Enlargement of Gold Nanoparticles on the Surface of a Self

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Anal. Chem. 2006, 78, 5227-5230

Enlargement of Gold Nanoparticles on the Surface of a Self-Assembled Monolayer Modified Electrode: A Mode in Biosensor Design Nandi Zhou,† Jing Wang,† Ting Chen,† Zhiguo Yu,‡ and Genxi Li*,†

Department of Biochemistry and National Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, P. R. China, and School of Life Science and Shanghai Key Laboratory of Bio-Energy Crops, Shanghai University, Shanghai 200444, P. R. China

Gold nanoparticle (Au-NP) seeds were adsorbed onto the surface of a self-assembled monolayer (SAM)-modified electrode. With the treatment of this modified electrode by Au-NPs growth solution containing different concentrations of H2O2 or cholesterol along with cholesterol oxidase (ChOx), the Au-NP seeds on the electrode surface were enlarged in varying degrees. As a result, the peak currents in corresponding cyclic voltammograms were inversely proportional to the concentration of H2O2 or cholesterol. ChOx was also further modified onto the surface of Au/ SAM/Au-NP electrode to prepare Au/SAM/Au-NP/ChOx electrode. Using the enzyme-modified electrode to detect cholesterol, which also utilized the enlargement of the NPs, an extraordinary low detection limit of 5 × 10-9 M was achieved and two linear dependence ranges of 7.5 × 10-8-1 × 10-6 and 1 × 10-6-5 × 10-5 M were obtained. Consequently, new kinds of H2O2 and cholesterol biosensors could be fabricated. Nanoparticles (NPs) have emerged as a new kind of inspiring material and have played important roles in a wide range of areas, such as electronics, catalysis,1,2 biomodeling,3,4 biolabeling,5,6 sensing,7 photonics,8 and optoelectronics,9 due to their special characteristics. Among all kinds of NPs, gold nanoparticles (AuNPs) have been extensively used in modification of various electrodes and fabrication of different kinds of biosensors. The excellent biological compatibility as well as large surface area caused by a small granule diameter has made Au-NPs good * Corresponding author. Telephone: +86-25-83593596. Fax: +86-25-83592510. E-mail: [email protected]. † Nanjing University. ‡ Shanghai University. (1) Lewis, L. N. Chem. Rev. 1993, 93, 2693-2730. (2) Haruta, M.; Date´, M. Appl. Catal., A 2001, 222, 427-437. (3) Mahtab, R.; Rogers, J. P.; Singleton, C. P.; Murphy, C. J. J. Am. Chem. Soc. 1996, 118, 7028-7032. (4) Mahtab, R.; Harden, H. H.; Murphy, C. J. J. Am. Chem. Soc. 2000, 122, 14-17. (5) Chan, W. C. W.; Nie, S. M. Science 1998, 281, 2016-2018. (6) Nicewarner-Pen ˜a, S. R.; Freeman, R. G.; Reiss, B. D.; He, L.; Pen ˜a, D. J.; Walton, I. D.; Cromer, R.; Keating, C. D.; Natan, M. J. Science 2001, 294, 137-141. (7) Haes, A. J.; Van Duyne, R. P. J. Am. Chem. Soc. 2002, 124, 10596-10604. (8) Maier, S. A.; Brongersma, M. L.; Kik, P. G.; Meltzer, S.; Requicha Ari, A. G.; Atwater, H. A. Adv. Mater. 2001, 13, 1501-1505. (9) Kamat,P. V. J. Phys. Chem. B 2002, 106, 7729-7744. 10.1021/ac0605492 CCC: $33.50 Published on Web 06/15/2006

© 2006 American Chemical Society

material to immobilize proteins and enzymes and to keep the biological activities of proteins (enzymes) for a long time. The ability of promoting electron transfer (eT) between the active centers of proteins and electrode makes it especially suitable to fabricate redox protein-modified electrodes. And the direct electrochemical response of several proteins have been reported, using Au-NPs as electron-transfer promoters.10-15 Recent studies revealed that Au-NPs could be enlarged in a solution containing tetrachloroauric acid (HAuCl4) as a gold source, cetyltrimethylammonium chloride (CTAC) as a surfactant, and H2O216 as a reductant. And, nicotinamide adenine dinucleotide (or nicotinamide adenine dinucleotide phosphate),17 glucose, together with glucose oxidase,18 or some small active molecules19,20 as reductants have also led to the enlargement of Au-NPs. Based on how the characteristics of UV-visible absorbance of Au-NPs changed with their sizes, optical sensors can be fabricated. In this paper, we report a new design mode of electrochemical biosensors by making use of the enlargement of Au-NPs. We have also combined the character of Au-NPs growth on the designed electrode surfaces with the biological catalytic reactions in the solutions to develop a cholesterol sensor. A cholesterol biosensor with extraordinarily lowered detection limit can be fabricated by further modifying cholesterol oxidase (ChOx) onto the modified electrode surface. EXPERIMENTAL SECTION Materials. Cholesterol oxidase (EC 1.1.3.6) from Pseudomonas fluorescens, cholesterol, and 2-mercaptoethylamine (cysteamine) were purchased from Sigma Chemical Co. Cetyltrimethylammo(10) Xiao, Y.; Ju, H. X.; Chen, H. Y. Anal. Biochem. 2000, 278, 22-28. (11) Feng, J. J.; Zhao, G.; Xu, J. J.; Chen, H. Y. Anal. Biochem. 2005, 342, 280286. (12) Zhang, J. D.; Oyama, M. J. Electroanal. Chem. 2005, 577, 273-279. (13) Di, J. W.; Shen, C. P.; Peng, S. H.; Tu, Y. F.; Li, S. J. Anal. Chim. Acta 2005, 553, 196-200. (14) Zhang, L.; Jiang, X. E.; Wang, E. K.; Dong, S. J. Biosens. Bioelectron. 2005, 21, 337-345. (15) Wang, L.; Wang, E. K. Electrochem. Commun. 2004, 6, 49-54. (16) Zayats, M.; Baron, R.; Popov, I.; Willner, I. Nano Lett. 2005, 5, 21-25. (17) Xiao, Y.; Pavlov, V.; Levine, S.; Niazov, T.; Markovitch, G.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 4519-4522. (18) Xiao, Y.; Pavlov, V.; Shlyahovsky, B.; Willner, I. Chem. Eur. J. 2005, 11, 2698. (19) Baron, R.; Zayats, M.; Willner, I. Anal. Chem. 2005, 77, 1566-1571. (20) Scampicchio, M.; Wang, J.; Blasco, A. J.; Arribas, A. S.; Mannino, S.; Escarpa, A. Anal. Chem. 2006, 78, 2060-2063.

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nium chloride and tetrachloroauric acid were obtained from Shanghai Sangon Biological Engineering & Technology and Service Co. Other reagents were of analytical grade. Water was purified with a Milli-Q purification system to a specific resistance (>16 MΩ/cm) and used to prepare all solutions. Preparation of Au-NP Seeds. Au-NP seeds were prepared by using NaBH4 as reductant and stabilized with sodium citrate according to the literature.21 In brief, 0.5 mL of 0.01 M HAuCl4 and 0.5 mL of 0.01 M sodium citrate were added to 18 mL of purified water and stirred. Then 0.5 mL of freshly prepared 0.1 M NaBH4 was added, and the solution color changed from colorless to orange. Stirring was stopped, and the solution was left undisturbed for 2 h. The diameter of the Au-NP seeds was ∼3.5 nm, which was measured by transmission electron microscopy (TEM). Electrode Modification. The substrate Au electrode was soaked in piranha solution (H2SO4:30% H2O2 ) 3:1) for 2 min (Caution! Piranha solution reacts violently with organic materials and should be handled with extreme care!) to eliminate the adsorbed organic matter and then rinsed with water. Then, the electrode was abraded with successively finer grades sand papers and further polished to a mirror smoothness with alumina powder (Al2O3) of various particle sizes (0.3 and 0.05 µm) on silk. Finally, it was sonicated for 5 min in water and ethanol, respectively. The cleaned Au electrode was soaked in 50 mM cysteamine aqueous solution for 2 h at room temperature in darkness. The resulting Au/cysteamine electrode was thoroughly rinsed with water to remove all physically adsorbed cysteamine. Then it was immersed in the solution containing Au-NP seeds for 6 h at 4 °C and thoroughly rinsed with water again. In this way, the modified electrode, Au/cysteamine/Au-NPs, was prepared. For further preparation of the Au/cysteamine/Au-NPs/ChOx electrode, Au/ cysteamine/Au-NP electrode was immersed in 5 mg/mL ChOx solution at 4 °C overnight. Growth of Au-NPs. According to ref 16 and the reaction conditions of our system, the Au-NPs growth solution consisted of 2.06 × 10-4 M HAuCl4, 2.0 × 10-3 M CTAC in 0.01 M phosphate buffer, pH 7.0, and different concentrations of either H2O2 or cholesterol (dissolved in 0.02 M phosphate buffer, pH 7.0, containing 10% Triton X-100) together with 0.5 mg/mL ChOx. The growth of Au-NPs was performed at room temperature of 25 °C in the case of H2O2, or 37 °C in the case of cholesterol with ChOx. The size of the Au-NPs would increase rapidly in the first 5 min and slowly later. After 15 min, the size of the Au-NPs remained unchanged. So, we have selected 15 min as the time for the growth of the Au-NPs. For the growth of Au-NPs on the surface of the Au/cysteamine/Au-NPs/ChOx electrode, we soaked the modified electrode with the growth solution containing different concentrations of cholesterol but no ChOx. Characterization of the Enlargement of Au-NPs. Au-NP seeds and the enlarged Au-NPs were characterized by JEM 2000EX transmission electron microscopy (JEOL) at 120 kV. The UV-visible absorbance spectra of the NPs were measured with a model UV-2201 spectrophotometer (Shimadzu). Electrochemical experiments were carried out with a model 263A potentiostat/ galvanostat (EG&G) and a three-electrode system. The working (21) Busbee, B. D.; Obare, S. O.; Murphy, C. J. Adv. Mater. 2003, 15, 414416.

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Figure 1. TEM images of (a) the Au-NP seeds and the enlarged NPs produced by the treatment of the Au-NP growth solution containing (b) 5 × 10-6 M H2O2, (c) 5 × 10-4 M H2O2, (d) 1 × 10-5 M cholesterol, and (e) 1 × 10-3 M cholesterol.

electrode was the modified electrode, Au/cysteamine/Au-NPs, or Au/cysteamine/Au-NPs/ChOx, with the NPs being enlarged in varying degree. A saturated calomel electrode (SCE) was used as the reference electrode, and all potentials reported in this work were versus SCE. A platinum wire electrode served as the counter electrode. RESULTS AND DISCUSSION Au-NPs can be enlarged in the Au-NP growth solution under reductive circumstances.16-20 H2O2 in the growth solution serves as reductant to convert AuIII to Au0. Au-NP seeds

HAuCl4 + 3/2H2O2 98 Au0 + 4HCl + 3/2O2 Here, the Au-NP seeds act as catalysts. The produced Au0 will then deposit on the surface of the seeds and the NPs are consequently enlarged. Figure 1a shows that the average diameter of the Au-NP seeds at the electrode surface is ∼3.5 nm. If the Au/cysteamine/AuNP-modified electrode is treated by the Au-NP growth solution, the Au-NP seeds will be enlarged. We have first performed experiments by employing the Au-NP growth solution containing H2O2. Experimental results have revealed that the Au-NPs on the electrode surface can be indeed enlarged. Moreover, the higher the concentration of H2O2 is in the growth solution, the larger the Au-NPs are. Panels b and c in Figure 1 show the enlarged Au-NPs upon treatment with 5 × 10-6 and 5 × 10-4 M H2O2, respectively. According to TEM analysis, though not very homogeneous, the grown NPs with the treatment of 5 × 10-6 M H2O2 have a mean diameter of ∼20 nm. And those NPs by the treatment of 5 × 10-4 M H2O2 have a mean diameter of ∼35 nm. The AuNP seeds can grow up to larger particles as the concentration of H2O2 in the system increases. Here, we report more interesting findings. When we use the Au/cysteamine/Au-NP-modified electrode to do electrochemical

Figure 2. Cyclic voltammograms of 5 mM K4Fe(CN)6/K3Fe(CN)6 solution obtained at Au/cysteamine/Au-NP electrodes upon treatment by the Au-NP growth solution consisted of 0, 1 × 10-7, 1 × 10-6, 1 × 10-5, 1 × 10-4 or 1 × 10-3 M H2O2 (from outer to inner). Scan rate 200 mV/s. Inset is the derived linear relationship between the cathodic peak current and the concentration of H2O2.

experiments, we notice that the cyclic voltammograms (CVs) will be changed with enlargement of the Au-NPs. The reason might be that the electric communication between the solution species and electrode will be somewhat blocked if the electrode surface is packed with the enlarged Au-NPs.22 Figure 2 shows the CVs for 5 mM K4Fe(CN)6/K3Fe(CN)6 solution obtained at the modified electrodes on which the Au-NPs are gradually enlarged by the treatment of the growth solution with different concentrations of H2O2. Obviously, successive decline of the peak currents and increase of the peak separations can be observed along with the increasing concentration of H2O2. Before treatment with the growth solution, the peak separation of the CV waves of the Au/ cysteamine/Au-NP electrode is 54 mV, and the cathodic peak current (Ipc) is 60.93 µA. Treated with growth solution containing 1 × 10-3 M H2O2, the cathodic peak current reduces to 33.53 µA and the peak separation rises to 110 mV. The more H2O2 exists in the growth solution, the smaller the peaks and the larger peak separation, which indicates bad reversibility is observed in CVs. However, detailed experimental results reveal that there is a linear dependence of the cathodic peak current (or anodic peak current) on the concentration of H2O2 in the range of 1 × 10-7-4 × 10-5 M (Figure 2, inset). The linear regression equation is y ) 58.1880.339x, r ) -0.9998. Therefore, an electrochemical H2O2 sensor can be developed by making use of the enlargement of the AuNPs and the decrease of the electrochemical wave obtained at the NP-modified electrode. It should be mentioned that the shape of the enlarged AuNPs is not perfect, as is shown in Figure 1. However, since we do not use the diameter or volume of the NPs as an analytical parameter, it does not matter whether the shape of the enlarged Au-NPs can be good or not. On the other hand, since we make use of the average effect of the enlargement of the NPs, it can still work even if the NPs are not homogeneous. We have also examined the possibility of developing an optical H2O2 sensor by employing our experimental system with reference to the literature.16 The linear concentration range for H2O2 detection is from 5 × 10-6 to 2.5 × 10-4 M (see Figure S1 in (22) Yang, M. L.; Zhang, Z. J. Electrochim. Acta 2004, 49, 5089-5095

Supporting Information). It has a lower detection limit than that reported in ref 26, which is probably due to the smaller size of the Au-NP seeds we have used (12-nm-diameter seeds were used in the reference). Nevertheless, compared with the proposed electrochemical technique, the optical H2O2 sensor cannot have a lower detection limit and wider ranges than the electrochemical sensor. H2O2 is an important metabolite of organisms. Numerous kinds of O2-dependent oxidases generate H2O2 upon catalytic oxidation of the related substrates. Thus, it suggests that the growth of AuNPs and the detection of H2O2 by making use of the Au-NP enlargement might be adopted to design biosensors with different kinds of oxidases for detection of their substrates. Cholesterol and its fatty acid esters are important compounds for human beings since they are components of biomembrane, nerve cells, and precursors of other biological molecules, such as bile acid and steroid hormones. The level of cholesterol is significant in the clinical diagnosis of diseases such as coronary heart disease, myocardial infarction, and arteriosclerosis.23 Therefore, a great many cholesterol biosensors have been fabricated to measure the content of cholesterol in human serum, food, etc.24-38 Among these biosensors, ChOx, a sort of O2-dependent oxidase catalyzing the oxidation of cholesterol by molecular O2 and producing H2O2 and cholest-4-en-3-one, is often used. Herein we employ ChOx to design a new kind of cholesterol sensor by making use of the growth of Au-NPs. Panels d and e in Figure 1 show the TEM images of the enlarged Au-NPs upon treatment with the Au-NP growth solution containing 0.5 mg/mL ChOx and 1 × 10-5 or 1 × 10-3 M cholesterol, respectively. Measured by TEM, though somewhat irregular-shaped, the mean diameter of the enlarged NPs for 1 × 10-5 M cholesterol is ∼10 nm. And the mean diameter of those enlarged NPs for 1 × 10-3 M cholesterol is ∼30 nm. Figure 3 shows the CVs for 5 mM K4Fe(CN)6/K3Fe(CN)6 solution, obtained at the Au/cysteamine/Au-NP-modified electrode treated by the Au-NP growth solution with 0.5 mg/mL ChOx and different concentrations of cholesterol. Similar to that shown in Figure 2, upon treatment by the Au-NP growth solution with (23) Nauck, M. Clin. Chem. 1997, 43, 1622-1629. (24) Nakaminami, T.; Ito, S.-I.; Kuwabata, S.; Yoneyama, H. Anal. Chem. 1999, 71, 1068-1076. (25) Singh, S.; Chaubey, A.; Malhotra, B. D. Anal. Chim. Acta 2004, 502, 229234. (26) Vidal, J.-C.; Espuelas, J.; Castillo, J.-R. Anal. Biochem. 2004, 333, 88-98. (27) Brahim, S.; Narinesingh, D.; Guiseppi-Elie, A. Anal. Chim. Acta 2001, 448, 27-36. (28) Charpentier, L.; El Murr, N. Anal. Chim. Acta 1995, 318, 89-93. (29) Tan, X.; Li, M.; Cai, P.; Luo, L.; Zou, X. Anal. Biochem. 2005, 337, 111120. (30) Bongiovanni, C.; Ferri, T.; Poscia, A.; Varalli, M.; Santucci, R.; Desideri, A. Bioelectrochemistry 2001, 54, 17-22. (31) Wang, H.; Mu, S. Sens. Actuators, B 1999, 56, 22-30. (32) Guo, M.; Chen, J.; Li, J.; Nie, L.; Yao, S. Electroanalysis 2004, 16, 19921998. (33) Shumyantseva, V.; Deluca, G.; Bulko, T.; Carrara, S.; Nicolini, C.; Usanov, S. A.; Archakov, A. Biosens. Bioelectron. 2004, 19, 971-976. (34) Ram, M. K.; Bertoncello, P.; Ding, H.; Paddeu, S.; Nicolini, C. Biosens. Bioelectron. 2001, 16, 849-856. (35) Pundir, S. C. S. Curr. Appl. Phys. 2003, 3, 129-133. (36) Vidal, J. C.; Garcia-Ruiz, E.; Espuelas, J.; Aramendia, T.; Castillo, J. R. Anal. Bioanal. Chem. 2003, 377, 273-280. (37) Garcia-Ruiz, E.; Vidal, J. C.; Aramendia, M. T.; Castillo, J. R. Electroanal. 2004, 16, 497-504. (38) Gobi, K. V.; Mizutani, F. Sens. Actuators, B 2001, 80, 272-277.

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Figure 3. Cyclic voltammograms of 5 mM K4Fe(CN)6/K3Fe(CN)6 solution obtained at Au/cysteamine/Au-NP electrode upon treatment by the Au-NP growth solution consisted of 0, 1 × 10-7, 1 × 10-6, 1 × 10-5, 1 × 10-4, or 1 × 10-3 M cholesterol (from outer to inner). Scan rate 200 mV/s. Inset is the derived linear relationship between the cathodic peak current and the concentration of cholesterol.

successively increasing concentrations of cholesterol, the apparent fall of the peak currents and the increase of peak separation are observed. Without treatment with the growth solution, the peak separation of Au/cysteamine/Au-NP electrode is 54 mV, and the cathodic peak current is 57.03 µA. Upon treatment by the Au-NP growth solution containing 2.5 × 10-4 M cholesterol, the cathodic peak current decreases to 29.98 µA and the peak separation rises to 124 mV. From the derived calibration curve, we can observe a successive decline of the cathodic peak current following the elevation of cholesterol content ranging from 1 × 10-7 to 2.5 × 10-4 M. The linear range is from 1 × 10-5 to 1 × 10-4 M (Figure 3 inset). For comparison, we have also used the UV-visible technique to examine the possibility of developing an optical cholesterol biosensor. The linear concentration range is from 5× 10-6 to 1 × 10-4 M, which is better than this proposal electrochemical sensor (see Figure S2 in Supporting Information). To improve the performance of the electrochemical biosensor and to reduce the consumption of ChOx, which is very important for sensor development, we have further modified the enzyme onto the surface of the Au/cysteamine/Au-NP-modified electrode to prepare the Au/cysteamine/Au-NPs/ChOx electrode. And the enzyme-modified electrode has also been treated with the Au-NP growth solution consisting of variable concentrations of cholesterol. As is shown in Figure 4, the peak currents of the CV curves can also be remarkably decreased and the peak separation has been increased, although ChOx has been further modified onto the surface of Au/cysteamine/Au-NPs. What is more, the peak currents of the CV curves will also continually fall off after the enzyme-modified electrode is treated by the growth solution containing successively elevated concentrations of cholesterol, same as the situation where the enzyme is in the growth solution instead of being modified onto the electrode surface. Further studies reveal that the successive decline of the cathodic peak current can be obtained following elevation of cholesterol content ranging from 5 × 10-9 to 5 × 10-5 M. Linear dependence is in the ranges of 7.5 × 10-8-1 × 10-6 (Figure 4, inset A) and 1 × 10-6-5 × 10-5 M (Figure 4, inset B). Compared with the case where ChOx is dissolved in the growth solution, the detection 5230 Analytical Chemistry, Vol. 78, No. 14, July 15, 2006

Figure 4. Cyclic voltammograms of 5 mM K4Fe(CN)6/K3Fe(CN)6 solution obtained at Au/cysteamine/Au-NP electrode (the outmost curve) and Au/cysteamine/Au-NPs/ChOx electrode upon treatment by the Au-NP growth solutions consisted of 0, 5 × 10-9, 1 × 10-8, 1 × 10-6, 1 × 10-5, or 1 × 10-4 M cholesterol (from outer to inner). Scan rate 200 mV/s. Insets A and B show the derived linear relationships between the cathodic peak current and the concentration of cholesterol.

limit is obviously very low. To the best of our knowledge, the detection limit of 5 × 10-9 M is the lowest detection limit that has ever been reported. Therefore, to combine the enlargement of Au-NPs on the electrode surface with the catalytic reaction of the substrate by the corresponding enzyme, we have fabricated new types of cholesterol biosensors. Furthermore, since the growth of the NPs on the electrode surface is due to the involvement of H2O2, the product of many kinds of O2-dependent oxidases, this study may provide a universal method to design biosensors for many kinds of substrates by using various corresponding oxidases. On the other hand, reports have indicated that not only H2O2 but also NADH (NADPH), reduced FAD-dependent enzymes with appropriate mediators, and even some small active molecules can also mediate the enlargement of Au-NPs.17-20 Therefore, we suggest that the sensor mode introduced in this paper may have potential applications in a wide range of fields. ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (Grants 90406005, 20575028) and the Program for New Century Excellent Talents in University, the Chinese Ministry of Education (NCET-04-0452). SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review March 27, 2006. Accepted May 15, 2006. AC0605492