RESULTS AND DISCUSSION Application of Planar Electrode Modified with Ferrocene to Glucose Biosensor Yi-Sheng Wang(汪乙生), Chung-Min Lien(連崇閔), Hau Lin(林浩) Department of Chemical and Materials Engineering, Southern Taiwan University ABSTRACT . A study was conducted to use the ferrocene to modify the screen printed planar electrode which was used as the working electrode to detect the responding current of reduction of hydrogen peroxide and then the concentration of hydrogen peroxide could be obtained from the responding current and consequently, the concentration of the glucose could be obtained. The results showed that the responding current for the carbon paste electrode modified with the ferrocene was elevated significantly. The optimum weight ratio for ferrocene : graphite carbon powders was 3 : 7 . At 30℃ , -0.2V operating potential, and in 0.05 M PBS buffer solution( pH = 7.4 ), when the screen printed planar electrode was modified with the ferrocene [ferrocene : graphite carbon powders = 3 : 7 ( weight ratio )] , the detection limit was 0.01 mM H2O2 , the linear range was 0.01～0.4 mM H2O2, R2=0.994, and the sensitivity was 238.27 μA/cm2ּmM H2O2 . For the glucose screen printed planar sensor, the detection limit was 0.06 mM C6H12O6 ; the linear range was 0.06 ~ 2.5 mM C6H12O6 ; R2 = 0.997 and the sensitivity was 5.12 μA/cm2 ．mMC6H12O6 . RESULTS AND DISCUSSION (A) (B) (A) (B) (C) (D) Fig. 2 TB graphs of the carbon paste electrodes with different ratios of ferrence to graphite carbon powders; the ferrence to graphite carbon powders were 〔(A) 3:7 (B) 2:8 (C) 1:9 (D) 0:10 (unmodified carbon paste electrode ) 〕; the operating potential = –0.2 V; in 0.1 M KCl of 5 mL 0.05 M PBS buffer solution (pH =7.4); stirring rate =500 rpm; after deoxygenating by purging nitrogen gas for 20 minutes,10 μL of 50mM H2O2 is injected per 100 seconds Fig 1. CV graphs for (A) carbon paste electrode modified with ferrocene (B) unmodified carbon paste electrode; after deoxygenating by purging nitrogen gas for 20 minutes; in 0.1 M KCl of 5 mL 0.05 M PBS buffer solution (pH =7.4) the range of scanning potential: -0.8～+0.8 V, scanning rate = 50 mV/s INTRODUCTION In recent years, diabetes has become 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 an 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(C5H5)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 and screen printed planar electrode which were used as the working electrodes 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 concentration of the glucose could be determined. EXPERIMENTAL Chemicals and Reagents Ferrocene(Fe(C5H5)2); Hydrogen Peroxide (H2O2); D(+)-Glucose Monohydrate(C6H12O6); Glucose Oxidase(EC 1.1.3.4, Type X-S: From Aspergillus Niger, 50000 units/mg); Graphite Carbon Powder; Carbon Paste; Cyclohexanone(C6H10O); Nafion; Potassium Dihydrogenphosphate (KH2PO4); Potassium Chloride (KCl). 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); Carbon Paste Electrode and Screen Printed Planar Electrode were used as the working electrodes, Coiled Platinum Wire was used as the counter electrode and Ag / AgCl was used as the reference electrode. Fig.4 The TB graph of screen printed planar electrodes for detection of the linear range of H2O2 (ferrocene : graphite carbon powders = 3 : 7) Fig. 3 The TB graph of screen printed planar electrodes for detection of the detection limit of H2O2 (ferrocene : graphite carbon powders = 3 : 7) Procedure 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. Then the ferrocene powders, graphite carbon powders and carbon paste were mixed with the appropriate ratio. 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 cm2 . 7 cm 0.5 cm 0.05 cm Fig. 6 The TB graph of screen printed planar electrode for detection of the linear range of glucose (ferrocene : graphite carbon powders = 3 : 7) Fig. 5 The TB graph of screen printed planar electrode for detection of the detection limit of glucose (ferrocene : graphite carbon powders = 3 : 7) Different weights of ferrocene Mixing with equal weight of carbon paste Different weights of carbon powders Coated on the nake-ended electric wire CONCLUSIONS The results showed that the responding current for the carbon paste electrode modified with the ferrocene was elevated significantly. The optimum weight ratio for ferrocene : graphite carbon powders was 3 : 7 . At 30℃ , -0.2V operating potential, stirring rate = 500 rpm and in 0.05 M PBS buffer solution( pH = 7.4 ), when the screen printed planar electrode ( the surface area of the screen printed planar electrode was 0.2827 cm2 )was modified with the ferrocene [ferrocene : graphite carbon powders = 3 : 7 ( weight ratio )] , the detection limit was 0.01 mM H2O2 , the linear range was 0.01～0.4 mM H2O2, R2=0.994, and the sensitivity was 238.27 μA/cm2ּmM H2O2 . For the glucose screen printed planar sensor, the detection limit was 0.06 mM C6H12O6 ; the linear range was 0.06 ~ 2.5 mM C6H12O6 ; R2 = 0.997 and the sensitivity was 5.12 μA/cm2 mM C6H12O6 . The above mentioned mixture with the appropriate ratio was evenly coated on the front side of screen plate and then the PE paper was placed under the screen plate and the plastic plate was used to print the mixture on the PE paper evenly. The electrode was dried in the oven. After the electrode was dried, the 2μL glucose oxidase solution was put onto the electrode and the electrode was dried at room temperature. Then 5μL of 1% Nafion solution was dropped onto the electrode evenly and after the electrode was dried at room temperature, we obtained the screen printed planar glucose electrode. REFERENCES 1. L. C. Clark and C. Lyons, “Electrode Systems for Continuous Monitoring in Cardiovascular Surgery, ” Annals New York Academy of Sciences, 102, 29 (1962). P. C. Pandey and S. Upadhyay, “Bioelectrochemistry of Glucose Oxidase Immobilized on Ferrocene Encapsulated Ormosil Modified Electrode,” Sensors and Actuators B, 76,193 (2001). 3.M. A. Kim and W.-Y. Lee, “Amperometric Phenol Biosensor Based on Sol-Gel Silicate/Nafion Composite Film,” Analytica Chimica Acta, 479, 143 (2003). 4. Y.-M. Uang and T.-C. Chou , “Fabrication of Glucose Oxidase/Polypyrrole Biosensor by Galvanostatic Method in Various pH Aqueous Solutions,” Biosensors and Bioelectronics, 19, 141 (2003). the 2μL glucose oxidase solution was put onto the electrode and the electrode was dried 1 2 5μL of 1% Nafion solution was dropped onto the electrode evenly