Ethylene Glycol Monolayer Protected Nanoparticles: Synthesis

Improved Methodology for the Preparation of Water-Soluble Maleimide-Functionalized Small Gold Nanoparticles. Pierangelo Gobbo and Mark S. Workentin...
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Ethylene Glycol Monolayer Protected Nanoparticles: Synthesis, Characterization, and Interactions with Biological Molecules† Ming Zheng, Zhigang Li, and Xueying Huang* Central Research and Development, Du Pont, Experimental Station, Wilmington, Delaware 19880 Received October 23, 2003. In Final Form: February 24, 2004 The usefulness of the hybrid materials of nanoparticles and biological molecules on many occasions depends on how well one can achieve a rational design based on specific binding and programmable assembly. Nonspecific binding between nanoparticles and biomolecules is one of the major barriers for achieving their utilities in a biological system. In this paper, we demonstrate a new approach to eliminate nonspecific interactions between nanoparticles and biological molecules by shielding the nanoparticle with a monolayer of ethylene glycol. A direct synthesis of di-, tri-, and tetra(ethylene glycol)-protected gold nanoparticles (Au-S-EGn, n ) 2, 3, and 4) was achieved under the condition that the water content was optimized in the range of 9-18% in the reaction mixture. With controlled ratio of [HAuCl4]/[EGn-SH] at 2, the synthesized particles have an average diameter of 3.5 nm and a surface plasma resonance band around 510 nm. Their surface structures were confirmed by 1H NMR spectra. These gold nanoparticles are bonded with a uniform monolayer with defined lengths of 0.8, 1.2, and 1.6 nm for Au-S-EG2, AuS-EG3, and Au-S-EG4, respectively. They have great stabilities in aqueous solutions with a high concentration of electrolytes as well as in organic solvents. Thermogravimetric analysis revealed that the ethylene glycol monolayer coating is ca. 14% of the total nanoparticle weight. Biological binding tests by using ion-exchange chromatography and gel electrophoresis demonstrated that these Au-S-EGn (n ) 2, 3, or 4) nanoparticles are free of any nonspecific bindings with various proteins, DNA, and RNA. These types of nanoparticles provide a fundamental starting material for designing hybrid materials composed of metallic nanoparticles and biomolecules.

Introduction Nanomaterials and their hybrid with biological molecules are recognized to have potential applications in electronics, optics, genomics, proteomics, and biomedical, and bioanalytical areas.1-6 As one of the most important class of nanomaterials, nanoparticles include metallic particles, such as Au, Ag, Cu, semiconducting particles, such as CdSe, ZnSe, and insulating particles, such as SiO2, TiO2. The utilization of these materials in biological applications depends on how well rational design can be achieved based on the specific binding between inorganic nanoparticles and biological molecules. To enable programmable assembly, a nanoparticle has to meet a few basic requirements: (1) It should be soluble in aqueous solution. (2) It should be stable without aggregation or agglomeration in the application media, such as highly concentrated electrolyte solutions. (3) The nonspecific interactions between the nanoparticle and biological entities should be eliminated. (4) It should be able to provide function or specific interaction with the target biological molecules. To meet all these criteria, the key is the surface property and the surface modification of the nanoparticle. Alkanethiolate monolayer protected metallic nanopar* Corresponding author. E-mail: [email protected]. † Publication No. 8473. (1) Kreibig, U.; Vollmer, M. Optical Properties of Metal Clusters; Springer Series in Material Science, No. 25; Springer-Verlag: Berlin, 1995; pp 187-201. (2) Motesharei, K.; Myles, D. C. J. Am. Chem. Soc. 1994, 116, 7413. (3) Mirkin, C. A.; Taton, T. A. Nature 2000, 405, 626-627. (4) Sastry, M.; Lala, N.; Patil, V.; Chavan, S. P.; Chittiboyina, A. G. Langmuir 1998, 14, 4138. (5) Fitzmaurice, D.; Connolly, S. Adv. Mater. 1999, 11, 1202-1205. (6) Mann, S.; Shenton, W.; Li, M.; Connolly, S.; Fitzmaurice, D. Adv. Mater. 2000, 12, 147-150.

ticles are the most widely studied system.7-10 However, the alkyl chains have strong hydrophobic binding with biological molecules, which is one type of nonspecific interaction. In addition, alkanethiolate-protected nanoparticles are not soluble in water, which makes them incompatible with biological molecules that require an aqueous environment for activity. Most of water-soluble nanoparticles reported in the literature are coated with charged ligands, which introduce another type of nonspecific binding with biomolecules through electrostatic interaction.11-14 The nonspecific interaction between nanoparticles and biological molecules is a fundamental issue that is not well addressed in the published literature.15-16 Poly(ethylene glycol) (PEG) has been widely studied and used as a biocompatible material that has good resistance to nonspecific bindings with biological mole(7) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. J. Chem. Soc., Chem. Commun. 1994, 801. (8) Brust, M.; Fink, J.; Bethell, D.; Schiffrin, D. J.; Kiely, C. J. J. Chem. Soc., Chem. Commun. 1995, 1655. (9) Hostetler, M. J.; Wingate, J. E.; Zhong, C.; 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. (10) Terrill, R. H.; Postlethwaite, T. A.; Chen, C.-H.; Poon, C.-D.; Terzis, A.; Chen, A.; Hutchison, J. E.; Clark, M. R.; Wignall, G.; Londono, J. D.; Superfine, R.; Falvo, M.; Johnson, C. S., Jr.; Samulski, E. T.; Murray, R. W. J. Am. Chem. Soc. 1995, 117, 12537. (11) Templeton, A. C.; Chen, S.; Gross, S. M.; Murray, R. W. Langmuir 1999, 15, 66. (12) Schaaff, T. G.; Knight, G. K.; Shafigullin, M. N.; Borkman, R. F.; Whetten, R. L. J. Phys. Chem. B 1998, 102, 10643. (13) Templeton, A. C.; Cliffel D. E.; Murray, R. W. J. Am. Chem. Soc. 1999, 121, 7081. (14) Chen, S.; Kimura, K. Langmuir 1999, 15, 1075. (15) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. Science 2002, 298, 1759-1762. (16) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016.

10.1021/la035981i CCC: $27.50 © 2004 American Chemical Society Published on Web 04/10/2004

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Figure 1. Gold nanoparticles are coated with (a) a monolayer of well-defined small molecules and a ligand, (b) polymer coils and a ligand, and (c) polymer coils and a ligand attached to a polymer coil.

cules.17-18 Small ethylene glycol molecules are also watersoluble and neutral in charge and have been demonstrated to resist protein binding when assembled on a flat gold surface.19-21 All of this previously accumulated knowledge has been naturally applied to nanoparticles. However, the synthesis of an ethylene glycol monolayer on a flat gold surface is very different from that on nanoparticles. A direct synthesis of PEG-protected gold nanoparticles has been developed and explored for biological applications.22-23 Very recently a thioalkylated oligo(ethylene glycol) ligand protected gold nanoparticle was directly synthesized in alcoholic solution.24 The monolayer with the structure of -S(CH2)11(OCH2CH2)4OH is composed of a hydrophobic block and a hydrophilic block. In our previous communication,25 we reported a direct synthesis of gold nanoparticles protected with small ethylene glycol molecules with defined lengths. These ethylene glycol monolayers on the nanoparticle surface eliminated the nonspecific interactions of nanoparticles with proteins. Small ethylene glycol molecule protected gold nanoparticles differ from PEG-protected gold nanoparticles in several aspects. First, small ethylene glycol molecules, HS(CH2CH2O)nCH3 (n ) 2, 3, and 4) have well-defined lengths, which are 8.5, 11.9, and 16 Å for di-, tri-, and tetra(ethylene glycol), respectively.26 PEG chains, on the other hand, form random coils on the nanoparticle surface. The conformation change of the polymer chain is reflected by its stretching or retracting, which depends on the polarity of the solvents. It is hard to predict the thickness when it is bonded on the nanoparticle surface. Second, small ethylene glycol molecules can form a densely packed monolayer on the surface of nanoparticles. A PEG polymer forms a loose, random structure on the surface. A cartoon comparing these surface structures is shown in Figure 1. Third, for further applications, such as programmable assembly with biological molecules, a mixed monolayer composed of an ethylene glycol molecule with well defined length and a functional ligand can be easily prepared and the ligand is readily accessible to target molecules. For a (17) Harris, J. M.; Zalipsky, S. Poly(ethylene glycol): Chemistry and Biological Applications; American Chemical Society: Washington, DC, 1997. (18) Deible, C. R.; Petrosko, P.; Johnson, P. C.; Beckman, E. J.; Russell, A. J.; Wagner, W. R. Biomaterials 1998, 19, 1885. (19) Prime, K. L.; Whitesides G. M. J. Am. Chem. Soc. 1993, 115, 10714-10721. (20) Roberts, C.; Chen, C. S.; Mrksich, M.; Martichonok, V.; Ingber, D. E.; Whitesides, G. W. J. Am. Chem. Soc. 1998, 120 (26), 6548-6555. (21) Zhang, M.; Desai, T.; and Ferrari, M. Biomaterials 1998, 19, 953-960. (22) Wuelfing, W. P.; Gross, S. M.; Miles, D. T.; Murray, R. W. J. Am. Chem. Soc. 1998, 120, 12696. (23) Otsuka, H.; Akiyama, Y.; Nagasaki, Y.; Kataoka, K. J. Am. Chem. Soc. 2001, 123, 8226. (24) Kanaras, A. K.; Kamounah, F. S.; Schaumburg, K.; Kiely, C. J.; Brust, M. J. Chem. Soc., Chem. Commun. 2002, 2294. (25) Zheng, M.; Davidson, F.; Huang, X. J. Am. Chem. Soc. 2003, 125, 7790. (26) The chain length data are calculated from the condition that these molecules are densely packed and fully stretched on the nanoparticle surface.

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polymer coil surface, a ligand bonded on nanoparticles could be buried or blocked in the coils. In order for the ligand to function properly, it has to be attached to the polymer coil, for example at the end of a polymer chain, as illustrated in Figure 1c. It is difficult for this structured material to be used for the specific and programmable assembly with biological molecules. A better surface structure for achieving the specific assembly with biological entities should be a mixed monolayer of ethylene glycol with well-defined length and a ligand molecule, as shown in Figure 1a. The ethylene glycol short chain as the major component functions as the background to minimize the nonspecific interaction between nanoparticles and biological molecules, whereas the ligand molecule as the minor component serves as a ligand to engage specifically with a target, such as a protein or cell. Therefore, a short chain ethylene glycol with a well-defined length is preferred to a long polymer chain for coating nanoparticles. In this paper, we extended our previous study on the direct synthesis of tetra(ethylene glycol)-protected gold nanoparticles by further investigation of the detailed synthesis of Au-S-EGn (n ) 2, 3, and 4) (Structures of free ethylene glycol molecules are shown below), their characterization and interactions with biological molecules.

Results and Discussion Synthesis of EG2-SH, EG3-SH, and EG4-SH. The starting material for synthesizing diethylene glycol thiol (EG2-SH) is 1-bromo-2-(2-methoxyethoxy)ethane. After refluxing with thiourea in ethanol, the bromo group was converted to an isothiourionium bromide and then to a -SNa group after a second refluxing with sodium hydroxide. Neutralization with dilute HCl generated diethylene glycol thiol. Distillation of the extracted EG2-SH produced pure compound with 57% yield. The pure EG2SH compound is colorless and has the typical thiol compound odor. Its structure was confirmed by 1H NMR spectrum. The synthesis of EG3-SH and EG4-SH was started with tri- and tetra(ethylene glycol)monomethyl ether, as shown in Scheme 1. In each case, the hydroxyl group was converted to the bromo group after adding phosphorus tribromide (PBr3). Since this step of reaction is exothermic, special care had to be taken when adding PBr3 into the reaction. The PBr3 solution needed to be added dropwise into the reaction solution which sat in a cold bath. If PBr3 was added too fast, the reaction could be so violent in local areas that EGn-OH (n ) 3 and 4) molecules could break into fragments. This side reaction may lead to byproducts with two thiol groups at the ends or a thiol group at one end and a hydoxyl group at the other end, which could not be easily removed by distillation due to their similar boiling points. These byproducts, though quite difficult to identify from 1H NMR spectra, will greatly interfere with the synthesis of the gold nanoparticles, resulting in aggregations in the process of the synthesis. After bromination of tri- and tetra(ethylene glycol)monomethyl ether, the remaining steps of the synthesis were the same as those of the synthesis of EG2SH: two steps of refluxing with thiourea and sodium hydroxide, followed by extraction, evaporation, and distil-

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lation. The final products EG3-SH and EG4-SH from distillation were pure, as confirmed by NMR spectra. Both EG3-SH and EG4-SH have the typical smell of thiol compounds. EG3-SH is colorless and EG4-SH is light yellow. With one more ethylene glycol unit, EG4-SH has higher solubility in water. This resulted in a higher loss of the final product in water during ether extraction; as a consequence, the yield of EG4-SH is quite low, only 28%. Synthesis of Ethylene Glycol Protected Gold Nanoparticles [Au-EGn (n ) 2, 3, or 4)]. In order for nanoparticles to be used in the biological assembly, the problem of nonspecific binding has to be overcome. Otherwise, the nanoparticles would not have any selectivity and specificity for biological entities. Due to the known fact that ethylene glycol materials have good resistance to protein nonspecific bindings as well as the desire for a defined thickness for a coating on the nanoparticle surface, small ethylene glycol molecules were the first choice for coating nanoparticles. A synthesis of short ethylene oxide chain protected gold nanoparticles by ligand exchange reaction was reported.27 The procedure involved synthesis of hexanethiol (C6) protected gold nanoparticle followed by two steps to replace C6 with ethylene glycol thiol molecules (EGn-SH, n ) 2, 3, and 4). Also utilizing the replacement reaction, we synthesized Au-S-EGn (n ) 2, 3, and 4) nanoparticles by starting with the synthesis of the tiopronin-protected gold nano-

particle11 and then followed by the ligand replacement reaction twice with a 20 times excess of EGn-SH (n ) 2, 3, and 4) each time. The replacement reaction was conducted in several different solvents, such as ethanol, water, THF, or a water/THF mixture. The experimental results showed that the Au-S-EGn (n ) 2, 3, or 4) nanoparticles indirectly synthesized by the replacement reaction with the Au-Tp nanoparticle could no longer be redissolved in water after centrifuging purification and lyophilization drying, and aggregation was seen after storage in aqueous solution for several weeks. In addition, the replacement reaction is tedious and difficult for further reactions to introduce a ligand for specific binding and assembly. Considering the practical utilities, we explored a direct synthesis method. First, we directly synthesized tetra(ethylene glycol)protected gold nanoparticle in water. In the aqueous phase, Au-S-EG4 could only be synthesized with a low molar ratio of reducing agent (NaBH4) and tetrachloroauric acid, for example 1:1. At the low ratio of [NaBH4]/[HAuCl4], the reduction reaction was not complete. With increasing [NaBH4]/[HAuCl4] ratio, the reaction mixture became more and more aggregated. So, the direct synthesis in aqueous phase results in very low yield, less than 10%. A similar problem was also observed by others.28 Then, a mixed solvent of methanol and water used by Brust et al. and Murray et al.7,11 was employed for direct synthesis of di-, tri-, and tetra(ethylene glycol) monolayer protected gold nanoparticles (Au-S-EGn, n ) 2, 3, and 4). The synthesis failed many times before it was discovered that the water content plays a key role. It was found that controlling the addition of water into the reaction mixture was critical.25 When the water content was either higher than 18% or lower than 9%, the reaction mixtures began to form agglomerates. When the water content was optimized in the range of 9%-18% (v/v), stable and water-soluble nanoparticles were formed. The reaction did not generate aggregates even though the reducing agent NaBH4 was 10 times (molar ratio) more prevalent than HAuCl4. The yield was much higher than the synthesis in aqueous solution, usually in the range of 4050%. It was also noticed that the reaction mixture became aggregated if the reagents contained a very small amount of impurity, such as HS-(CH2CH2O)n-OH. When diluted, these directly synthesized ethylene glycol protected gold nanoparticles became red-purple and clear. The directly synthesized ethylene glycol gold nanoparticles are very stable in aqueous solution, even with a high concentration of salt, for example, 1.0 M NaCl. After 12 month of storage in aqueous solution, no agglomeration was seen. After drying in a lyophilizer for a week, they can be readily redissolved in water to form a red-purple, clear solution. All three nanoparticles, Au-S-EGn (n ) 2, 3, and 4), are soluble and stable in water. It has been noticed that dried Au-S-EG2 sometimes did not redisperse in water. For comparison, Au-S-EG2 synthesized by ligand replacement reaction was reported to be insoluble in water.27 This suggests that ethylene glycol protected nanoparticles prepared by the direct synthesis and by the ligand replacement reaction behave somehow differently. In addition, all Au-S-EGn (n ) 2, 3, or 4) nanoparticles have very good solubility in most organic solvents, such as acetone, methanol, DMSO, chloroform, DMF, and THF. Solvent Effect on Direct Synthesis of Au-S-EGn (n ) 2, 3, or 4) Nanoparticles. We also synthesized AuS-EGn (n ) 2, 3, and 4) using methanol as the solvent.

(27) Foos, E. E.; Snow, A. W.; Twigg, M. E.; Ancona, M. G. Chem. Mater. 2002, 14(5), 2401-2408.

(28) Bartz, M.; Kuther, J.; Nelles, G.; Weber, N.; Seshadri, R.; Tremel, W. J. Mater. Chem. 1999, 9, 1121.

Scheme 1. Synthesis of Triethylene Glycol Thiol Molecule (EG3-SH).

Ethylene Glycol Monolayer Protected Nanoparticles

Reagents of HAuCl4 and EG3-SH, for example, were dissolved in a mixture of methanol and acetic acid and then a freshly prepared NaBH4 solution in methanol was added into the mixture with rapid stirring. As soon as the NaBH4 methanol solution was added, Au-S-EG3 nanoparticles were formed. After purifying and drying, the nanoparticle is readily redissolved in water to form a clear, red-purple solution. However the yield is very low, less than 10%. No precipitation was observed in the reaction mixture, meaning that the reduction reaction of HAuCl4 was not complete. The mechanism of the partial reduction in methanol solvent is not investigated in this paper. A literature survey showed that the choice of the solvent is critical for the synthesis of water-soluble nanoparticles. Even though the study on the mechanism of the solvent effect on the synthesis of water-soluble nanoparticles is beyond the scope of this paper, a summary of literature findings should be beneficial to future studies on this issue. The ligands for capping the metallic nanoparticles can be classified into three categories: strongly ionic, weakly ionic, and neutral molecules. For strongly ionic ligands, water as the single solvent is sufficient for synthesizing water-soluble nanoparticles. Examples include the synthesis of gold nanoparticles capped with coenzyme A,11 N,N-trimethyl(undecylmercapto)ammonium,29 5-mercapto2-benzimidazolesulfonic acid sodium salt,30 and the zwitterionic ligand cysteine.31 For weakly ionic ligands, a mixture of water and a water-miscible organic solvent, such as methanol, is used for the synthesis. Those examples include the synthesis of the gold nanoparticles protected with tiopronin,11 glutathione,12 and mercaptosuccinic acid.14 In the specific example of synthesis of tiopronin-protected gold nanoparticle, using water as the only solvent leads to a water-insoluble product.11 For the synthesis of GSH-protected gold nanoparticle, a 2:3 water: methanol medium is used to prevent an uncontrolled reduction reaction.12 We hypothesized that the ratio of water and the organic solvent in the mixed solvent system is related to the structure and the polarity of the ligand. A quantitative determination of this ratio could be very important and extremely useful, even though it is beyond of the investigation in this paper. Neutral ligands for coating nanoparticles include ethylene glycol small molecules and polymers. With the increase of the units of the ethylene glycol from 2 to 4 to 70,23 the synthesis of the nanoparticles could be achieved in a solvent from a mixture of water and methanol to pure water. Apparently, the solubility of the ligand molecules plays a big role in choosing the right solvent for the synthesis. Although the mechanistic reasons for this correlation are not clear, this summary should nevertheless provide a useful guidance for the synthesis of new types of water-soluble nanoparticles. 1 H NMR Spectroscopy. Parts a and b of Figure 2 show the NMR spectrum for the free EG3-SH molecule and the Au-S-EG3 nanoparticle, respectively. In Figure 2b, disappearance of the proton signal from -SH (t, 1.59 ppm) of free EG3-SH clearly supported that the gold nanoparticle is bonded with the -S-EG3 monolayer. More evidence is that a double triplet peak for -S-CH2- from free EG3-SH became a single triplet peak after the thiol group was bonded onto the gold nanoparticle. The chemical shift of -S-CH2- moved from 2.70 ppm in the free (29) Cliffel, D. E.; Zamborini, F. P.; Gross, S. M.; Murray, R. W. Langmuir 2000, 16, 9699. (30) Li, X.-M.; Paraschiv, V.; Huskens, J.; Reinhoudt, D. N. J. Am. Chem. Soc. 2003, 125, 4279. (31) Naka, K.; Itoh, H.; Tampo, Y.; Chujo, Y. Langmuir 2003, 19, 5546.

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EG3-SH up to 2.84 ppm for the bonded Au-S-CH2-. This can be explained by the fact that the electron density of the sulfur atom in Au-S-CH2- is decreased compared with that in free EG3-SH. The chemical shifts and the integrals of the remaining protons confirmed that the bonded triethylene glycol molecule have the same structure as the free one. In the same way, the structure of Au-S-EG4 was confirmed by the 1H NMR spectra of Au-S-EG4 and free EG4-SH.25 For the Au-S-EG2 nanoparticle, the drying by lyophilization could make the particle aggregated sometimes. Its 1H NMR measurement was not recorded. The 1H NMR spectra of both Au-SEG3 and Au-S-EG4 showed a small quantity of water, even though the nanoparticle samples were dried by lyophilization for a week and the DMSO-d6 was treated with molecular sieves dried by baking at 500 °C. It was very difficult to remove the trace water inside the AuS-EGn nanoparticle. This could be due to the huge surface area of the nanoparticles and the good water solubility of the ethylene glycol coatings. Transmission Electron Microscopy (TEM). Highresolution transmission electron microscopy (HRTEM) was used to provide the information on the shape, size, and the size distribution of the nanoparticles. Parts a, b, and c of Figure 3 show the images and the particle size distribution of Au-S-EG2, Au-S-EG3, and Au-S-EG4, respectively. Figure 3b also shows the atomic resolution image of a single Au-S-EG3 nanoparticle, indicating that the particle is a single crystal and the core shape is a truncated polyhedron. Under the synthetic condition that the molar ratio of [EG3-SH]/[AuCl4-] was controlled at 0.5, the average gold core size is 3.5, 3.5, and 3.3 nm for Au-S-EG2, Au-S-EG3, and Au-S-EG4 nanoparticles, respectively. Au-S-EG2 nanoparticles have broader core size distribution than those of Au-S-EG3 and Au-SEG4. This could be due to the limited solubility of diethylene glycol, which has the tendency to cause the aggregation of Au-S-EG2 nanoparticles in water. The gold core size of Au-S-EGn (n ) 2, 3, and 4) nanoparticles can be roughly tuned by adjusting the ratio of [EGn-SH]/ [AuCl4-]. The general trend is that the gold core size increases upon decreasing the ratio of [EGn-SH]/[AuCl4-], which is similar to what is seen for other ligand-protected gold nanoparticles.11 Thermogravimetric Analysis (TGA). It is welldocumented that Au-S bond is not stable at high temperature. The organic monolayer on the gold nanoparticle decomposes thermally, resulting in metallic gold with the release of volatile disulfides. TGA has been conveniently used to monitor the decomposition temperature and acquire the organic composition in monolayerprotected nanoparticles. Figure 4 shows the weight loss of Au-S-EG2, Au-S-EG3, and Au-S-EG4 under thermal decomposition up to 500 °C. The decomposition onset temperature depends on how it is defined as well as many other factors, including purge gas, heating rate, and sample geometry (i.e., exposed surface area-to-volume ratio). In this study, it is defined as the temperature at which 0.5% weight loss is reached. Under this definition, the onset of decomposition for Au-S-EG2, Au-S-EG3, and Au-S-EG4 is 150, 175, and 205 °C, respectively. With the increase of the chain length of ethylene glycol, the monolayer bonded on the gold nanoparticle is thermally more stable. Each additional ethylene glycol unit roughly increases by 25 °C the onset of the decomposition temperature. Foos et al. reported the decomposition temperature at 148, 183, and 190 °C for Au-S-EG2, AuS-EG3, and Au-S-EG4 prepared by replacement reaction, respectively.27 The variation of the decomposition

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Figure 2.

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H NMR spectra of (a) free EG3-SH and (b) Au-S-EG3 nanoparticle.

1

temperature could be caused by the different synthesis approach as well as the definition of the decomposition temperature. In comparison with the alkanethiolate monolayer protected gold nanoparticles with the same number of atoms on the ligand chain, the decomposition temperature of Au-S-EGn is much lower. For example, the decomposition temperature of Au-S-C6H13, Au-SC8H17, and Au-S-C12H23 is 200, 230, and 260 °C, respectively.27 The reason for the lower decomposition temperature could be the presence of the electronwithdrawing oxygen atom on the chain, which lowers the electron density of the sulfur atom and weakens the Au-S bond. The lower decomposition temperature of Au-SEGn might be of great interest for some special applications, such as printable electronics. These water-soluble nanoparticles can be printed on the surface and converted to conductive lines or thin films by heating. The organic composition of the Au-S-EGn (n ) 2, 3, and 4) is ca. 14% or, more specifically, 14.8%, 13.5%, and 14.2% for AuS-EG2, Au-S-EG3, and Au-S-EG4, respectively. For the simplicity of the calculation, the core shape of the

nanoparticles is assumed to be spherical. The ligand number on each of the nanoparticles is calculated to be 335, 227, and 162 for Au-S-EG2, Au-S-EG3, and AuS-EG4, respectively. The average surface area occupied by one ligand is 0.12, 0.17, and 0.24 nm2 for EG2, EG3, and EG4, respectively. These are the very crude values. However, they point out that, with the increase of the molecular weight, the footprint is also increased. For comparison, the literature reported 0.21 nm2 for the surface area footprint of a linear structured molecule, dodecanethiol,32 on flat gold surface and 0.12 nm2 for the mercaptosuccinic acid bonded on the gold nanoparticle.33 UV-Vis Spectroscopy. The optical response of a nanoparticle is closely related with its electromagnetic property.1 The size of the nanoparticle plays a critical role in its optical response. UV-visible spectroscopy has been widely used to characterize the relationship between the (32) Sellars, H.; Ulman, A.; Schnidman, Y.; Eilers, J. E. J. Am. Chem. Soc. 1993, 115, 9389. (33) Yao, H.; Momozawa, O.; Hamatani, T.; Kimura, K. Chem. Mater. 2001, 13, 4692.

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Figure 3. TEM images of nanoparticles prepared by drop-casting aqueous solution onto the carbon grid: (a) Au-S-EG2 with an average particle size of 3.5 nm counted from 493 particles; (b) Au-S-EG3 an the average particle size of 3.5 nm counted from 262 particles; and (c) Au-S-EG4 an the average particle size of 3.4 nm counted from 179 particles. The upper right corner of Figure 3b is the atomic resolution image of a single Au-S-EG3 nanoparticle.

particle size and its optical properties.34,35 Metallic nanoparticles with diameter larger than 3 nm typically show a broad surface plasma (SP) band around 520 nm, and the SP intensity increases with the nanoparticle size up to ∼4.4 nm.9 Figure 5 shows the UV-vis spectra of AuS-EG2, Au-S-EG3, and Au-S-EG4 in an aqueous phase. (34) Whetten, R. L.; Khoury, J. T.; Alvarez, M. M.; Murthy, S.; Vezmar, I.; Wang, Z. L.; Stephen, P. W.; Cleveland, C. L.; Luedtke, W. D.; Landman, U. Adv. Mater. 1996, 5, 428-433. (35) Schaaff, T. G.; Shafigullin, M. N.; Khoury, J. T.; Vezmar, I.; Whetten, R. L.; Cullen, W.; First, P. N.; Gutierrez-Wing, C.; Ascensio, J.; Jose-Yacaman, M. J. Phys. Chem. B 1997, 101, 7885.

The peak wavelength of the SP band is about 510 nm for the three ethylene glycol protected gold nanoparticles, which is shorter than that of most of the monolayer protected gold nanoparticles reported in the literature, for example 518 nm for dodecanethiolate-protected gold nanoparticles in hexane solvent,9 522 nm for tioproninprotected gold nanoparticles in aqueous solution,11 and 530 nm for mercaptosuccinic acid protected gold nanoparticles in aqueous solution.14 The reason for the shift of λmax of the SP band for ethylene glycol protected nanoparticles is not studied in this paper. It is well-known

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Figure 4. Weight loss of (a) Au-S-EG2, (b) Au-S-EG3, and (c) Au-S-EG4 nanoparticles is monitored as a function of temperature up to 500 °C under dry nitrogen purge.

Figure 5. UV-vis spectra of (a) Au-S-EG2, (b) Au-S-EG3, and (c) Au-S-EG4 nanoparticles in aqueous solution.

that the SP band is sensitive to the bonded ligand and the solvent. It is noticed from Figure 5 that the intensity of the SP band for Au-S-EG2 nanoparticles is lower than that for Au-S-EG3 and Au-S-EG4, even though the average core size of these three nanoparticles are similar, which is about 3.5 nm in diameter. It is speculated that this is related with (1) the bonding ligand and (2) the inhomogeneity of particle size distribution. Gel Electrophoresis. Protein binding to Au nanoparticles can be conveniently monitored by gel electrophoresis, since protein-nanoparticle complexes are expected to migrate differently than the free Au particles. Protein-binding reactions were done by mixing 10 µL of ∼50 µM Au particles with 1.0 µL of 10 mg/mL protein in sodium phosphate buffer solution (50 mM, pH 7.3). After incubation at room temperature for 10 min, the entire reaction mixture was loaded on a 1% agarose gel. Five proteins were chosen for the binding test: lysozyme, cytochrome C, bovine serum albumin (BSA), ribonuclease A, and myoglobin. These are representative of positive and negatively charged proteins at neutral pH as well as

Figure 6. Gel image of nanoparticles binding with (a) lysozyme, (b) BSA, (c) DNA, and (d) RNA. Lanes 1, 3, 5, and 7 are Au-Tp, Au-S-EG2, Au-S-EG3, and Au-S-EG4 nanoparticles, respectively. Lanes 2, 4, 6, and 8 are the mixtures of protein, DNA, or RNA with Au-Tp, Au-S-EG2, Au-S-EG3, and AuS-EG4 nanoparticles, respectively.

of various degrees of hydrophobicity. DNA and RNA were also chosen for testing the nonspecific binding of AuS-EGn (n ) 2, 3, and 4) nanoparticles. For comparison, tiopronin-protected gold nanoparticle (Au-Tp), a water soluble nanoparticle, was synthesized and tested for biological binding. The results of nanoparticle binding with lysozyme, BSA, Escherichia coli chromosomal DNA, and total RNA are shown in parts a, b, c, and d, repectively, of Figure 6. Lanes 1, 3, 5, and 7 are 10 µL of ∼50 µM Au-Tp, Au-S-EG2, Au-S-EG3, and Au-S-EG4, respectively. Lanes 2, 4, 6, and 8 are the same amount of Au particles mixed with 1.0 µL of 10 mg/mL lysozyme (Figure 6a), BSA (Figure 6b), DNA (Figure 6c), or RNA sample (Figure 6d) in sodium phosphate buffer (50 mM, pH 7.3), respectively. When Au-Tp particles were mixed with lysozyme and BSA, different degrees of nonspecific binding were observed, as indicated by the band shifts. When the same experiment was done with Au-EGn (n ) 2, 3, and 4) nanoparticles, no band shift was observed, indicating no binding. The most dramatic contrast was

Ethylene Glycol Monolayer Protected Nanoparticles

Figure 7. Gel image of cell extract binding assay with the particles: Au-S-EG2 (a), Au-S-EG3 (b), and Au-S-EG4 (c). Lane 1, 10 µL of Au-S-EGn (n ) 2, 3 or 4) particles at ∼100 µM; Lane 2-5, the same amount of Au particles mixed with 5 µL of the prepared E. coli cell extract (∼108 cells), 10 µg of total RNA (1 µL), 10 µg of chromosomal DNA (1 µL), and 1 µL of 50 mM (final concentration) of GSH, respectively.

provided by the binding between Au-Tp particles and lysozyme. Addition of lysozyme into the Au-Tp particle solution causes an immediate color change from pinkish red to blue, indicating that Au particles formed aggregates, presumably because the positively changed lysozyme (pI ) 11) molecules cross-linked negatively charged Au-Tp particles (-COOH). However, when lysozyme was mixed with Au-S-EGn (n ) 2, 3, and 4) solutions, no color change was observed. Neither Au-Tp nor Au-S-EGn (n ) 2, 3, and 4) nanoparticles bind with the DNA and RNA tested. Since both DNA and RNA molecules are negatively charged, it is reasonable that the negatively charged AuTp does not have binding with them. Binding tests with other proteins, cytochrome C, ribonuclease A, and myoglobin, showed the same results: Au-S-EGn (n ) 2, 3, and 4) nanoparticles do not have any nonspecific interaction, while Au-Tp nanoparticle has various degree of binding. We further performed a binding test with cell extract, as shown in Figure 7. The E. coli cell extract was incubated with Au-S-EGn (n ) 2, 3, and 4) for 10 min at the room temperature, and then the mixture was loaded on 1% agorose gel. It was found that Au nanoparticles incubated with the cell extract migrated toward positive electrode, as shown in lane 2 in Figure 7a-c. We hypothesize that the observed effect of the cell extract is due to the exchange reaction that happened between the EGnS- (n ) 2, 3, and 4) molecules on the surface of the particles and the glutathione (GSH) and other molecules with thiol groups presented in the cell extract, since it is known that a high concentration (∼mM) of GSH or similar small thiol molecules are found ubiquitously in all types of cells. To test the hypothesis, we examined the effect of pure GSH molecule on the mobility of the nanoparticles. In lane 5, Au-S-EGn (n ) 2, 3, and 4) nanoparticles mixed with GSH solution indeed migrated to positive electrode. However, it is interesting to notice that the migration speed of nanoparticles in GSH solution was in the order of Au-S-EG2 > Au-S-EG3 > Au-S-EG4. This result suggests that the replacement with GSH is fastest for Au-S-EG2 and slowest for Au-S-EG4, which means that the gold nanoparticle protected with a longer chain on the surface is more stable than that with a short chain. This is due to the bulky effect of the densely packed longer chain that slows down the penetration of a glutathione or other thiol molecules to the gold surface to conduct a replacement reaction. If we look at the nanoparticle migration order in the mixture with the cell lysate in lane 2, it does not show the same trend as that in the mixture with GSH in lane 5. This indicates that the effect of cell lysate is probably more complex than a simple replacement reaction with GSH. It is conceivable that gold nanoparticles resume nonspecific binding with a variety of proteins in the cell lysate after some of the ethylene glycol molecules were replaced by GSH or other small thiol molecules. This presents a general problem for any nanoparticle that use

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thiols to form the protection monolayer. The solution to such a problem is very important for the nanoparticles to be practically utilized in the biological cells. We are working on the issue right now. Ion Exchange Chromatography Test. Ion exchange chromatography analysis of protein binding to Au-SEGn (n ) 2, 3, and 4) and Au-Tp nanoparticles was carried out using a BioCAD/SPRINT HPLC system (PerSeptive Biosystems, Framingham, MA). Both a cation exchange column (HS20) and an anion exchange column (HQ20) (PerSeptive Biosystems) were used to test the binding of the nanoparticles. For cation exchange separation, all the nanoparticles [Au-S-EGn (n ) 2, 3, and 4) and Au-Tp] eluted at void volume. Lysozyme was retained due to ionexchange interaction between the positively charged protein and the negatively charged resin. For anion exchange separation, all three nanoparticles [Au-S-EGn (n ) 2, 3, and 4)] were eluted at the void volume, while Au-Tp nanoparticles were retained. The retention of AuTp nanoparticles is due to the ion-exchange interaction between the carboxylic acid groups (-COOH) on Au-Tp and the positively charged anion resin. Au-S-EGn (n ) 2, 3, and 4) nanoparticles were eluted at the void volume from both cation and anion exchange columns, indicating that the surface of Au-EG4 particles is charge neutral and does not bind to either column. This is consistent with the results of Au-S-EGn (n ) 2, 3, and 4) particles’ migration in agarose gel. Conclusion In conclusion, we discovered that with the water content optimized in the range of 9-18% in the reaction mixture, di-, tri-, and tetra(ethylene glycol)-protected gold nanoparticles Au-S-EGn (n ) 2, 3, and 4) could be directly synthesized. When the feeding ratio of [HAuCl4]/[EGnSH] is controlled at 2, the particle size of Au-S-EGn (n ) 2, 3, and 4) is about 3.5 nm in diameter and the surface organic monolayer takes ca. 14% weight. These gold nanoparticles are bonded with a uniform monolayer with a defined length varying from 0.8 to 1.6 nm. They have great stability in aqueous, electrolyte, and organic solutions. These nanoparticles are less thermally stable than the corresponding alkanethiolate-protected nanoparticles with the same number of atoms on the backbone. The lower thermal stability could be an advantage for converting the complex to a metal for formation of a thin metallic film. Its potential application includes printable electronics. Ion-exchange chromatography and gel electrophoresis experiments demonstrated that these AuS-EGn (n ) 2, 3 or 4) nanoparticles have neutral and hydrophilic surfaces and are completely resistant to nonspecific interactions or bindings with proteins, DNA, and RNA. These types of nanoparticles provide a fundamental starting material for designing hybrid materials composed of metallic nanoparticles and biomolecules. One of the advantages for the direct synthesis method is that it allows the synthesis of a mixed monolayer of ethylene glycol and a functional ligand to achieve elimination of biological nonspecific interaction, providing specific interaction at the same time. From the aspect of industrial applications, these biologically inert gold nanoparticles from the direct synthesis will potentially have commercial utilities in electronic, sensor, and biomedical applications. Some of those works are under way. Experimental Section Chemicals and Materials. If not mentioned, all the reagents were purchased from Aldrich and used without further purification.

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Synthesis of Diethylene Glycol Thiol (EG2-SH). 1-Bromo2-(2-methoxyethoxy)ethane (90%, 10.0 g) and thiourea (99%, 8.3 g) were added into a 250 mL dried round-bottom flask. Then 80 mL of ethanol (99.9%) was added into the flask. The mixture was refluxed for 6 h. After the mixture was cooled to room temperature, EtOH was removed by rotary evaporation. Then 150 g of 20% NaOH was added and the mixture refluxed for 3 h. The mixture was cooled to the room temperature and poured to a 500 mL beaker. HCl (15%, prepared from the concentrated HCl) was slowly added into the mixture with stirring until the pH reached 2. The mixture was extracted four times with 200 mL of ether each time. The liquid in the ether phase was collected in a 1000 mL beaker and the ether phase was extracted with 200 mL of deionized water to further remove the salt and other impurities. The ether was removed by rotary evaporation and the crude product was distilled at 41-42 °C under 1.2 mmHg pressure. The final product was colorless and weighed 6.0 g, giving a yield of 57%. The structure was confirmed by NMR measurements. 1H NMR (500 MHz, CDCl3) δ: 1.60-1.65 (t, 1H), 2.70-2.80 (m, 2H), 3.45 (s, 3H), 3.55-3.75 (m, 6H). Synthesis of Triethylene Glycol Thiol (EG3-SH). As shown in Scheme 1, a typical example is the following. Phosphorus tribromide (21.92 g, 81.0 mmol) (PBr3, 99%) in 50 mL of dry CH2Cl2 was slowly added to a stirred mixture of 20.0 g (122.0 mol) of tri(ethylene glycol) monomethyl ether (98%, Alfa Aesar), 30 mL of dry CH2Cl2, and 7.0 mL of pyridine in a 250 mL round-bottom flask at 0 °C. The reaction mixture became cloudy and generated gas as soon as the PBr3 solution was added. The resulting mixture was stirred at room temperature for 16 h. After reaction, the mixture was extracted successively with 50 mL of saturated sodium carbonate aqueous solution, 50 mL of 5% sulfuric acid, and 2 × 30 mL of deionized water. The organic phase was further dried over anhydrous magnesium sulfate and the solvent was removed by rotary evaporation. The crude product was further used for synthesis of EG3-SH. The following procedures were the same as that for the synthesis of EG2-SH. The yield was ∼34%. The structure was confirmed by NMR spectra. 1H NMR (500 MHz, CDCl3) δ: 1.56-1.62 (t, 1H), 2.662.74 (dt, 2H), 3.38 (s, 3H), 3.54-3.58 (m, 2H), 3.60-3.68 (m, 8H). Synthesis of Tetraethylene Glycol Thiol (EG4-SH). One example of the synthesis of tetraethylene glycol thiol (EG4-SH) is briefly described here. Phosphorus tribromide (99%, 9.0 g, 0.033 mol) was slowly added to a stirred mixture of 10.0 g (0.048 mole) of tetra(ethylene glycol) monomethyl ether (98%, Alfa Aesar) and 2.0 g of pyridine at 0 °C. The resulting mixture was stirred at room temperature for 16 h. Then 10 mL of deionized water was added. The mixture was extracted three times with 40 mL of carbon tetrachloride for each. The combined organic extracts were rinsed successively with 25 mL of 10% sodium carbonate aqueous solution, 5% sulfuric acid, and deionized water and further dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation. The crude product, CH3O(CH2CH2O)3CH2CH2Br (5.432 g, ∼0.021 mmol), thiourea (99%, 3.05 g, 40.0 mmol), and 45 mL of ethanol (99.9%) were mixed in a 100 mL round-bottom flask and refluxed for 6 h. After the mixture was cooled to room temperature, EtOH was removed by rotary evaporation. Then 45.0 g of 20% NaOH was added and the mixture refluxed for 3 h. The mixture was cooled to the room temperature and acidified with 15% HCl until the pH reached 2.0. The mixture was extracted four times with 50 mL of ether for each. The ether phase was extracted with 50 mL of deionized water to further remove the salt and other impurities. The ether was removed by rotary evaporation and the crude product was distilled to give the final product (EG4-SH) with a yield of 28%. 1H NMR (500 MHz, CDCl ) δ: 1.50-1.55(t, 1H), 2.60-2.65 (dt, 3 2H), 3.31(s, 3H), 3.45-3.50 (m, 2H) and 3.53-3.62 (m, 12H). Direct Synthesis of Au-S-EG2, Au-S-EG3, and AuS-EG4 in a Medium of Water and Methanol. In a typical synthesis of Au-S-EG2, 30 mL of MeOH (HPLC grade) and 5.0 mL of acetic acid (HPLC grade) were mixed in a 150 mL Erlenmeyer flask by stirring for 2-5 min. Then, 78.0 mg (0.2 mmol) of tetrachloroauric acid (HAuCl4‚xH2O) (99.99%) and 13.6 mg (0.1 mmol) of diethylene glycol thiol (EG2-SH) were added to the above mixed solvents and dissolved by stirring for 5 min, which gave a clear, yellow solution. Next, 75.0 mg (2.0 mmol) of sodium borohydride (NaBH4, 99%) was dissolved in 5.0 mL of

Zheng et al. Nanopure water. The NaBH4 solution was dropwise added into the above solution with rapid stirring. With the first drop of NaBH4 added, the HAuCl4 solution immediately turned to dark brown from yellow. Rapid stirring was continued for 2 h. The reaction mixture was transferred into a 50 mL plastic centrifuging tube and centrifuged at 2500 rpm for 30 s to remove large particles, if there were any. The supernatant was then transferred into 15 mL filter tubes with 30K MW cutoff (Centriplus YM-30, Millipore), purified by centrifuging at 3500 rpm and washing with Nanopure water four times, and then dried in a lyophilizer for 2 days. In a typical synthesis of Au-S-EG3, 45 mL of methanol (HPLC grade) and 7.5 mL of acetic acid (HPLC grade) were mixed in a 150 mL Erlenmeyer flask by stirring for 2-5 min. Then, 0.236 g (0.6 mmol) of tetrachloroauric acid (HAuCl4‚xH2O) (99.99%) and 54.0 mg (0.3 mmol) of triethylene glycol thiol (EG3-SH) were added to the above mixed solvents and dissolved by stirring for 5 min, which gave a clear, yellow solution. Next, 0.225 g (6.0 mmol) of sodium borohydride (NaBH4, 99%) was dissolved in 7.5 mL of Nanopure water. The NaBH4 solution was dropwise added into the above solution with rapid stirring. With the first drop of NaBH4 added, the HAuCl4 solution immediately turned to dark brown from yellow. Rapid stirring was continued for 2 h. The purification method is the same as that for Au-S-EG2. In a typical synthesis of Au-S-EG4, 45 mL of methanol (HPLC grade) and 5.0 mL of acetic acid (HPLC grade) were mixed in a 150 mL Erlenmeyer flask by stirring for 2-5 min. Then, 0.236 g (0.6 mmol) of tetrachloroauric acid (HAuCl4‚xH2O) (99.99%) and 67.2 mg (0.3 mmol) of tetraethylene glycol thiol (EG4-SH) were added to the above mixed solvents and dissolved by stirring for 5 min, which gave a clear, yellow solution. Next, 0.225 g (6.0 mmol) of sodium borohydride (NaBH4, 99%) was dissolved in 5.0 mL of Nanopure water. The NaBH4 solution was dropwise added into the above solution with rapid stirring. With the first drop of NaBH4 added, the HAuCl4 solution immediately turned to dark brown from yellow. Rapid stirring was continued for 2 h. The purification method is the same as that for Au-S-EG2. Direct Synthesis of Au-S-EG3 in Methanol. In a typical synthesis of Au-S-EG3, 30 mL of methanol (HPLC grade) and 5.0 mL of acetic acid (HPLC grade) were mixed in a 150 mL Erlenmeyer flask by stirring for 2-5 min. Then, 0.118 g (0.3 mmol) of tetrachloroauric acid (HAuCl4‚xH2O) (99.99%) and 27.0 mg (0.15 mmol) of triethylene glycol thiol (EG3-SH) were added to the above mixed solvents and dissolved by stirring for 5 min, which gave a clear, yellow solution. Next, 0.113 g (3.0 mmol) of sodium borohydride (NaBH4, 99%) was dissolved in 5.0 mL methanol. The NaBH4 methanol solution was dropwise added into the above solution with rapid stirring. With the first drop of NaBH4 added, the HAuCl4 solution immediately turned to dark brown from yellow. Rapid stirring was continued for 2 h. The purification method is the same as that in the above. Synthesis of Tiopronin Monolayer Protected Gold Nanoparticles (Au-Tp). The detailed procedure is described in the literature.11 In a typical synthesis, 60 mL of methanol (HPLC grade) and 10 mL of acetic acid (HPLC grade) were mixed in a 250 mL Erlenmeyer flask by stirring for 2-5 min. Then, 0.39 g (1.0 mmol) of tetrachloroauric acid (HAuCl4‚xH2O) (99.99%) and 16.3 mg (0.1 mmol) of N-(2-mercaptopropionyl)glycine (Tiopronin, 99%) were added to the above mixed solvents and dissolved by stirring for 5 min, which gave a clear, yellow solution. Next, 0.6 g (16.0 mmol) of sodium borohydride (NaBH4, 99%) was dissolved in 30 mL of Nanopure water. The NaBH4 solution was dropwise added into the above solution with rapid stirring. With the first drop of NaBH4 added, the HAuCl4 solution immediately turned to dark brown from yellow. Rapid stirring was kept for 2 h. Synthesized Au-Tp nanoparticles were transferred into a 15 mL filter tube with 30K MW cutoff (Centriplus YM-30, Millipore), purified by centrifuging and washing with Nanopure water four times, and then dried in the lyophilizer for 3 days. The average particle size of Au-Tp nanoparticles is 3.5 nm, as measured by TEM. Synthesis of Di- and Tetra(ethylene glycol)-Protected Nanoparticles (Au-S-EG2 and Au-S-EG4) by Replacement Reactions. A typical example is given here. 5.0 mL of aqueous Au-Tp nanoparticle (20.0 mg, ∼5.0 × 10-3 mmol of tiopronin) was mixed with 0.75 mL of ethanol containing 30.0

Ethylene Glycol Monolayer Protected Nanoparticles mg of EG2-SH (0.22 mmol, ∼40 times of tiopronin on gold nanoparticle surface) or 50.0 mg of EG4-SH (0.22 mmol, ∼40 times of tiopronin) in a 25 mL round-bottom flask. After the mixture was stirred for 24 h at room temperature, the nanoparticles were purified either by dialysis or centrifuging. For dialysis, the reaction mixture was transferred to a 10 cm long membrane tubing (Spectrum, MWCO 3500). The membrane was then stirred in 2 L of Nanopure water for 24 h with the water changed five times. The nanoparticles could also be purified by centrifugation. The reaction mixture was transferred into a 15 mL filter tube with a 30K MW cutoff (Millipore) and purified by centrifuging and washing with Nanopure water four times. The purified nanoparticles underwent a second replacement reaction with the same reaction conditions and purification as the first time and were then dried in the lyophilizer for 3 days. 1H NMR Spectroscopy. 1H NMR spectra of free EG -SH (n n ) 2, 3, and 4) were recorded with a Bruker 500 MHz at room temperature in CCl3D. 1H NMR spectra of Au-S-EG3 and AuS-EG4 nanoparticles were recorded with a Varian Inova 400 MHz at room temperature in DMSO-d6. The sample was run with a one-pulse experiment using a 90° pulse with a 20 s delay. The baseline was flattened by using a spline baseline corrector. 1H NMR (Au-S-EG ) δ: 1.14 (s) and 1.24 (s) from DMSO 4 impurity; 2.32(s), 2.5(s), and 2.68(s) from DMSO; 2.90 (t, 2H from -Au-S-CH2-); 3.22 (s, 3H from -OCH3); 3.3 (s, broad) from H2O; 3.43 (m, 2H from Au-S-CH2CH2-) and 3.5 (m, 10H from -OCH2CH2O-) and 3.64 (t, 2H from -CH2OCH3). 1NMR (Au-S-EG3) δ: 2.28 (s), 2.44 (s), and 2.658 (s) from DMSO-d6; 2.80-2.85 (t, 2H from Au-S-CH2-); 3.17 (s, 3H from -OCH3); 3.22 (s, broad) from H2O; 3.34-3.38 (m, 2H from Au-SCH2CH2-) and 3.42-3.48 (m, 6H from -OCH2CH2O-) and 3.543.60 (t, 2H from -CH2OCH3). Transmission Electron Microscopy (TEM). The samples were prepared by dropping nanoparticle aqueous solution by a pipet onto a carbon-coated Cu grid substrate until water evaporated. The nanoparticle images were obtained by JEOL2011 transmission electron microscope operating at 200 kV with the resolution better than 0.18 nm. TEM images of Au-EG2, Au-EG3, and Au-EG4 nanoparticles are shown in Figure 3. UV-Vis Spectroscopy. UV-vis spectra were taken with a Varian Cary UV-vis spectrometer. A 100 µL sample of 0.1 mg/ mL nanoparticle in water was removed into a 1 cm quartz cell by a pipet. The wavelength was scanned from 200 to 700 nm with the resolution of 1 nm. Thermogravimetric Analysis (TGA). Thermogravimetric analysis (TGA) was performed with a TA Instruments, Inc. Model 2950 TGA system. From 8 to 25 mg of dry nanoparticle sample was used for the analysis. The reversible moisture uptake/loss (between 0% and ∼67% RH), the underlying weight loss (after 1600 min), and the weight loss at 500 °C were recorded. For each sample, the weight change was monitored at 30 °C while the purge gas (100 mL/min) was switched from dry to ∼67% RH and back to dry. Then, the weight was monitored as a function of temperature up to 500 °C when the sample was heated at 10 °C/min under 100 mL/min dry nitrogen purge.

Langmuir, Vol. 20, No. 10, 2004 4235 Gel Electrophoresis Analysis of Protein Binding to AuEG4. Protein binding reaction was done by mixing 10 µL of ∼50 µM Au particles with 1.0 µL of 10 mg/mL protein in sodium phosphate buffer solution (50 mM, pH 7.3). After incubation at room temperature for 10 min, the entire reaction mixture was loaded on 1% agarose gel. Gel electrophoresis was run in 1X TBE buffer (Tris borate-EDTA) at 90 V constant voltage for 20 min. Gel pictures were taken by directly scanning the gel on a HP ScanJet 6300C. Cell Extract Binding Assay. Bacterial E. coli wild type cell MG1655 was chosen for this assay. Cell extract was prepared as following. A single colony, picked from an agar plate previously streaked with cells, was used to inoculate 5 mL of LB rich medium. Cells were allowed to grow overnight at 37 °C to the stationary phase. The overnight culture was then diluted (1:100) into 25 mL LB rich medium, and was allowed to grow to OD600 ) 0.2. The pellet from 6 mL of cell culture collected by centrifugation was washed and resuspended in 50 µL water in a 1.5 mL Eppendorf tube. Cell membrane was broken by three freezeand-thaw cycles. Cell extract was then collected after centrifugation to remove cell membrane debris and other nonsoluble components. Total RNA and chromosomal DNA from the E. coli cells were purified following standard molecular biology protocols. The binding assay was done by mixing the indicated amount of Au-EG4 particles with cell extract, RNA, DNA, and GSH solutions at room temperature. After 10 min of incubation, the total volume of mixtures were subject to gel-electrophoresis, following procedures described in previous experiments. Ion Exchange Chromatography. Ion exchange chromatography analysis of protein binding to Au-EG4 and Au-Tp particles was carried out using BioCAD/SPRINT HPLC system (PerSeptive Biosystems, Framingham, MA) with cation exchange column HS20 (PerSeptive Biosystems). In all cases, 0.1 M MES (2-(N-morpholino)ethanesulfonic acid) buffer (pH 7) with 0-2 M NaSCN salt gradient were used, with a flow rate of 10 mL/min. In a typical run, 0.3 mL of either Au-EG4 or Au-Tp (concentration ∼2 µM) in NaH2PO4(43 mM)/K2HPO4 (14.7 mM) buffer (pH ) 7.3) was mixed with an equal volume of 1 mg/mL protein or the phosphate buffer, incubated at room temperature for 2 h. The reaction mixture was centrifuged at 13 000 rpm in an Eppendorf 5415C for 10 min, loaded on to the column, and then eluted. For anion exchange, 1 mL of a Au-EG4 and Au-Tp mixture (∼1:5) at a concentration of ∼1 µM was loaded onto a HQ20 column and eluted with 0.1 M MES buffer at pH 7 and a salt gradient from 0 to 2 M NaSCN.

Acknowledgment. This work comes from the Du Pont CR&D Bioelectronics group. The authors thank Mr. Kevin Sheran for some of the synthesis work, Dr. Fredric Davidson and Mr. Steven F. Krakowski for their support with 1H NMR spectroscopy, Dr. John Coburn for the thermal analysis, and Mr. Ray Richardson for the UV-vis spectroscopy. LA035981I