1. 基于纳米金和硫堇固定酶 的过氧化氢生物传感器 答辩人：陈贤光（ 03 应化） 指导老师：童叶翔 教授 邹小勇 教授 2006.6 广州

2. Hydrogen Peroxide Biosensor Based on Immobilizing Enzyme by Gold Nanoparticles and Thionine ① CHEN, Xian-Guang ZOU, Xiao-Yong TONG, Ye-Xiang* School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, China ① This paper will submit to Acta Chim. Sinica.

3. 1. Introduction 1.1 Mechanism of Hydrogen Peroxide Biosensor Guilbault G. G., et. al. Anal. Chim. Acta., 1973, 64: 439-445.

4. 1. Introduction 1.2 Fabrication of hydrogen peroxide biosensor made in our work

5. 1. Introduction 1.3 Catalytic reaction of H 2 O 2 appear in the system

6. 2. Results and Discussion 2.1 Prepare GNs by sol-gel method Fig. 1 TEM image of GNs. ( d = 20 nm )

7. 2. Results and Discussion 2.2 Electrochemical impedance spectrum (EIS) Fig. 2 Nyquist plot of EIS of the electrodes (a) and equivalent circuit (b) of the biosensor. (a) (b) R app : electrode apparent resistance; R sol : solution resistance; R ct : charge transfer resistance. Electrode R app / Ω R sol / Ω R ct / Ω a1179 93261724 b2208 96236334 c3536 98823780 d5016 144.67580 Table 1 Electrochemistry parameters

8. 2. Results and Discussion 2.3 Atomic force microscopy (AFM) images Fig. 3 Three-dimensional AFM images of bare Pt (a), Pt/GNs (b), Pt/GNs/Thio (c) and Pt/GNs/Thio/(HRP-GNs) (d) electrode.

9. 2. Results and Discussion 2.4 Cyclic voltammgrams (CVs) (b) Fig. 5 CVs of electrocatalysis of the biosensor to H 2 O 2 in pH 7.0 PBS containing 1×10 -3 mol/L hydroquinone. (a) Fig. 4 CVs (a) of the biosensor at different scan rates in blank pH 7.0 PBS and the linear calibration curve between scan rates and cathode peak currents (b).

10. 2. Results and Discussion 2.5 Some factors effect on the response currents Fig. 6 Effect of applied potential on the response currents of the biosensor to 2×10 -4 mol/L H 2 O 2. Other conditions are as Fig. 5. Fig. 7. Effect of pH of PBS on the response currents of the biosensor to H 2 O 2 at -0.15V. Other conditions are as Fig. 6.

11. 2. Results and Discussion 2.5 Some factors effect on the response currents (b) (a) Fig. 8. Effect of temperature on the response currents of the biosensor to H 2 O 2 at -0.15V (a) and linear calibration curve between lni and T -1 (b). Other conditions are as Fig. 7. (Activation energy for enzymatic reaction of the biosensor was 12.40 kJ/mol.)

12. 2. Results and Discussion 2.6 Spectra study of the effect between HRP and GNs Fig. 9 UV-Vis spectra of HRP in the absence (a) and presence (b) of GNs. Fig. 10 FT-IR spectra of HRP in the absence (a) and presence (b) of GNs.

13. Fig. 11 Circular dichroism spectra of HRP in the absence (a) and presence (b) of GNs. Other conditions are as Fig. 10. Fractions of secondary structure: α : α-helix ; β : β-sheet ; t : turn ; r : random. System α/ ％α/ ％ β/ ％β/ ％ t / % r/ %r/ % HRP28.5 029.941.6 HRP + GNs27.7 031.241.1 Table 2 Secondary structures of HRP 2. Results and Discussion 2.6 Spectra study of the effect between HRP and GNs

14. 2. Results and Discussion 2.7 Applied performance of the biosensor to H 2 O 2 (b) (a) Fig. 12. Typical current-time response curve (the inset is calibration curve ) for successive addition of 1×10 -4 mol/L H 2 O 2 (a) and linear calibration curve between i -1 and C H 2 O 2 -1 (b). Other conditions are as Fig. 7. (Apparent Michaelis-Menten constant of the biosensor was 6.5×10 -4 mol/L.)

15. 2. Results and Discussion 2.8 Applied performance of the bienzymebased biosensor to glucose Scheme 1 The possible catalytic reaction mechanism of the bienzymebased biosensor to glucose

16. 2. Results and Discussion 2.8 Applied performance of the bienzymebased biosensor to glucose Fig. 13. Typical current-time response curve and calibration curve (inset) for successive addition of 5×10 -4 mol/L glucose at -0.1V in pH 7.0 PBS containing 1×10 -3 mol/L hydroquinone.

17. 3. Conclusion The covalent bond and electrostatic adsorption effect between GNs and Thio could immobilize enzyme firmly, retaining the biology structure and catalytic ability of enzyme. Followed by adding a Chit membrane on outmost surface of the electrode, a sensitive and stabile H 2 O 2 biosensor was obtained. The results of EIS and AFM test confirmed the fabrication procedure was effective. And a bienzymebased glucose biosensor had been constructed successfully by this method.

18. 4. Production [1] Chen Xianguang, Zhao Guofang, Zou Xiaoyong*. An amperometric glucose biosensor based on gold nanoparticles (in Chinese). (Submitted to Chin. J. of Anal. Chem.) [2] Chen Xianguang, Zou Xiaoyong, Tong Yexiang*. Hydrogen peroxide biosensor based on immobilizing enzyme by gold nanoparticles and thionine (in Chinese). (Submitting to Acta Chim. Sinica.)

19. This project was financially supported by the Foundation for Innovative Chemical Experiment Research of School of Chemistry and Chemical Engineering, Sun Yat-Sen University (2006-15); the Foundation of Students Research Training of Sun Yat-Sen University (2005); the Open Laboratory Foundation of Sun Yat-Sen University (2005); and the National Natural Science Foundation of China (Nos. 20573136, 20575082). 5. Acknowledgement

20. 致谢 感谢童叶翔教授和邹小勇教授 在本课题研究期间对本人的指导。