.
(21) Soon, K.-M.; Mosley, D. W.; Peelle, B. R.; Zhang, S.; Jacobson, J. M. J. Am. Chem. Soc. 2004, 126, 5064-5065. (22) Hainfeld, J. F.; Powell R. D. J. Histochem. Cytochem. 2000, 48, 471-480. (23) (a) Zanchet, D.; Micheel, C. M.; Parak, W. J.; Gerion, D.; Alivisatos, A. P. Nano. Lett. 2001, 1, 32-35. (b) Zanchet, D.; Micheel, C. M.; Parak, W. J.; Gerion, D.; William, S. C.; Alivisatos, A. P. J. Phys. Chem. B 2002, 106, 11758-11763.
Experimental Section Chemicals, Solvents, and Materials. All solvents and organic chemicals (ACS reagents) were purchased from Aldrich (24) Shaffer, A. W.; Worden, J. G.; Huo, Q. Langmuir, published online Aug. 20, 2004, http://dx.doi.org/10.1021/la049308k.
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Table 1. Nanoparticle Samples Prepared in the Present Study experimental conditions
sample
solid support (functional group density)
AuNP1 1% DVB crossAuNP2 linked Wang resin (1.4 mmol/g) AuNP3 1% DVB crossAuNP4 linked Wang resin AuNP5 (1.0 mmol/g) AuNP6 2% cross-linked JandaJel resin (1.0 mmol/g) AuNP7 Whatman filter paper
OH:SHa 2:1 2:1 1:1 3:1 5:1 2:1 N/A
reaction solvent CH2Cl2 hexane/CH2Cl2 (2/1) hexane/CH2Cl2 (2/1) hexane/CH2Cl2 (2/1) CH2Cl2
% trimers, tetramers, and oligomersb 20-40 15-25 20-30 5-10 0-5 >60 0-5
a Ligand loading ratio. b Percentage of nanoparticles appearing as trimers, tetramers, and oligomers as determined by TEM analysis of the diamine-coupling product
(Milwaukee, WI) or VWR (West Chester, PA), except the following items. The gold salt HAuCl4‚xH2O was purchased from Strem Chemicals (Newburyport, MA). The acetylprotected 6-mercaptohexanoic acid was prepared according to the reported procedure.25 One percent divinylbenzene (DVB) cross-linked Wang resin with particle size around 100-200 mesh and a hydroxyl group density of 1.4-3.2 mmol/g was obtained from Advanced ChemTech (Louisville, KY). This resin swells 5-10 times in dichloromethane. One percent DVB crosslinked Wang resin with a hydroxyl group density of 1.0 mmol/g used in the study was purchased from AnaSpec Inc. (San Jose, CA). This resin has the same swellability as the Wang resin from Advanced ChemTech. Two percent 1-(4-(4-vinylphenoxy)butoxy)-4-vinylbenzene cross-linked JandaJel resin was purchased from Aldrich with a functional group density of 1.0 mmol/g. This resin is reported to swell two times better than 1% DVB cross-linked Wang resin in dichloromethane. Solidphase synthesis was conducted manually using a “homemade” shaking and vacuum manifold system. The Sephadex gel used in gel permeation chromatography (GPC) is a lipophilic dextrin gel LH-20 from Sigma with a separation limit of 7000 Da molecular weight. The gel was preincubated in the corresponding eluent solvent overnight prior use. The column used for nanoparticle purification has a length of 15 cm and a diameter of 1.5 cm. The C18 reverse phase TLC plates were purchased from VWR and had a pore size of 60 Å. The C18-modified silica gel for reverse phase chromatography (RPC) was purchased from Sorbent Technology (Atlanta, GA) with a pore size of 100 Å. Physical Characterization Methods. For TEM studies, approximately 1 µL of sample in appropriate solvents was placed on a 300 mesh Formvar-coated grid using an Eppendorf micropipet and immediately wicked off using filter paper. After allowing the sample to dry in air for 5-10 min, images were obtained using a JEOL 100CX transmission electron microscope at 80 keV. The TEM images were analyzed manually. When analyzing the nanoparticle monomer, dimer, trimer, and other oligomers in the diamine-coupled product, four or five areas with a size around 300 × 300 nm were randomly chosen from more than one TEM image. When choosing the analysis regions, one should avoid the solvent line areas and overly concentrated regions. The total number of nanoparticles that appeared in the whole analysis area was counted first. Then the total number of nanoparticles that appeared in trimers and oligomers was counted. The percentage of nanoparticles that appeared in trimers and oligomers was then calculated to estimate the purity of monofunctionalized nanoparticles in the product, which is summarized in Table 1 for all the samples. 1H NMR spectra were obtained on a Varian Mercury 300 MHz spectrometer using a line-broadening factor of 1 Hz and (25) Svedhem, S.; Hollander, C. A.; Shi, J.; Konradsson, P.; Lierberg, B.; Svensson, S. C. T. J. Org. Chem. 2001, 66, 4494-4503.
relaxation delay of 5 s. X-ray diffraction analysis of nanoparticle samples was performed with a Philips automated diffractometer (PW3040 multipurpose diffractometer). Samples were ground into a fine powder, pipetted onto a quartz slide as a slurry in hexane, and air-dried. Crystalline phases were identified and analyzed using an MDI Jade Software version 3.1. FT-IR spectra were acquired using a Nicolet Magna-IR 850 Series II spectrometer in transmission mode. Samples were prepared by dropping about 100 µL of sample in appropriate solvent onto a NaCl salt plate and allowing the solvent to evaporate off. The sample chamber was purged with nitrogen gas for 15 min prior to acquisition. Spectra were analyzed using OMNIC software version 5.1 and were baseline adjusted. 1. Solid-Phase Synthesis of AuNP1 to AuNP7. The synthesis of nanoparticle products is only summarized very briefly here. Detailed experimental conditions and procedures for the solid-phase synthesis and chromatographic purification of each sample are included in the Supporting Information. For AuNP1 to AuNP6, polystyrene resin, such as Wang resin or JandaJel resin with different functional group densities, was used as solid support. Briefly, acetyl-protected 6-mercaptohexanoic acid was coupled to the solid support using the standard procedure of loading the first amino acid to polymer resin.26,27 After deprotection of the acetyl group by either 3 M ammonia solution in dioxane/water (4/1) or 33% piperidine in DMF, the free thiol group was allowed to undergo place exchange reaction in different solvents with butanethiolprotected gold nanoparticles (BtAu), which were prepared according to the Schiffrin reaction.15 For different samples, the loading ratio of the bifunctional thiol ligands to the solid support and the solvents used for place exchange reaction were varied according to the conditions listed in Table 1. At the end of the place exchange reaction, the resin-bound nanoparticles were cleaved off using 20% trifluoroacetic acid (TFA) in dichloromethane. After blowing off the solvents with a N2 stream, the crude product was first cleaned by washing with copious amounts of petroleum ether and then further purified by GPC. Some samples were further analyzed and purified by C18 reverse phase chromatography (RPC). The eluting solvents used in the chromatography varied slightly from one sample to the other. See the Supporting Information for detailed conditions. The yield of each purified product varies from 1 to 10 mg per gram of polymer resin for different samples. The AuNP7 sample was prepared using Whatman #1 filter paper as the solid support. The loading of 6-mercaptohexanoic acid to the support was done under similar conditions as used in polymer resin-based synthesis. The deprotection of the acetyl group was achieved by suspending the filter paper in aqueous ammonia solution (0.5-1.0 M) for 3 min. The bound nanoparticle product was cleaved off from filter paper by bubbling anhydrous ammonia gas to the filter papers suspended in dichromethane and swirling for a few minutes. The crude product was purified by GPC. The yield of this product is less than 1 mg per gram of filter paper. 2. Coupling Reaction of Solid-Phase Synthesis Nanoparticle Product with Alkyldiamine. Each nanoparticle product was coupled together with ethylenediamine (EDA) or 1,7-heptanediamine (HDA) using either the N-hydroxysuccinimide activation method or an in-situ coupling method with diisopropylcarbodiimide (DIPCDI) in appropriate solvents. The coupling reaction was allowed to continue for 24-72 h. The alkyldiamine and the activation agents or coupling agents were used in large excess versus the nanoparticles to drive the reaction to completion. More detailed coupling conditions can be also found in the Supporting Information. The coupled nanoparticle products were subjected to TEM analysis without further purification. (26) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161-214. (27) Fields, G. B. Methods in Enzymology Volume 289: Solid-Phase Peptide Synthesis; Academic Press: New York, 1997.
Solid-Phase Synthesis of Modified Au Nanoparticles
Results and Discussion The solid-phase synthesis technique has become an extremely powerful tool for combinatorial library synthesis since it was first reported by Merrifield in 1963.28 The most notable advantage of solid phase versus solution phase synthesis is the largely simplified purification procedure and easy handling of multiple reaction vessels. Between reaction steps, the unreacted reagents and impurities can be easily filtered off from the reaction mixture, leaving the pure product attached to the solid support. Because of this advantage, solid-phase synthesis has become an essential tool in combinatorial chemistry research for accelerated drug discovery.29-31 As demonstrated in our preliminary study,20 solidphase synthesis can be used as an efficient tool to control the chemical functionalization of nanoparticle materials. However, much work must still be conducted before the full potential of such an approach can be demonstrated. Through our preliminary study, we found a few major challenges in solid-phase reactions involving nanoparticles. First, nanoparticles are much larger than typical organic and inorganic molecules used in standard solid-phase synthesis. The solubility and mobility of nanoparticles in solution is at least a few orders of magnitude lower than small molecules. Given the example of alkanethiolate-protected gold nanoparticles, the average solubility of these materials in organic solvent is typically around a few milligrams per mL, equivalent to 10-5 M, assuming a molecular weight of 60 000 g/mol for a gold nanoparticle with an average core diameter of 1.8 nm.15 This is a very low concentration for a typical organic reaction. Another important issue is the control of the functional group density of the solid support. The commonly used solid support for small molecular library synthesis is a slightly crosslinked polymer such as polystyrene resin. These resins are designed to have excellent swellability in reaction solvents to allow efficient loading of molecules to the support, so as to maximize the yield of the final product. However, such a high swellability is undesired in nanoparticle synthesis. As the swellability of the solid support increases, the control over the functional group density decreases, since the polymer chains become more mobile. Consequently, it will become more difficult to control the number of functional groups eventually attached to the nanoparticles surface. The following describes in detail how these problems affect the solidphase reaction of nanoparticles and how we obtained monofunctionalized nanoparticles with optimum yield and high purity by choosing appropriate solid supports and experimental conditions. 1. AuNP1: General Experimental Procedure and Conditions. The first polymer support that we chose for the solid phase modification of nanoparticles was 1% DVB cross-linked polystyrene Wang resin with a hydroxyl group density of 1.4 mmol/g. This is one of the (28) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149-2154. (29) Czatnik, A. W., DeWitt, S. H., Eds. A Practical Guide to Combinatorial Chemistry; American Chemical Society: Washington, DC, 1997. (30) Jung, G., Ed. Combinatorial Peptide and Nonpeptide Library; VCH: New York, 1996. (31) Chaiken, I. M.; Janda, K. D., Eds. Molecular Diversity and Combinatorial Chemistry; American Chemical Society: Washington, DC, 1996.
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most commonly used and studied supports for peptide and other organic molecule library synthesis. According to the swellability data of this polymer resin, there should be less than one -OH group per 10 nm3 volume of polymer beads.32 For a nanoparticle with a 2.8 nm diameter (including the core and the monolayer ligands), the volume of an individual nanoparticle is approximately 11.5 nm3. To ensure one-to-one place exchange reaction between immobilized thiol ligands and the nanoparticles, the functional group density was further lowered down by loading only 0.5 equiv of 6-mercaptohexanoic acid to the resin (Table 1). That brings the functional group density (now it is actually the thiol ligand density) further down to less than one functional group per 20 nm3 volume of polymer beads. Statistically such a functional group density of the polymer resin beads should be low enough to ensure one-to-one reaction between the resin-bound thiol ligands and the nanoparticles. The successful loading of 6-mercaptohexnoic acid to the Wang resin and the following deprotection of acetyl group were confirmed by testing with Ellman’s agent, 5,5′-dithiobisnitrobenzoic acid (DTNB). The deprotected Wang resin appeared as bright yellow in DTNB solution. Originally the deprotection was accomplished by using a 3 M ammonia solution in mixed solvent dioxane/water (4/1, v/v) for overnight. Later it was found that the deprotection could be completed in less than 30 min by 33% piperidine in DMF. Following the deprotection, the resin-bound thiol groups were then allowed to undergo place exchange reaction with BtAu nanoparticles with a core diameter around 1.8 nm. Dichloromethane was initially used as the solvent for the place exchange reaction, because the nanoparticles have fairly good solubility in this solvent and the polymer resin also has good swellability. It was originally assumed that with better swellability, the functional groups on the polymer resin would be further apart from each other, therefore, making the functional group density even lower. During the place exchange reaction, the nanoparticle solution became clearer and the polymer beads became darker. At room temperature, it takes 3-5 days for the place exchange reaction to complete. When the place exchange reaction was conducted at 40 °C, the beads turned dark black in about 8 h. According to this visual examination, the loading efficiency of nanoparticles to the solid beads is close to 100%. Higher temperature should be avoided because the nanoparticles become unstable at temperatures above 50 °C. After washing away the unattached nanoparticles, the resin-bound nanoparticles were cleaved off from the resin using a 20% trifluoroacetic acid solution in dichloromethane. During the cleavage reaction, approximately half the particles were collected back and the other half was always trapped within the beads. We have tested many solvents and solvent mixtures, but with no (32) Advanced ChemTech Handbook of Combinatorial & SolidPhase Organic Chemistry; Advanced ChemTech, Inc.: Louisville, KY, 1998; pp 100-101. The Wang resin (1% DVB cross-linked polystyrene) used in this study has a functionality (-OH group) of 1.4-3.2 mmol/g and density of 1.05 g/cm3. This resin swells about 5-10 times its dry volume in CH2Cl2. A swellability of 10 times was used for the calculation, since the solid-phase place exchange reaction was conducted at 40-45 °C. According to these numbers, it is calculated that there is approximately one hydroxyl group per 10 nm3 in CH2Cl2 solution.
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Figure 1. Cartoon illustrating how multiple thiol ligands may become attached to gold nanoparticles due to the flexible polymer chain.
success. It was hypothesized that the nanoparticles were trapped in the polymer resin beads by two possible mechanisms. The first possibility is that the nanoparticles entered into the nanoscale pores within the resin and could not diffuse out. However, this possibility was eliminated by a control experiment. In this experiment, polymer resin beads with no thiol ligands attached were incubated in BtAu nanoparticle solution under the same conditions used for solid-phase place exchange reaction. After 24-48 h of incubation time, the polymer beads were washed with dichloromethane and no particles were found to retain in the beads after washing. This experiment suggests that if nanoparticles were retained in the beads after incubation, the nanoparticle must have been covalently bound to the polymer resin. Therefore, we believe the second possibility is most appropriate in explaining the observed results, which is attributed to a cross-linking effect of the nanoparticles to the polymer resin. Since the polystyrene resin is only slightly cross-linked, the polymer chains still have a fairly large amount of flexibility in solution, as demonstrated by the 5-10 times swellability in dichloromethane. It is possible that thiol ligands from different sites of the flexible polymer chain are attached to the nanoparticle, as shown in Figure 1. When this occurs, the nanoparticles are covalently trapped inside the polymer beads and function as cross-linker to the polymer chains. Such a cross-linking reaction further lowered the swellability of the polymer resin, making the polymer resin less accessible to solvents and the reaction less likely to occur. After cleaving off from the beads, the nanoparticle solution was collected and concentrated en vacuo. The nanoparticle residual was then washed with a large amount of petroleum ether to eliminate unreacted thiol ligands and TFA residue. The petroleum ether solution was monitored by thin-layer chromatography until no organic ligands were visible under the UV lamp. However, NMR analysis of the product usually revealed the presence of a small percentage of free thiol ligands in the sample, as shown in Figure 2a. In the NMR spectrum, the two sets of sharp peaks, one triplet at 2.7 ppm and one quartet at 2.3 ppm, can be attributed to the free thiol ligand. The significantly broadened peak sets from 0.8 to 2.0 ppm are due to the nanoparticle-bound thiol ligands, including butanethiol
Figure 2. 1H NMR spectrum of gold nanoparticle crude product AuNP1 (a) and 1H NMR spectrum (b) and IR spectrum (c) of GPC-purified AuNP1.
and 6-mercaptohexanoic acid. More detailed results and discussion on the NMR and FT-IR study of solid-phase and solution-phase place exchanged nanoparticle product have been described in a separate report.24 The small percentage of free ligands was then further eliminated from the nanoparticle sample by GPC using a methanol/dichloromethane (1/9) solvent with a trace amount of trifluoroacetic acid (less than 0.1%). Indeed, after gel permeation chromatography, the final product AuNP1 is free of unattached thiol ligands, as indicated by the NMR spectrum in Figure 2b. A small triplet at 2.2 ppm is attributed to the trace amount of oxidized disulfide ligands, which appears in essentially all the spectra of gold nanoparticles. TEM and X-ray diffraction analysis of this clean product has shown that the nanoparticles have the same average core diameter of 1.8 nm as the original BtAu nanoparticles, indicating that the nanoparticle structure remains intact after the solid-phase reactions. In the NMR spectrum of the cleaned product shown in Figure 2b, a small broad peak at 1.8 ppm is of
Solid-Phase Synthesis of Modified Au Nanoparticles
particular interest for the product analysis. It is attributed to the R-protons from the 6-mercaptohexanoic acid ligands that are newly attached to the nanoparticles. In our previous work, we proposed an equation to calculate the amount of bifunctional ligands attached to nanoparticles based on the integration ratio between the R-proton peak from 6-mercaptohexnoiac acid versus all the other methylene and methyl protons from both butanethiol and 6-mercaptohexanoic acid ligands.24 Using this equation, we estimated that the monolayer of the solid-phase-modified nanoparticles contains less than 5% bifunctional ligands. Such a percentage indicates that only a few limited numbers of carboxylic groups were attached to the nanoparticle through solidphase synthesis. FT-IR analysis of the purified nanoparticle product also supports this result. The IR spectrum of the GPC-purified nanoparticle product as shown in Figure 2c exhibits a carbonyl stretching band at 1741 cm-1, indicative of successful attachment of 6-mercaptohexanoic acid to the nanoparticles. This high wavenumber of carbonyl stretching band also reveals the lack of hydrogen bonding between surface-bound carboxylic groups, suggesting that very few carboxylic groups were attached to each nanoparticle by solidphase place exchange reaction. Although both NMR and FT-IR analysis could give a semiquantitative estimation on the average percentage of functional groups attached to nanoparticles, they do not provide direct evidence of nanoparticles with single functional groups, because the sensitivity of 1H NMR spectroscopy is not sufficient for such quantitative analysis. For this reason, we designed a diaminecoupling reaction to confirm the presence of nanoparticles with single surface functional groups.20 It is assumed that if there is only one carboxylic group present on the nanoparticle surface, the coupling of two nanoparticles with one alkyl diamine molecule will lead to the formation of a nanoparticle dimer. If more than one functional group is present, then the coupling reaction will lead to the formation of nanoparticle trimers, tetramers, oligomers, and larger aggregates. By examining the number of nanoparticle oligomers versus dimers using a transmission electron microscope, the relative percentage of nanoparticles with single functional groups in the product should be obtained. Initially we used N-hydroxysuccinimide at the presence of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) to activate the carboxylic groups from the nanoparticles and a large excess of diamine (approximately 100-1000 equiv) for the coupling reaction. Later the activation agent was changed to diisopropycarbodiimide (DIPCDI), because it was noticed that EDC could cause nanoparticle ripening under acidic conditions. The TEM image of diamine-coupled AuNP1 product is shown in Figure 3a. From this image, a large amount of nanoparticle dimers can be clearly identified, indicating that the majority of the product is monofunctionalized nanoparticles. Nanoparticle trimers and larger aggregates are also present, which suggests that the product also contains multifunctionalized nanoparticles. In addition, individual nanoparticles were also found in the image. The chemical functionality of the individual nanoparticles is more complicated. These nanoparticles could be nonfunctionalized particles or func-
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Figure 3. TEM images of diamine-coupling product of AuNP1 (a) and nanoparticles functionalized from solution-phase place exchange reaction (b) Circles represent nanoparticles pairs and arrows indicate nanoparticles trimers.
tionalized nanoparticles not coupled with other particles. The possibility for these individual nanoparticles to be nonfunctionalized is unlikely, as confirmed by the control experiment discussed in previous section. From this control experiment, we concluded that if nanoparticles were retained in the beads after extensive washing, the nanoparticles must have been covalently attached to the polymer beads, which means the nanoparticles obtained after solid-phase synthesis contain at least one carboxylic group. However, obtaining the precise yield of monofunctionalized nanoparticles from TEM analysis of the coupling product is not as direct as it appears to be. Two major difficulties are present in the quantitative analysis: first, the individual nanoparticles that appeared in the TEM images could be monofunctionalized or multifunctionalized, and second, the nanoparticle trimers, tetramers, and oligomers could contain monofunctionalized nanoparticles as well. In addition, to increase the reactant concentration, a significantly imbalanced stoichiometric ratio of diamine versus nanoparticles was used in the coupling reaction. For this reason, it is impossible to obtain completely coupled nanoparticle product. However, statistically, it is always true that
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under the same coupling conditions, the smaller the amounts of nanoparticle trimers and oligomers that appeared in the coupling product, the fewer the multifunctionalized nanoparticles the sample should contain. Such a statistical analysis can give indirect evidence on the percentage of monofunctionalized nanoparticles in the solid-phase synthesis product. For AuNP1, approximately 20-40% nanoparticles were found in trimers and higher order oligomers (Table 1), indicating the presence of a fairly significant amount of multifunctionalized product in the sample. Even so, the solidphase place exchange reaction is still a much better method in controlled monofunctionalization of nanoparticles than solution-phase place exchange reaction, which resulted in an even distribution of nanoparticle monomers, dimers, trimers, and oligomers in the coupling product, as shown in Figure 3b. The percentage of nanoparticles that appeared as trimers and oligomers in this sample is more than 60%. Although monofunctionalized nanoparticles appeared as the major component in the solid-phase synthesis product, the percentage of multiple functional group modified nanoparticles from this sample is also quite high. Even after further lowering the loading level of the 6-mercaptohexanoic acid, there was still a significant amount of trimers and tetramers appearing in the coupled nanoparticle product. There is an obvious discrepancy between the calculated and the experimentally obtained results. This difference could be explained again by the flexibility of polymer chains in the swelled resin, as shown in Figure 1. The flexibility of the polymer chain allowed the thiol ligands from different polymer chains to move around and to be attached to the same nanoparticles, which lead to the formation of nanoparticles with multiple functional groups. To prevent this from occurring, the polymer resin should be rigid with minimal swelling. This is in contradiction to a conclusion made by Jacobson et al.21 that the higher swellability of the resin, the better the control of the number of functional groups attached to the nanoparticles. The experimental results discussed below will further support our conclusion. 2. AuNP2: Place Exchange Reaction Conducted in Mixed Hexane/Dichloromethane Solvents. For polymers, lower swellability means lower mobility of the polymer chains. The swellability of Wang resin in dichloromethane is approximately 5 times and the swellability in hexane is only 1.1.33 We surmised that if a mixed solvent of hexane and dichloromethane was used for the place exchange reaction, the swellability of the resin would decrease substantially, as would the mobility of the polymer chain. Using a mixed hexane/ dichloromethane (2/1, v/v) solvent for the place exchange reaction, we prepared a second nanoparticle product, AuNP2. It was noticed in the synthesis that a smaller amount of nanoparticles was trapped inside the polymer beads after cleavage reaction. TEM analysis of the diamine-coupled product of this nanoparticle sample (images included in Supporting Information) showed that the number of nanoparticle trimers, tetramers, and oligomers is significantly lower compared to the coupling product of AuNP1. More accurate counting of different (33) Do¨rwald, F. Z. Organic Synthesis on Solid Phase; Wiley-VCH: Weinheim, 2000.
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images gave the average percentage of nanoparticles appearing in trimers, tetramers, and oligomers of around 15-25% (Table 1). This comparison study confirmed our hypothesis that polymer chains should have minimum mobility to ensure good control over the functional group density of the polymer beads and oneto-one place exchange reaction between the immobilized thiol ligands and the nanoparticles in solution. 3. AuNP3 to AuNP5: The Ligand Loading Ratio Effect. Aside from controlling the swellability of the solid support, one can also vary the loading ratio of bifunctional thiol ligands to the polymer beads to control the functional group density. By loading a smaller amount of 6-mercaptohexanoic acid to the beads, the number of thiol groups available for place exchange reaction per unit volume will decrease accordingly. To examine this effect, we prepared three additional samples AuNP3, AuNP4, and AuNP5 by controlling the ligand loading ratio to 1:1, 3:1, and 5:1 (-OH groups from polymer beads versus the -SH groups from thiol ligands), respectively. Mixed hexane/dichloromethane (2/1) solvent was used again for the place exchange reaction for all three samples. During the chromatographic purification process, some noticeable differences were observed from these three samples. For the AuNP3 sample, a single band was eluted from the GPC column using 10% methanol/ dichloromethane with trace amounts of TFA (less than 0.1%). If TFA is not added to the solvent, the nanoparticles will be stuck in the column. Similar results were observed for AuNP1 and AuNP2 samples. For AuNP4 and AuNP5, two bands were eluted out of the column using 10% methanol/dichloromethane, with the first band eluted in less than 2 min and the second band eluted about 20 min later. For AuNP4, the second band is the major product, which accounts for 80% of the sample, and for AuNP5, the first band is the major product, which makes about 60% of the sample. The separation of AuNP4 and AuNP5 into two bands by GPC is quite interesting. GPC is also referred to as size exclusion chromatography, which means the sample is separated into fractions according to size effect. Larger molecules or entities will be eluted out of the column because of a shorter retention time, and vice versa. In our case, we examined the two bands of the product with TEM, and no size difference was found from the first band and the second band nanoparticles. This result suggests that some other mechanisms are involved in the separation process, which was revealed through further analysis of the sample by reverse phase chromatography (RPC). The RPC analysis of AuNP3 to AuNP5 was conducted on a C18 silica gel-coated TLC plate with a pore size of 6 nm using 20% methanol/dichloromethane as the developing solvent. For AuNP3, two bands appeared on the plate. The first band has an Rf value of essentially 1.0 with significant tailing, and the second band has an Rf value of almost 0. The second band can only become mobile after the addition of a trace amount of TFA in the developing solvent. For the first fraction product of AuNP4 and AuNP5 eluted from GPC column, a single spot was developed on the TLC plate with an Rf value close to 1.0, and for the second fraction from GPC column, the sample appeared on the plate with a
Solid-Phase Synthesis of Modified Au Nanoparticles
zero Rf value. This sample became mobile after the addition of a trace amount of TFA in the developing solvent. As a comparison, the original BtAu nanoparticle was also analyzed by reverse phase TLC and the sample appeared as a single spot on the TLC plate with an Rf value of 1.0 using the same developing solvent. Combining all these results, we believe that the first band eluted out of the GPC column with an Rf value of 1.0 on the reverse phase TLC plates is the nanoparticles with a single or a minimum number of carboxylic groups, while the second band from GPC column with an Rf value of zero on the TLC plate is mainly the multifunctionalized nanoparticles. Nanoparticles with multiple carboxylic groups tend to form hydrogenbonded aggregates. The silica gel used to prepare the reverse phase TLC plates has a pore size of 6 nm. That means anything significantly larger than 6 nm, such as the hydrogen-bonded nanoparticle aggregates, would be immobilized at the spotting point on the TLC plates or trapped in the GPC column. With the addition of a trace amount of TFA in the developing solvent, such a hydrogen-bonding network was destroyed and the nanoparticles were able to move along the solvent line on the TLC plates or elute out of the GPC column. This conclusion was further supported by the analysis of diamine-coupling product. The coupling product of AuNP3 (purified by GPC only) appeared to be quite similar to AuNP2, as revealed from TEM analysis (image included in the Supporting Information). The percentage of nanoparticles appearing in trimers and other oligomers is around 20-30%, indicating the presence of a significant amount of multifunctionalized nanoparticles in the product. For AuNP4 and AuNP5, while the coupling product of the first eluted band from GPC column remained in solution, a large amount of precipitates was formed in the coupling product of the second eluted band, suggesting that the second band is mainly multifunctionalized nanoparticles. The TEM images of coupled product from the first band of AuNP4 and AuNP5 are shown in parts a and b of Figure 4, respectively. Both images contain almost only nanoparticle pairs and individual nanoparticles, with no or a minimum number of trimers, tetramers, and other higher order oligomers. For AuNP4, the percentage of nanoparticles appearing in nanoparticle trimers, tetramers, and oligomers is 5-10%, and for AuNP5, this percentage is further decreased to less than 5% (Table 1). Because of this low percentage (or more precisely, low probability) of nanoparticle trimers and other higher order oligomers in both samples, it is reasonable to believe that all the individual particles appearing in the images are also monofunctionalized nanoparticles. This means that the purity of the monofunctionalized nanoparticles of these two purified samples is close to 9095%. Recalling that the relative quantity of the first band obtained from GPC for AuNP5 is much higher than AuNP4, it is very clear that with decreased ligand loading ratio, the yield of monofunctionalized nanoparticles is increased significantly. In the example of AuNP5, approximately 6 mg of purified monofunctionalized nanoparticles was obtained from 2 g of polymer resin. This enhanced yield and high purity has made the large-scale production of such nanoparticle materials feasible.
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Figure 4. TEM image of diamine-coupling product of AuNP4 (a) and AuNP5 (b).
It should be explained here why we were not able to obtain pure product from the other three samples, AuNP1 to AuNP3, even though there is a high percentage of monofunctionalized nanoparticles in those samples, as indicated by TEM and TLC analysis. When the sample contains a significantly large amount of multifunctionalized nanoparticles, all the nanoparticles, whether they are monofunctionalized or multifunctionalized, will aggregate together after being loaded into the chromatography column (both GPC and RPC). The sample could not be eluted out of the column until the addition of a trace amount of TFA to break the hydrogenbonding network. Only when the percentage of multifunctionalized nanoparticles in the sample is extremely small and most nanoparticles were not aggregated together, such as the case of AuNP4 and AuNP5, monofunctionalized particles could be separated from other products by the two chromatographic techniques as discussed here. 4. AuNP6: Solid-Phase Synthesis Using 2% CrossLinked JandaJel Resin. To further confirm our conclusion on the effect of polymer resin swellability on the nanoparticle functionalization, we have used another polymer resin, 2% cross-linked polystyrene JandaJel resin, to prepare AuNP6 sample by solid-phase reaction. Because the 1-(4-(4-vinylphenoxy)butoxy)-4-
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Figure 5. TEM image of diamine-coupling product of AuNP6 from JandaJel synthesis.
Figure 6. TEM image of diamine-coupling product of AuNP7 from Whatman filter paper synthesis.
vinylbenzene cross-linking molecule is rather flexible compared to divinylbenzene, it is known that this resin swells 2 times better than 1% DVB cross-linked Wang resin (product catalog information). According to our hypothesis, this resin will yield more multiple functionalized nanoparticles rather than the single functional group modified nanoparticles. Indeed, after the addition of ethylenediamine and DIPCDI to the AuNP6 nanoparticle solution, the majority of the nanoparticles precipitated out within a few hours. These aggregates were dissolved in mixed dichloromethane/methanol solution with the addition of a trace amount of TFA. TEM analysis of this sample clearly shows the presence of large nanoparticle oligomers and aggregates (Figure 5). The number of nanoparticles that appeared in nanoparticle trimers, tetramers, and oligomers is higher than 60%. 5. AuNP7: Solid-Phase Synthesis Using Whatman Filter Paper. Filter paper is made with polysaccharides and has been reported in some occasions for peptide and other combinatorial library synthesis.34,35 Compared to other solid supports, filter paper is of low cost and addressable combinatorial libraries can be made with a simple equipment setup. Using Whatman #1 filter paper as the solid support, we prepared another sample, AuNP7, by solid-phase place exchange reaction. Following the same procedure as loading amino acids on to filter paper,33,34 we loaded the bifunctional 6-mercaptohexnoic acid on to the Whatman filter paper. The acetyl group was then deprotected by diluted aqueous ammonia solution in a few minutes. The place exchange reaction between butanethiol-protected gold nanoparticles and the paper-bound thiol ligands was clearly observed from the darkening of the filter paper after a few hours of reaction. We also conducted a control experiment by incubating nanoparticles with unmodified filter paper in dichloromethane. The filter paper remained white after incubation. The attached nanoparticle product was cleaved off from the filter paper by purging ammonia gas in the dichloromethane solution of the filter paper. The nanoparticle product was
recovered after the same washing and purification cycle as used in other solid-phase synthesis products. During the GPC purification process of the obtained crude product, we noticed that only a single band was eluted from the column with a 10% methanol/dichloromethane solvent at a retention time of less than 2 min. The coupling product of this nanoparticle sample with an excessive amount of diamine gave nanoparticle dimers and individual nanoparticles as the major product, almost completely free of nanoparticle trimers and other oligomers, as shown in Figure 6. According to the probability of nanoparticle trimers and other oligomers appearing in the TEM image, the percentage of single functional group-modified nanoparticles is estimated to be above 95%. This result further confirms our previous conclusion that a better control on the monofunctionalization of nanoparticles will be achieved if a more rigid polymer is used as the solid support. Cellulose (filter paper) is a highly cross-linked polysaccharide with very little swellability in organic and aqueous solutions. Due to this low swellability, there is very little chance for the attachment of multiple ligands onto the surface of the nanoparticles from different polymer chains. The obtained nanoparticle product is strictly the result of a one-to-one place exchange reaction between nanoparticles and bifunctional thiol ligands. On the other hand, the low swellability of cellulose in both aqueous and organic solvents makes the number of active sites available for reaction extremely limited, which in turn results in an extremely low yield of the product compared to the products obtained from polymer resin, such as AuNP4 and AuNP5.
(34) Frank, R. D.; Do¨ring, R. Tetrahedron Lett. 1988, 44, 60316040. (35) Hudson, D. J. Comb. Chem. 1999, 1, 403-457.
Conclusion Through this systematic study, we succeeded in preparing monofunctional group modified gold nanoparticles with enhanced yield and high purity by choosing an appropriate solid support and fine-tuning experimental conditions. Clearly this is a superior synthetic strategy for the controlled monofunctionalization of nanoparticles compared to solution-phase place exchange reaction. The solid support is no doubt the most
Solid-Phase Synthesis of Modified Au Nanoparticles
critical part of the synthesis. If the solid support is too soft and the polymer chains are too flexible, a large percentage of nanoparticles with multiple functional groups will be produced. On the other hand, if the solid support is too rigid with low swellability, such as the case of cellulose filter paper, the loading efficiency of the nanoparticles to the solid support will decrease substantially. The macroporous polymer resin35 may be a much more appropriate candidate for nanoparticle reactions, because these polymer materials are highly rigid (with 10% cross-linking density), allowing good control over the functional group density, and also highly porous, providing sufficient reactive sites. Further studies along these lines are being conducted and will be reported in due course.
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Acknowledgment. This work is supported by the National Science Foundation CAREER award #DMR0239424, the NSF/North Dakota State EPSCoR, and the North Dakota State University Research Foundation. We also thank Scott Payne of the USDA Electron Microscopy Laboratory for assistance in TEM analysis. Supporting Information Available: Detailed experimental conditions used in the synthesis and purification of each nanoparticle samples, diamine-coupling reaction conditions, and full-scale TEM images of each sample. This material is available free of charge via the Internet at http://pubs.acs.org. CM048907+
