Clickable Gold Nanoparticles as the Building Block of Nanobioprobes

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Clickable Gold Nanoparticles as the Building Block of Nanobioprobes Ming-Xi Zhang, Bi-Hai Huang, Xiao-Yu Sun, and Dai-Wen Pang* Key Laboratory of Analytical Chemistry for Biology and Medicine, Ministry of Education, College of Chemistry and Molecular Sciences, and State Key Laboratory of Virology, Wuhan University, Wuhan 430072, PR China Received January 22, 2010. Revised Manuscript Received April 5, 2010 A new method of fabricating “clickable” gold nanoparticles that could be used as the building block of nanobioprobes was described. On the basis of a well-developed strategy of encapsulating hydrophobic nanoparticles with a layer of amphiphilic polymers, cheap, easily prepared graft polymer was used as a modifier to prepare monodisperse azidefunctionalized gold nanoparticles (AuNPs), which showed good stability in physiological solution. By conjugation with alkyne functional horseradish peroxidase (HRP) via click chemistry under mild conditions, the azide-AuNPs have demonstrated their potential in the fabrication of stable, bioactive nanobioprobes. Some critical problems in the fabrication of nanobioprobes, such as how to detect the number of bound biomolecules on nanoparticles and evaluate the bioactivities of nanobioprobes, are discussed in detail.

Introduction In the past decade, gold nanoparticles, quantum dots, and magnetic nanoparticles have greatly activated the development of bioscience as a powerful new tool with unique physical and chemical properties.1 In general, high-quality nanoparticles with a uniform size were hydrophobic and were synthesized in the organic phase.2-6 Therefore, the surface modification and functionalization of nanoparticles have become the bottleneck in transferring nanoparticles from the organic to the aqueous phase for biosystems. Surface-modification strategies can be classified as the following: (1) ligand exchange with hydrophilic ligands with functional groups via coordination7-10 and (2) encapsulation by a layer of amphiphilic micelles or polymers.11-13 Both of them have their advantages and disadvantages. Ligand exchange was easy to carry out, but it was very hard to obtain the degree of substitution of hydrophilic ligands on the surface and the coordination of hydrophilic ligands with inorganic cores was relatively weak. Nanoparticles modified via the second strategy utilizing multiple hydrophobic interactions of amphiphilic ligands with original surface hydrophobic ligands had good stability in aqueous solution, but the layer-by-layer assembly resulted in large hydrodynamic radii of nanoparticles, which limited their applications under certain conditions. *Corresponding author. E-mail: [email protected]. (1) Rosi, N. L.; Mirkin, C. A. Chem. Rev. 2005, 105, 1547. (2) Hiramatsu, H.; Osterloh, F. E. Chem. Mater. 2004, 16, 2509. (3) Peng, Z. A.; Peng, X. J. Am. Chem. Soc. 2001, 123, 183. (4) Sun, S.; Zeng, H. J. Am. Chem. Soc. 2002, 124, 8204. (5) Jana, N. R.; Peng, X. J. Am. Chem. Soc. 2003, 125, 14280. (6) Jana, N. R.; Gearheart, L.; Murphy, C. J. Langmuir 2001, 17, 6782. (7) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016. (8) Xu, C.; Xu, K.; Gu, H.; Zheng, R.; Liu, H.; Zhang, X.; Guo, Z.; Xu, B. J. Am. Chem. Soc. 2004, 126, 9938. (9) De, M.; You, C.-C.; Srivastava, S.; Rotello, V. M. J. Am. Chem. Soc. 2007, 129, 10747. (10) Palma, R. D.; Peeters, S.; Bael, M. J. V.; Rul, H. V. d.; Bonroy, K.; Laureyn, W.; Mullens, J.; Borghs, G.; Maes, G. Chem. Mater. 2007, 19, 1821. (11) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. Science 2002, 298, 1759. (12) Fan, H.; Leve, E. W.; Gabaldon, C. S. J.; Tallant, D.; Boyle, S. B. T.; Wilson, M. C.; Brinker, C. J. Nano Lett. 2005, 5, 645. (13) Grancharov, S. G.; Zeng, H.; Sun, S.; Wang, S. X.; O’Brien, S.; Murray, C. B.; Kirtley, J. R.; Held, G. A. J. Phys. Chem. B 2005, 109, 13030.

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At present, the most widely used way to functionalize nanoparticles is to conjugate biomolecules to nanoparticles via amide bonds mediated by a kind of carbodiimide activator: N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC).14 However, EDC chemistry was not suitable for nanoparticles that were less stable at relatively high EDC and salt concentrations in aqueous solution. How to rationally design high-quality, stable bioconjugates that integrate the activities of biomolecules and the intrinsic properties of nanoparticles is still a big problem in applications of nanoparticles to bioassays, biolabeling, biosensors, and diagnosis. Recently, click chemistry,15 the copper(I)-catalyzed 1,2,3-triazole formation of azide and alkyne groups, has been applied for surface engineering and the fabrication of nanomaterials because of its specificity, high thermodynamic force (>30 kcal/mol), compatibility of various media, relatively mild experimental conditions, and good stability of final products.16-25 Herein, we describe a method for fabricating “clickable” gold nanoparticles (AuNPs) functionalized with azide groups, which can be used as the building block for conjugation with biomolecules via click chemistry (Scheme 1). By surface modification, hydrophobic AuNPs were transferred to aqueous solution with azide groups on the surface. To demonstrate the potential of the resulting azide-AuNPs further, horseradish peroxidase (HRP) was used to covalently (14) Hermanson, G. T. Bioconjugate Techniques; Academic Press: San Diego, CA, 1996. (15) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004. (16) O’Reilly, R. K.; Joralemon, M. J.; Wooley, K. L.; Hawker, C. J. Chem. Mater. 2005, 17, 5976. (17) Brennan, J. L.; Hatzakis, N. S.; Tshikhudo, T. R.; Dirvianskyte, N.; Razumas, V.; Patkar, S.; Vind, J.; Svendsen, A.; Nolte, R. J. M.; Rowan, A. E.; Brust, M. Bioconjugate Chem. 2006, 17, 1373. (18) Nandivada, H.; Jiang, X.; Lahann, J. Adv. Mater. 2007, 19, 2197. (19) Guo, Z.; Lei, A.; Zhang, Y.; Xu, Q.; Xue, X.; Zhang, F.; Liang, X. Chem. Commun. 2007, 2491. (20) Sommer, W. J.; Weck, M. Langmuir 2007, 23, 11991. (21) Boisselier, E.; Salmon, L.; Ruiz, J.; Astruc, D. Chem. Commun. 2008, 5788. (22) Maltzahn, G. v.; Ren, Y.; Park, J.-H.; Min, D.-H.; Kotamraju, V. R.; Jayakumar, J.; Fogal, V.; Sailor, M. J.; Ruoslahti, E.; Bhatia, S. N. Bioconjugate Chem. 2008, 19, 1570. (23) Gole, A.; Murphy, C. J. Langmuir 2008, 24, 266. (24) Zhang, Y.; He, H.; Gao, C.; Wu, J. Langmuir 2009, 25, 5814. (25) Kim, Y.-P.; Daniel, W. L.; Xia, Z.; Xie, H.; Mirkin, C. A.; Rao, J. Chem. Commun. 2010, 76.

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Scheme 1. Conjugation of Clickable AuNPs with Biomolecules via Click Chemistry

of the organic phase, the aqueous phase was extracted with diethyl ether (3  20 mL). The collected organic solution was dried with Na2SO4 and concentrated to give an oily liquid. 1H NMR (CD3OD): 1.791 (2H, m), 2.772 (2H, t), 3.439 (2H, t). FTIR: 2100 cm-1 (azide group).

Synthesis of Octylamine-Modified Poly(acrylic acid) (OPA). Certain amounts of acetone and ethyl acetate were added bind with them to prepare HRP-AuNP conjugates. There are some key problems in the fabrication of nanoparticle-based bioprobes: How many biomolecules were conjugated with nanoparticles? Did these biomolecules retain their activity after conjugation? HRP is an ideal model protein because it has a strong absorption peak at 402 nm (ε402 nm: 105 L 3 mol-1 3 cm-1) for quantitative analysis. Moreover, the activity of HRP could be detected by a facile catalytic activity assay. In this work, the azideAuNPs were covalently conjugated with alkyne enzyme via click chemistry and the conjugation was quantitatively confirmed. As evaluated by an enzyme-catalyzed colorimetric reaction, the enzyme-AuNPs conjugates retained their catalytic activity, exhibiting their ability to be an ideal nanobioprobe.

Experimental Procedures Materials. Hydrogen tetrachloroaurate(III) (HAuCl4), sodium borohydride (NaBH4), sodium azide and poly(acrylic acid) (PA, MW 800-1000) were purchased from Sinopharm Chemical Reagent Co. Ltd. Didodecyldimethylammonium bromide (DDAB), 3-chloropropyl-1-amine, propiolic acid (PLA), N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), and 2,2-azinobis(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS) were purchased from Sigma. Dodecylamine (DDA) was purchased from Acros. Peroxidase horseradish (HRP) was purchased from Roche. Amicon and Microcon centrifugal filter units were purchased from Millipore. Other reagents were of analytical grade. Ultrapure water was used in all needed experiments. Synthesis of DDA-Capped AuNPs. DDA-capped AuNPs were prepared by following Peng’s method.5 At first, the seed solution was prepared as following: 4 mL of a 1% HAuCl4 aqueous solution was mixed with 10 mL of 25 mM DDAB in toluene. After Au was transferred to the toluene layer, the toluene layer was mixed with 500 mg of DDA in 5 mL of toluene. Then 0.054 g of NaBH4 in 2 mL of H2O was added to the mixture under vigorous stirring. The mixture turned dark brown, which indicated the formation of gold nanoparticles. Gold nanoparticles were precipitated by adding ethanol, followed by centrifugation, and then were redissolved in 15 mL of toluene. Then, 3 mL of 1% HAuCl4, 200 mg of DDAB, and 370 mg of DDA were mixed in 10 mL of toluene to get a precursor gold salt solution. The seed solution (1.4 mL) was mixed with the precursor solution, and finally 4 mL of a 0.2 M hydrazine aqueous solution was added dropwise to the mixture within 5 min under vigorous stirring. The color of the solution turned deep purple, as indicated by the formation of the final products. Gold nanoparticles were precipitated by adding ethanol, followed by centrifugation, and then were redispersed in 15 mL of chloroform to obtain a mean diameter of around 5 nm. Synthesis of 3-Azidopropyl-1-amine. According to previous reports,26,27 a solution of 3-chloropropyl-1-amine (4 g, 0.043 mol) and sodium azide (8.4 g, 0.129 mol) in 30 mL of water was held at 80 °C for 15 h. After half of the water was removed by distillation under vacuum, the reaction solution was cooled in an ice bath. Diethyl ether (20 mL) and KOH (1.6 g) were added to the solution, keeping the temperature under 10 °C. After separation

to 10 mL of a crude poly(acrylic acid) (PA, MW 800-1000) solution and then concentrated under vacuum to remove the solvent to get a white PA solid. Octylamine-modified poly(acrylic acid) (OPA) was synthesized using a modified published method.28 PA (0.3 mg) and EDC (0.6 mg) were dissolved in 13.5 mL of DMF and then transferred into a 50 mL roundbottomed flask. After dissolution, 0.2 mL of octylamine was added to the flask dropwise. The solution was stirred for 16 h. Most DMF was removed under vacuum, 1 mL of 1 mol/L HCl and 60 mL of H2O were added to the reaction solution, and a yellowish precipitate was separated by centrifugation (9000 rpm for 10 min). The precipitate was washed with H2O (2  60 mL) and redissolved in acetone. The acetone was removed under vacuum, and the yellowish solid was collected. Synthesis of Azide-OPA. PA (0.3 mg) and EDC (0.6 mg) were dissolved in 13.5 mL of DMF and then transferred to a 50 mL round-bottomed flask. After dissolution, 0.2 mL (1.2 μmol) of octylamine and 0.06 mL (0.6 μmol) of 3-azidopropyl-1-amine were added to the flask dropwise. The solution was stirred for 16 h. Most DMF was removed under vacuum, 1 mL of 1 mol/L HCl and 60 mL of H2O were added to the reaction solution, and a yellowish precipitate was separated by centrifugation (9000 rpm for 10 min). The precipitate was washed with H2O (2  60 mL) and redissolved in acetone. The acetone was removed under vacuum, and the yellowish solid was collected. Preparation of Azide-AuNPs. Azide-OPA (6 mg) was dissolved in 1 mL of methanol and mixed with 2 mL of an as-prepared DDA-capped AuNP chloroform solution. The solution was stirring for several minutes, and the solvent was removed under vacuum. The precipitate was redissolved in a 0.05 mol/L NaOH solution to get a clear pink solution. The crude solution of azide-AuNPs was purified by an Amicon centrifugal filter unit (MWCO 100 kDa) and washed with H2O (4  2 mL) to remove excess azide-OPA. The final products were dissolved in 2 mL of H2O. The molar concentration of azide-AuNPs was determined by UV-vis spectrometry. Preparation of Alkyne-HRP. The preparation of alkyneHRP was carried out according to a modified EDC method by Brennan et al.17 Typically, 2.4 mL of HRP (1 mg/mL in a 0.02 mol/L pH 7 phosphate buffer solution (PBS)), 27 μL of propiolic acid (100 mM in THF), and 26 μL of EDC (100 mM in PBS) were mixed in 10% v/v THF/PBS and then reacted for 10 h at 25 °C. The crude alkyne-HRP was purified by a Microcon centrifugal filter unit (MWCO 30 kDa) and washed with PBS (3  3 mL). Purified alkyne-HRP was freeze dried to obtain the powder for further use. The molar concentration of HRP was determined by UV-vis spectrometry.

Preparation of HRP-AuNP Conjugates via Click Chemistry. To 1 mL of a PBS solution of azide-AuNPs (20 nmol/L)

were added an alkyne-HRP solution and 1 μL of catalyst (10 mM CuSO4 3 5H2O, 50 mM ascorbic acid in H2O). The reaction solution was stirred overnight at 25 °C. The products were purified by an Amicon centrifugal filter unit (MWCO 100 kDa) and washed with PBS (6  2 mL). The purified HRP-AuNP conjugates were redissolved in 100 μL of PBS and stored at 4 °C. The molar concentration of the conjugates was determined by UV-vis spectrometry.

Calculation of the Theoretical Maximum Number of Bound HRPs per Au Particle. The radius of the Au particle was 2.7 nm (r) as determined by TEM. The thickness of the

(26) Carboni, B.; Benalil, A.; Vaultier, M. J. Org. Chem. 1993, 58, 3736. (27) Vercillo, O. E.; Andrade, C. K. Z.; Wessjohann, L. A. Org. Lett. 2008, 10, 205.

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(28) Zhou, M.; Nakatani, E.; Gronenberg, L. S.; Tokimoto, T.; Wirth, M. J.; Hruby, V. J.; Roberts, A.; Lynch, R. M.; Ghosh, I. Bioconjugate Chem. 2007, 18, 323.

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Article Scheme 2. Fabrication of Clickable AuNPs

modification layer was about 1.5 nm (l1) as calculated by the length of the capping molecules. HRP is a water-soluble globular protein with a diameter of about 5.2 nm (l2). Therefore, the theoretical maximum number of bound HRPs per Au particle N (eq 1) could be obtained by dividing V (eq 2) by the volume of HRP, V0. V V0

ð1Þ

3 π½ðr þ l1 þ l2 Þ3 - ðr þ l1 Þ3  4

ð2Þ

N ¼

V ¼

Catalytic Activity Assay of HRP-AuNP Conjugates. The catalytic activity of HRP-AuNP conjugates was determined by a colorimetric reaction. HRP can catalyze the oxidation of ABTS mediated by H2O2 to produce the colored radical anion, which has a distinct absorption at 418 nm. Briefly, 1 μL of 0.5 μmol/L HRPAuNP conjugates were added to a 200 μL solution containing 2 mmol/L ABTS and 2 mmol/L H2O2. The assay was performed in the kinetic mode of a UV-vis spectrophotometer by detecting the time-resolved absorption change at 418 nm from 0 to 180 s. Characterization Methods. Nuclear magnetic resonance (NMR) analysis was conducted on a Varian Mercury VX-300 NMR spectrometer operating at 300 MHz. The data were obtained with samples dissolved in CD3OD. Infrared spectra were recorded on a Nicolet Nexus 670 Fourier transform infrared spectrometer. Transmission electron microscopy (TEM) images were obtained on a JEOL JEM-1230 transmission electron microscope at 100 kV. UV-vis spectra and kinetic spectra of a catalytic activity assay were recorded on Shimadzu UV-2550 UV-vis spectrophotometer, and the data were corrected for water background absorption. Dynamic light scattering (DLS) data were recorded on a Malvern Nano-ZS ZEN3600 zetasizer.

Results and Discussion Fabrication of Clickable AuNPs. On the basis of a welldeveloped strategy of encapsulating hydrophobic nanoparticles with a layer of amphiphilic polymers or surfactant molecules,7,12,29 a new route for fabricating azide-functionalized AuNPs is proposed here in Scheme 2.Graft polymers based on poly(acrylic acid) (PA) were chosen for the modification of DDA-capped AuNPs because the abundant carboxylic groups in poly(acrylic acid) provide a controllable platform for conveniently conjugating (29) Gao, X.; Cui, Y.; Levenson, R. M.; Chung, L. W. K.; Nie, S. Nat. Biotechnol. 2004, 22, 969. (30) Bentzen, E. L.; Tomlinson, I. D.; Mason, J.; Gresch, P.; Warnement, M. R.; Wright, D.; Sanders-Bush, E.; Blakely, R.; Rosenthal, S. J. Bioconjugate Chem. 2005, 16, 1488. (31) Ballou, B.; Lagerholm, B. C.; Ernst, L. A.; Bruchez, M. P.; Waggoner, A. S. Bioconjugate Chem. 2004, 15, 79.

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Figure 1. Infrared spectra of OPA and azide-OPA. The peak at 2100 cm-1 in the dashed box clearly shows the existence of the azide group.

different functional molecules via an amide bond.28,30,31 In this work, by simultaneously conjugating octylamine and synthesized 3-azidopropyl-1-amine to the main chain of a PA molecule, a new kind of graft polymer;azide-octyl-poly(acrylic acid) (azide-OPA);was produced. Compared to octyl-poly(acrylic acid) (OPA), the infrared spectrum of synthesized azide-OPA displayed a strong peak at 2100 cm-1, clearly reflecting the existence of azide groups in this graft polymer (Figure 1). The coupling of δO-H and νC-O resulted in the appearance of absorption bands at 1380 and 1260 cm-1, indicating that azide-OPA still retained a part of the unmodified carboxyl groups. Because of the hydrophobic octyl chains grafted onto the PA chain, azide-OPA could encapsulate dodecylamine(DDA)-capped hydrophobic AuNPs via hydrophobic interaction. Therefore, AuNPs could be decorated with carboxyl groups, which facilitated dissolution in aqueous solution, and azide groups for further functionalization. In the organic phase, azide-OPA assembled on the surface of a DDA-capped AuNP under vacuum via the hydrophobic interaction between the octylamine chain of azide-OPA and the DDA chain on the AuNP. In a typical TEM image (Figure 2a), the azide-AuNPs were monodisperse without any aggregation, confirming that AuNPs were modified with azide-OPA separately. It also excluded the possibility that this graft polymer formed large micelles that may encapsulate more than one particle. The evident surface plasmon band (SPB) at 524 nm in the UV-vis spectrum (Figure 2b) clearly agreed with the above result. Some groups have reported the fabrication of azide-functionalized AuNPs for click chemistry, but the fabrication methods based on ligand exchange required uncommon mecapto compounds17,20,32 or used complicated in situ modification of (32) Zhou, Y.; Wang, S.; Zhang, K.; Jiang, X. Angew. Chem., Int. Ed. 2008, 47, 7454.

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Figure 2. (a) Typical TEM image of azide-AuNPs. (b) UV-vis spectrum of azide-AuNPs dissolved in ultrapure water at room temperature.

AuNPs with an azide compound.21 Our method adopted a cheap, easily prepared graft polymer to modify AuNPs via facile hydrophobic interactions. Therefore, it could be a versatile azidefabrication method for hydrophobic nanoparticles in the organic phase. Stability of Azide-AuNPs in Aqueous Solution. As mentioned above, nanoparticles fabricated for bioapplications should satisfy some requirements. Good stability in an aqueous solution was of primary importance. In terms of quantum size effect, the specific surface energy increased remarkably as the diameter of the nanoparticles decreased. Therefore, the surfaces of nanoparticles needed to be tightly protected by ligands to avoid aggregation. Because the octylamine group with a favorable length (C8) in the graft polymer interacted hydrophobically with DDA, a synthesized azide-AuNP whose surface was capped with a compact polymer shell could be stored in water at room temperature for months. In practical applications, the properties of functional groups on the surfaces of nanoparticles also determined their stability in different buffer solutions. On the basis of Mie theory, the shape and position of the absorption peak, namely, the surface plasmon resonance (SPR) band, directly reflected the stability of AuNPs in solution. If the AuNPs were unstable or if they aggregated, then the SPR band would broaden and shift to longer wavelength. The stability of azide-AuNPs under physiological conditions was analyzed by UV-vis spectrometry. The absorption curves of azide-AuNPs in a neutral or weakly alkaline Britton-Robinson (BR) buffer solution (pH 6-10) were almost identical with an SPR band evident at 528 nm (Figure 3a), suggesting that the products had good stability in such a pH range. The carboxylic group on the surface is negatively charged at pH 6-10, which makes azide-AuNPs water-soluble and provides enough electrostatic force to disperse AuNPs in the buffer solution. However, AuNPs aggregated when the pH decreased to 5, which could be reflected by the red-shifted SPR band in the UV-vis spectrum. The protonation of carboxylic groups in the acidic range reduced the electrostatic force between individual particles, resulting in the aggregation of AuNPs. The TEM images and DLS spectra of azide-AuNPs in BR buffer solution at different pH values are shown in Supporting Information. The ions could also affect the stability of nanoparticles in buffer solution by breaking the surface double electric layer of nanoparticles. The absorption curves of azide-AuNPs in ultrapure water at different NaCl concentrations are shown in Figure 3b. The relatively low ionic strength (below 0.1 mol/L NaCl) could not destroy the stability of azide-AuNPs. When the concentration of NaCl reached 0.15 mol/L, the shape of the absorption curve broadened slightly and AuNPs tended to aggregate. Considering that the ion strength under physiological conditions was about 10174 DOI: 10.1021/la100315u

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Figure 3. UV-vis spectra of AuNPs (a) in BR buffer solution at different pH values and (b) in ultrapure water at different NaCl concentrations at room temperature.

Figure 4. Infrared spectra of azide-AuNPs and HRP-AuNPs conjugates prepared in different reactant molar ratios of HRP/AuNPs (sample 1, 10:1; sample 2, 20:1; sample 3, 50:1; sample 4, 100:1).

that of 0.136 M NaCl, azide-AuNPs could be applied under normal physiological conditions to maintain good stability. The TEM images and DLS spectra of azide-AuNPs in ultrapure water under different NaCl concentrations are shown in Supporting Information. Conjugation of Alkyne-HRP with Azide-AuNPs via Click Chemistry. Before conjugation, HRP molecules were functionalized with alkyne groups via conjugation with propiolic acid using the EDC method. In the presence of a small amount of Cu(I) (5 μmol/L), the conjugation of alkyne-HRP with azide-AuNPs was performed in 0.02 M phosphate buffer solution (pH 7) at room temperature overnight. Unbound 44 kDa HRP molecules were removed from HRP-AuNPs conjugates by ultracentrifugation six times with the Amicon centrifugal filter unit (MWCO 100 kDa). The filtrate from the last ultracentrifugation of the HRP-AuNP solution did not exhibit catalytic activity, excluding the possibility of the existence of free HRP molecules in the solution of HRP-AuNP samples. Different reactant molar ratios of HRP/AuNPs (10:1, 20:1, 50:1, and 100:1) were adopted in the conjugation experiments for comparison. As shown in the infrared spectra (Figure 4), it is clear that the relative intensity of the azide group at 2100 cm-1 in AuNPs (yellow region) decreased after conjugation with alkyneHRP via the click reaction. Because of the presence of porphyrin in HRP, the vibration of C-N in the aryl ring in the region around 1100 cm-1 increased relatively (blue region). The above results are strong evidence for the successful conjugation of AuNPs with HPR via click chemistry. The sizes of HRP-AuNP conjugates in solution were characterized by dynamic light scattering (DLS). In Figure 5, the DLS diameters of HRP-AuNP conjugates are larger than those of azide-AuNPs. The particles were well-dispersed without any aggregation in the TEM images of conjugates (Supporting Information), confirming that the increased size resulted from the conjugation of AuNPs with HRP. Furthermore, the DLS Langmuir 2010, 26(12), 10171–10176

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Article Table 2. Summary of the Conjugation of HRP to AuNPs sample 1 sample 2 sample 3 sample 4 reactant ratio of HRP/AuNPs number of bound HRPs/particle effective number of HRPs/particle

10:1 3.8 0.04

20:1 8.5 0.08

50:1 15.4 0.25

100:1 23.3 0.49

Figure 5. DLS data of azide-AuNP and HRP-AuNP conjugates at the same molar concentrations in PBS at room temperature. Table 1. DLS Diameters of Azide-AuNPs and HRP-AuNPs Conjugates at the Same Molar Concentrations in PBS at Room Temperature azide-AuNPs sample 1 sample 2 sample 3 sample 4 DLS diameter (nm)

10.44

13.5

15.02

16.64

18.4

Figure 6. UV-vis spectra of azide-AuNPs and HRP-AuNP conjugates at the same molar concentrations in PBS at room temperature.

diameters of conjugates increased with the increasing ratio of HRP/AuNPs (Table 1), suggesting that more HRP molecules conjugated with AuNPs. The HRP-AuNP conjugates were also characterized by UV-vis spectroscopy (Figure 6). Compared to bare azide-AuNPs, the conjugates at the same molar concentrations exhibited evident absorption peaks at 402 nm, which could be attributed to the absorption of bound HRP on AuNPs. This was direct evidence of the successful conjugation of HRP with AuNPs. In addition, the absorption intensity increased as the reactant molar ratio increased, which meant that more HRP molecules were bound to AuNPs. By subtracting the background of pure AuNPs and comparing the absorbance HRP at 402 nm to that of AuNPs at 524 nm, the molar ratio of HRP/AuNPs in conjugates, equal to the number of bound HRPs per Au particle, could be calculated. The results of conjugates prepared in different reactant ratios are listed in Table 2. A model of monolayer binding of HRP on a single Au particle is demonstrated in Figure 7. The theoretical maximum number of bound HRPs per Au particle was 43. Because the intervening space between spherical HRP molecules was not considered in this calculation model, we speculated that the number of bound HRPs per Au particles (23.3 in sample 4) was close to the actual maximum number of bound HRPs per Au particle in this experiment. An important criterion for the evaluation of nanobioprobes is that the conjugates must keep the original activities of bound biomolecules. The activity of HRP-AuNPs was detected by the HRP-catalyzed Langmuir 2010, 26(12), 10171–10176

Figure 7. Schematic representation of the monolayer binding of HRP molecules to a single Au nanoparticle.

Figure 8. (a) Time-dependent curves of absorption intensity at 418 nm of ABTS oxidation in the presence of equimolar amounts of azide-AuNP and HRP-AuNP conjugate samples 1-4 (2.6  10-7 mol/L). (b) Time-dependent curves of absorption intensity at 418 nm of ABTS oxidation in the presence of alkyne-HRP at different concentrations. The inset is the linear dependence of the baseline-corrected absorbance at 418 nm after 180 s on the concentration of alkyne-HRP. (The horizontal axis is the molar concentration of alkyne-HRP divided by 1.56  10-8 mol/L.)

oxidation of ABTS by H2O2. As soon as the HRP-AuNPs conjugates were added to the substrate solution, the absorption intensity at 418 nm evidently increased. The time-dependent curves of absorption change from 0 to 180 s in the presence of HRP-AuNP samples are shown in Figure 8a. ΔA418 nm of HRP-AuNP conjugates for samples 1-4 are 0.059, 0.126, 0.391, and 0.756, respectively. In contrast, no change in absorption intensity at 418 nm was observed for azide-AuNPs, showing that azide-AuNPs could not catalyze the oxidation. These results suggest that the catalytic activity was from the HRP molecules bound to AuNPs, not from the Au particle itself. Subsequently, alkyne-HRP was adopted as the standard for calculating the effective number of HRPs per Au particle. In a typical enzymatic reaction, if the concentration of the substrate was much greater than that of the enzyme, then there was a linear correlation between the reaction rate and the concentration of enzyme. In this experiment, the reaction rate was reflected by the absorbance change at 418 nm (ΔA418 nm) from 0 to 180 s. As shown in Figure 8b, the catalytic activities of a series of alkyneHRPs at different concentrations were detected via the same method (1, 1.56  10-8 mol/L; 2, 3.12  10-8 mol/L; 4, 6.24  10-8 mol/L; 8, 1.25  10-7 mol/L; and 16, 2.5  10-7 mol/L). ΔA418 nm values in Figure 8b were 0.082 for 1, 0.183 for 2, 0.393 for 4, 0.739 for 8, and 1.473 for 16, respectively. A linear equation of ΔA418 nm against the concentration of DOI: 10.1021/la100315u

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Article

Zhang et al.

alkyne-HRP was obtained. Therefore, the effective concentration of HRP in HRP-AuNP conjugates and the effective number of HRPs/Au particle could be calculated using this equation. As listed in Table 2, the effective numbers of HRP/particle for the samples were much less than the numbers of bound HRP/ particle. The activity of HRP was inhibited after conjugation with AuNPs. To evaluate the influence of the Cu(I) catalyst on the activity of HRP, an alkyne-HRP solution was treated with 50-fold Cu(I) catalyst overnight at 25 °C. The activity of the alkyne-HRP sample treated with Cu(I) catalyst was determined by the colorimetric reaction mentioned above. The alkyne-HRP sample treated with the Cu(I) catalyst retained 65.6% of the activity of Cu(I)untreated alkyne-HRP (Supporting Information). This result implied that the influence of the Cu(I) catalyst was not the main reason for the loss of activity of HRP. It is well known that the activities of some biomolecules are site-specific and largely related to their conformation. Taking nanoparticle-based bioprobes, for example, biomolecules are first covalently bound to nanoparticles via functional groups, so the conformation of biomolecules may be affected. Second, biomolecules stick together on the surfaces of nanoparticles and cannot rotate freely in solution. The steric hindrance effect may greatly restrict the activities of biomolecules. The above results indicated that it was inaccurate to evaluate the bioactivities of nanobioprobes only by calculating how many biomolecules were bound to nanoparticles. Establishing respective methods of evaluate the actual activities of products is also necessary in the fabrication of an ideal nanobioprobe.

Conclusions A new kind of clickable AuNP was synthesized for the fabrication of nanobioprobes using a cheap, easily prepared graft

10176 DOI: 10.1021/la100315u

polymer as a modifier. The resulting azide-AuNPs have good monodispersity and stability in physiological solution. In the presence of Cu(I), azide-AuNPs could react with alkyne-HRP via click chemistry under mild conditions, producing HRP-AuNP conjugates. The number of HRPs bound on the surfaces of AuNPs could be controlled by adjusting the molar ratio of reactants. As confirmed by a colorimetric reaction catalyzed by HRP, such HRP-AuNP conjugates still retained the catalytic activity of HRP molecules bound to AuNPs. This strategy was based on the hydrophobic interaction between alkyl chains in graft polymers and hydrophobic ligands on the surfaces of particles, which is applicable to any kind of particles. Therefore, this method could be versatile for hydrophobic nanoparticles synthesized in an organic phase. Acknowledgment. This work was supported by the National Key Scientific Program (973)-Nanoscience and Nanotechnology (2006CB933100), the Science Fund for Creative Research Groups of the NSFC (20621502 and 20921062), the National Natural Science Foundation of China (20833006), and the Ministry of Public Health (2009ZX10004-107 and 2008ZX10004-004). We also thank Mr. Weihan Wang and Dr. Zhimou Guo for helpful discussions. Supporting Information Available: TEM images and DLS spectra of azide-AuNPs in a BR buffer solution at different pH values and in ultrapure water at different NaCl concentrations. TEM images of HRP-AuNPs conjugates. Experimental details of the evaluation of HRP activity after click reaction. This material is available free of charge via the Internet at http://pubs.acs.org.

Langmuir 2010, 26(12), 10171–10176