Anal. Chem. 2005, 77, 6102-6104
Correspondence
Hemoglobin-Based Hydrogen Peroxide Biosensor Tuned by the Photovoltaic Effect of Nano Titanium Dioxide Hui Zhou, Xin Gan, Jin Wang, Xiaoli Zhu, and Genxi Li*
Department of Biochemistry and National Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 210093 Nanjing, P. R. China
The photovoltaic effect of titanium dioxide (TiO2) nanoparticles, induced by ultraviolet light, can greatly improve the catalytic activity of hemoglobin as a peroxidase, with the sensitivity increased nearly 3-fold and the detection limit lowered 2 orders, in contrast to the catalytic reactions in the dark, which indicates a possible method to tune the properties of proteins for development of photocontrolled protein-based biosensors. The protein-based biosensor, as the device to detect special physiological signals as well as realize accurate environmental and medical monitoring, has made great progress with the inherent enzymatic sensitivity and selectivity of proteins to unique substrates. However, the efficient electrical communication between redox proteins and solid electrode surfaces still exists as the “bottleneck”. Until now, many methods have been tried and adopted, and it has been a feasible way to obtain direct electrochemical responses of proteins embedded in some films.1-9 Together with some other groups, our laboratory has explored lots of materials, and recently nanomaterials have been employed.10-16 * To whom correspondence should be addressed. E-mail:
[email protected]. (1) Wollenberger, U.; Drungiliene, A.; Stocklein, W.; Kulys, J. J.; Scheller, F. W. Anal. Chim. Acta 1996, 329, 231-237. (2) Lu, Z.; Lvov, Y.; Jansson, I.; Schenkman, J. B.; Rusling, J. F. J. Colloid Interface Sci. 2000, 224, 162-168. (3) Fan, C.; Wang, H.; Sun, S.; Zhu, D.; Wagner, Gerhard; Li, G. Anal. Chem. 2001, 73, 2850-2854. (4) Han, X.; Huang, W.; Jia, J.; Dong, S.; Wang, E. Biosens. Bioelectron. 2002, 17, 741-746. (5) Lei, C.; Wollenberger, U.; Bistolas, N.; Guiseppi-Elie, A.; Scheller, F. W. Anal. Bioanal. Chem. 2002, 372, 235-239. (6) Rusling, J. F.; Forster, R. J. J. Colloid Interface Sci. 2003, 262, 1-15. (7) Zhang, Y.; He, P.; Hu, N. Electrochim. Acta 2004, 49, 1981-1988. (8) Zhang, W.; Huang, Y.; Dai, H.; Wang, X.; Fan, C.; Li, G. Anal. Biochem. 2004, 329, 85-90. (9) Shumyantseva, V. V.; Ivanov, Y. D.; Bistolas, N. F.; Scheller, W.; Archakov, A. I.; Wollenberger, U. Anal. Chem. 2004, 76, 6046-6052. (10) Liu, T.; Zhong, J.; Gan, X.; Fan, C.; Li, G.; Matsuda, N. ChemPhysChem 2003, 4, 1364-1366. (11) Gan, X.; Liu, T.; Zhong, J.; Liu, X.; Li, G. ChemBioChem 2004, 5, 16861691. (12) Joshi, P. P.; Merchant, S. A.; Wang, Youdan; Schmidtke, D. W. Anal. Chem. 2005, 77, 3183-3188. (13) Li, Q.; Luo, G.; Feng, J. Electroanalysis 2001, 13, 359-363.
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Actually, nanoparticles (NPs) or colloids are attracting growing attention owing to their specific characteristics of large specific surface area, high surface adsorption, and good biocompatibility. And nano titanium dioxide (TiO2) is a promising material that has been used in many fields.17-31 Here we report that hemoglobin (Hb) can exhibit direct electrochemistry and catalytic activity toward hydrogen peroxide after the protein is entrapped in the TiO2 NPs, and the photovoltaic effect of the nanomaterials can play an interesting role in electron-transfer reactivity and catalytic ability. The peroxidase activity of Hb can be interestingly influenced by the photovoltaic effect, and what is more interesting, a technique to fabricate a novel protein-based biosensor might be developed. EXPERIMENTAL SECTION Chemicals and Apparatus. Bovine Hb was obtained from Sigma and used without further purification. TiO2 NPs with a (14) Topoglidis, E.; Astuti, Y.; Duriaux, F.; Gratzel, M.; Durrant, J. R. Langmuir 2003, 19, 6894-6900. (15) Liu, S.; Ju, H. Analyst 2003, 128, 1420-1424. (16) Zhou, H.; Gan, X.; Liu T.; Yang, Q.; Li, G. J. Biochem. Biophys. Methodol., in press. (17) Honda, K.; Fujishima, A. Nature 1972, 238, 37-38. (18) O’Regan, B.; Gratzel, M. Nature 1991, 353, 737-740. (19) Henglein, A. Chem. Rev. 1989, 89, 1861-1873 (20) Kamat, P. V. Chem. Rev. 1993, 93, 267-300. (21) Alivisatos, A. P. J. Phys. Chem. 1996, 100, 13226-13239. (22) Yamazaki-Nishida, S.; Cervera-March, S.; Nagano, K. J.; Anderson, M. A.; Hori, K. J. Phys. Chem. 1995, 99, 15814-15821. (23) Vinodgopal, K.; Stafford, U.; Gray, K. A.; Kamat, P. V. J. Phys. Chem. 1994, 98, 6797-6803. (24) Wang, R.; Hashimoto, K.; Fujishima, A.; Chikuni, M.; Kojima, E.; Kitamura, A.; Shimohigoshi, M.; Watanabe, T. Nature 1997, 388, 431-432. (25) Wang, R.; Hashimoto, K.; Fujishima, A.; Chikuni, M.; Kojima, E.; Kitamura, A.; Shimohigoshi, M.; Watanabe, T. Adv. Mater. 1998, 10, 135-138. (26) Li, X.; Liu, H.; Cheng, L.; Tong, H. Environ. Sci. Technol. 2003, 37, 39893994. (27) Topoglidis, E.; Lutz, T.; Willis, R. L.; Barnett, C. J.; Cass, A. E. G.; Durrant, J. R. Faraday Discuss. 2000, 116, 35-46. (28) Wamer, W. G.; Yin, J.; Wei, R. Free Radical Biol. Med. 1997, 23, 851858. (29) Hidaka, H.; Horikoshi, S.; Serpone, N.; Knowland, J. J. Photochem. Photobiol. A: Chem. 1997, 111, 205-213. (30) Horikoshi, S.; Serpone, N.; Zhao, J.; Hidaka, H. J. Photochem. Photobiol. A: Chem. 1998, 118, 123-129. (31) Horikoshi, S.; Serpone, N.; Yoshikawa, S.; Hidaka, H. J. Photochem. Photobiol. A: Chem. 1999, 120, 63-74. 10.1021/ac050924a CCC: $30.25
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diameter of 35 nm were obtained from Nanjing High Technology Nano Co. Ltd. Other reagents were all of analytical grade. Water was purified with a Milli-Q purification system (Branstead) to a specific resistance of >16 MΩ cm-1 and was used to prepare all solutions. Electrochemical experiments were carried out with a VMP potentiostat (PerkinElmer) in a three-electrode system. A saturated calomel electrode (SCE) and a platinum wire electrode served as the reference and counter electrodes. All the potentials reported in this work were versus SCE. Preparation of Electrodes. The substrate pyrolytic graphite (PG) electrode was prepared by putting a PG rod into a glass tube and fixing with epoxy resin. Electrical contact was made by adhering a copper wire to the rod with the help of a Wood alloy. The PG electrode was first polished on rough and fine sandpapers. Then its surface was polished to mirror smoothness with an alumina (particle size of ∼0.05 µm)/water slurry on silk. Eventually, the electrode was thoroughly washed by ultrasonicating in both double-distilled water and ethanol for ∼5 min. Ten milligrams of TiO2 NPs was added into 10 mL of doubledistilled water, and the resultant mixture was ultrasonicated for 10-15 min. Then this TiO2 colloid was bioconjugated with Hb solution (6 mg mL-1, pH 8.0), in the volume proportion of 1/5, and spread evenly onto the surfaces of the PG electrodes. The electrode surfaces were covered with Eppendorf tubes in the first 2 h to prepare uniform films and dried overnight in the air. The modified electrodes were thoroughly rinsed with pure water and dried again. Finally, they were covered with Eppendorf tubes directly facing UV-visible light and were irradiated at a distance of 10 cm. Electrochemical Measurements. Prior to the experiments, the test buffer solution was first bubbled thoroughly with highpurity nitrogen for at least 5 min. Then a stream of nitrogen was blown gently across the surface of the solution in order to maintain an anaerobic solution throughout the experiment. Cyclic voltammetry was carried out in the scan range from 200 to -800 mV. All experiments were carried out at a temperature of 25 ( 0.5 °C. RESULTS AND DISCUSSION Figure 1 shows that a pair of well-defined and reproducible peaks, which belong to the redox process of Hb, can be observed at the PG electrode modified with TiO2 NPs and Hb bioconjugates, with a formal potential at -0.353 V, in a pH 7.0 phosphate buffer solution (PBS). Further studies reveal that, after addition of H2O2 in the test solution, the reduction peak current of Hb will increase, accompanied by the decrease and even disappearance of the oxidation peak, as demonstrated in Figure 2. The reduction peak current increases linearly with the H2O2 concentration ranging from 9 × 10-6 to 1 × 10-4 M (R ) 0.99), with a reactive sensitivity of 9.53 µA mM-1, which makes it possible to fabricate an Hbbased H2O2 biosensors. The detection limit is 3 × 10-6 M. It should be mentioned that the above experiments are performed in the dark with no photovoltaic effect of nano TiO2 being activated, although the nanomaterial has provided a favorable environment for the electron-transfer reactions of Hb. However, when the catalytic reaction takes place after the ultraviolet (UV) irradiation on the Hb-TiO2 NP film for some time, the current of the catalytic reduction peak will increase much more
Figure 1. Cyclic voltammograms obtained at TiO2 NP-modified PG electrode (dashed curve) and Hb-TiO2 NP-comodified PG electrode (solid curve) in 0.1 M phosphate buffer with pH 6.0. Scan rate: 200 mV/s.
Figure 2. Cyclic voltammograms obtained at Hb-TiO2 NP bioconjugate-modified PG electrode for 0.1 M pH 7.0 PBS in the presence of 0 (the solid curve), 1 × 10-7, 5 × 10-7, 1 × 10-6, 1 × 10-5, 5 × 10-5, 1 × 10-4, and 3 × 10-4 M (from inner to outer) H2O2 concentration. The experiments are performed in the dark.
significantly along with the increase of H2O2 concentration, compared to that in the dark. As shown in Figure 3, after UV irradiation for 1 h, the sensitivity of catalysis increases to 12.51 µA mM-1 with a detection limit of 2 × 10-7 M. After irradiation for a period of 5 h, the sensitivity keeps increasing to 25.02 µA mM-1, and the detection limit decreases to 1 × 10-8 M (RSD ) 2.5%, n ) 5). So the sensitivity increases nearly 3-fold and the detection limit lowers by 2 orders, in contrast to the catalytic reactions in the dark. The greatly enhanced enzymatic activity of Hb is related to the extent of UV irradiation. The longer the UV irradiation time, the higher the catalytic reduction currents. And this phenomenon is due to the photovoltaic effect of TiO2 NPs. Comparative experiments have revealed that Hb embedded in other films, such as poly(ethylene glycol), nano cadmium sulfide and nano zinc oxide cannot exhibit an increased catalytic activity after UV irradiation. On the contrary, after UV irradiation, both the direct electrochemical response of the protein and the catalytic reduction peak will be decreased by using poly(ethylene glycol) and nano Analytical Chemistry, Vol. 77, No. 18, September 15, 2005
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Figure 3. Linear fitting relationship between the catalytic reduction peak and H2O2 concentration after the UV irradition on the Hb-TiO2 NP film for (I) 0, (II) 1, and (III) 5 h.
follows. After irradiation of UV light on TiO2 NPs, a series of reactions will take place, which will produce hydroxyl radicals, superoxide anion, ozone, and electron pairs.32 The highly reactive molecules, such as hydroxyl radicals and superoxide anions, may have an effect on the heme rings of Hb as well as some amino acid residues of the protein framework, which may finally lead to the enhancement of catalytic activity of hemoglobin. Certainly, more studies should be carried out to get to know the detailed reasons for the great enhancement of catalytic activity caused by the photovoltaic effect on Hb. Nevertheless, we have demonstrated that Hb, taking advantage of the photovoltaic effect of TiO2 NPs, can catalyze the H2O2 reduction more efficiently, and behave as a nice peroxidase with a higher sensitivity, reactivity, and a lower detection limit, which is very important in biosensor fabrication. The photovoltaic effect of TiO2 NPs can greatly promote the catalytic ability of Hb by controlling the extent of UV irradiation, which indicates a possible method to develop new kinds of photocontrolled protein-based biosensors. ACKNOWLEDGMENT
cadmium sulfide, while UV irradiation has no apparent effect with nano zinc oxide. On the other hand, titanium oxide powder cannot give this result either. Therefore, the photoexcited TiO2 NPs play an important role in the promotion of the catalytic ability of this nonnatural enzyme. The TiO2 NPs themselves cannot work, since no redox peak related to H2O2 can be observed at TiO2 NPs-only modified electrode. We have also tested the photovoltaic effect with some other proteins, but Hb has proved to be much more sensitive to the influence of the photovoltaic effect. So there should be some specific interaction between TiO2 NPs and Hb. Currently, it is not very clear how the photovoltaic effect of TiO2 NPs works. A preliminary explanation can be addressed as
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This work is supported by the National Natural Science Foundation of China (Grant 90406005) and the Program for New Century Excellent Talents in University, the Chinese Ministry of Education.
Received for review May 26, 2005. Accepted August 4, 2005. AC050924A
(32) Al-Ekabl, H.; Serpone, N. J. Phys. Chem. 1988, 92, 5726-5731.
