1. The Detection of Hydrogen Peroxide and Glucose for the Electrode Modified with Ferrocene Yi-Sheng Wang ( 汪乙生 ), Ting-Li Lin ( 林庭立 ), Hau Lin ( 林浩 ) Department of Chemical and Materials Engineering, Southern Taiwan University 南台科技大學化學工程與材料工程系 ABSTRACT A study was conducted to use the ferrocene to modify the carbon paste electrode. The TB (Time Base) graphs for different operating potentials, and pH values were plotted to determine the optimum operating conditions. The optimum weight ratio for carbon paste : ferrocene : graphite carbon powders was carbon paste : ferrocene : graphite carbon powders =1 : 0.3 : 0.7. The results showed that the responding current for the carbon paste electrode modified with ferrocene was elevated significantly. The optimum operating conditions are -200mV operating potential, 500rpm stirring rate and in 0.05 M PBS buffer solution( pH = 7.4 ). For the glucose biosensor, the detection limit was 0.06 mM C 6 H 12 O 6 ; the linear range was 0.06 ~ 2 mM C 6 H 12 O 6 ; R 2 = 0.992 and the sensitivity was 83.1 μA/ cm 2 ． mM C 6 H 12 O 6. INTRODUCTION Nowadays, diabetes is one of the top ten causes of death for the people in Taiwan. Therefore, developing a rapid, convenient, and economical glucose biosensor for detecting the glucose is a very important research subject. The glucose and oxygen can be catalyzed by the glucose oxidase and the glucose is oxidized to gluconic acid and the oxygen is reduced to hydrogen peroxide. Because the ferrocene (Fe(C 5 H 5 ) 2 ) possesses the excellent conductivity and catalytic characteristic, it can be used to elevate the responding current for detection of reduction of hydrogen peroxide. A study was conducted to use the ferrocene to modify the carbon paste electrode which was used as the working electrode to detect the responding current of reduction of hydrogen peroxide in the phosphate buffer solution(PBS) and then the concentration of hydrogen peroxide could be obtained from the responding current and consequently, the sensitivity of the glucose biosensor could be determined. EXPERIMENTAL SECTION Equipment Electrochemical Analyzer (BAS 100W) was used to measure the activity of electrode by Cyclic Voltammetry ( CV ) and Time Base ( TB ) mode ; pH meter (Metrohm 731); Constant Temperature Thermal Bath (Wisdom BC-2DT 10L); Oven (DENG YNG) ; Electric Stirrer (Fargo); Carbon Paste Electrode was used as the working electrode, Coiled Platinum Wire was used as the counter electrode and Ag / AgCl was used as the reference electrode. Chemicals and Reagents Ferrocene(Fe(C 5 H 5 ) 2 ); Hydrogen Peroxide (H 2 O 2 ); D(+)-Glucose Monohydrate(C 6 H 12 O 6 ); Glucose Oxidase(EC 1.1.3.4, Type X-S: From Aspergillus Niger, 50000 units/mg); Graphite Carbon Powder; Carbon Paste; Cyclohexanone(C 6 H 10 O); Nafion ; Potassium Dihydrogenphosphate (KH 2 PO 4 ); Potassium Chloride (KCl). Procedure (1) Take one section of 7 cm electric wire with 0.05 cm inside diameter. After depriving the coating 0.5 cm length from both ends, the nake-ended wire was washed, dried and ready for use. 7 cm 0.05cm 0.5 cm (2) Then the ferrocene powders, graphite carbon powders and carbon paste were mixed with the appropriate ratio (ferrocene : graphite carbon powders : carbon paste = 0.3 : 0.7 : 1). After the mixing was complete, the mixture was evenly coated on the nake- ended electric wire and dried in the oven and then we obtained the carbon paste electrode. The surface area of the carbon paste electrode was 0.0805 cm 2. Ferrocene Mixing with carbon paste Graphite carbon powders Coated on the nake- ended electric wire glucose oxidase1 % Nafion PVC cover (3) After the above mentioned carbon paste electrode was dried, the 2μL glucose oxidase solution( 3mg of glucose oxidase was dissolved in 500μL PBS buffer solution ) was put onto the surface of electrode and the electrode was dried at room temperature. Then 5μL of 1% Nafion solution(in 95% alcohol) was dropped onto the electrode evenly and after the electrode was dried at room temperature, we obtained the glucose biosensor. RESULTS AND DISCUSSION Fig 1. CV graphs for (A) carbon paste electrode modified with ferrocene (B) unmodified carbon paste electrode; the range of scanning potential: -0.8 ～ +0.8 V, scanning rate = 50 mV/s Fig. 3 The TB graphs of carbon paste electrode for detection of H 2 O 2 at different pH values of PBS buffer solution (ferrocene : graphite carbon powders = 3 : 7); the pH values are [ (A) pH = 6.0 (B) pH = 7.0 (C) pH = 7.4 (D) pH = 8.0 ] Fig. 4 TB graphs of the carbon paste electrodes with different ratios of carbon paste to ferrocene to graphite carbon powders; the carbon paste to ferrocene to graphite carbon powders are 〔 (A) 1 : 0.3 : 0.7 (B) 2 : 0.3 : 0.7 (C) 3: 0.3 : 0.7 〕 Fig.5 The TB graph of glucose biosensor for detection of the detection limit of glucose (ferrocene : graphite carbon powders = 3 : 7). Fig. 6 The TB graph of glucose biosensor for detection of glucose (ferrocene : graphite carbon powders = 3 : 7); At 30 ℃ ; the operating potential = –200 mV; in 0.1 M KCl of 5 mL 0.05 M PBS buffer solution ( pH=7.4 ); stirring rate=500 rpm; 20μL of 100mM glucose is injected per 100 seconds CONCLUSIONS The results showed that the responding current for the carbon paste electrode modified with the ferrocene was elevated significantly. Because at –300mV operating potential, it might cause the interference of the interfering substances in the human blood, –200mV operating potential was used in this research. Also because the pH of human blood is about 7.4, pH = 7.4 PBS buffer solution was used in this research. The optimum operating conditions are -200mV operating potential, 500rpm stirring rate and in 0.05 M PBS buffer solution ( pH = 7.4 ). The optimum weight ratio for carbon paste : ferrocene : graphite carbon powders was1 : 0.3 : 0.7. For the glucose biosensor, the detection limit was 0.06 mM C 6 H 12 O 6 ; the linear range was 0.06 ~ 2 mM C 6 H 12 O 6 ; R 2 = 0.996 and the sensitivity was 83.1 μA/cm 2 ． mM C 6 H 12 O 6. REFERENCES [1] M. A. Kim and W.-Y. Lee, “Amperometric Phenol Biosensor Based on Sol-Gel Silicate/Nafion Composite Film,” Analytica Chimica Acta, 479, 143 (2003). [2] L. Qian and X. Yang, “Composite Film of Carbon Nanotubes and Chitosan for Preparation of Amperometric Hydrogen Peroxide Biosensor,” Talanta, 68, 721(2006). [3] M. M. Musameh, R. T. Kachoosangi, L. Xiao, A. Russell and R. G. Compton,, “Ionic Liquid-Carbon Composite Glucose Biosensor,” Biosensors and Bioelectronics, 24, 87 (2008). (A) (B) Fig. 2 The TB graphs of carbon paste electrode for detection of H 2 O 2 at different operating potentials (ferrocene : graphite carbon powders = 3 : 7); the operating potentials are [ (A) –300mV (B) –200mV (C) –100mV ]