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The Effect of the pH of Phosphate Buffer Solution on the Detection of Hydrogen Peroxide for the Carbon Paste Electrode Modified with Ruthenium Hexacyanoferrate Kuo-Hsiang Liao (廖國翔) , Chung-Min Lien (連崇閔) , 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： Hydrogen peroxide sensor is an important research subject . In recent years, diabetes has become one of the top ten causes of death for the people in Taiwan. Therefore, developing a glucose biosensor which can detect glucose rapidly and conveniently is also an important research subject. The glucose and oxygen can be catalyzed by the glucose oxidase to produce the gluconic acid and hydrogen peroxide. Because the ruthenium hexacyanoferrate(Ⅱ) 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 ruthenium hexacyanoferrate(Ⅱ) to modify 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( pH = 7.4 )and the sensitivity of detection of hydrogen peroxide was determined from the responding current and consequently, the concentration of the glucose could be determined. The results showed that the responding current for the carbon paste electrode modified with the ruthenium hexacyanoferrate(Ⅱ) was elevated significantly. At 30℃, -200 mV operating potential, 600 rpm stirring rate and in 0.05 M phosphate buffer solution (PBS), the TB (Time Base) graphs for the carbon paste electrode at different pH of buffer solution were plotted to evaluate the effect of pH of buffer solution on the responding current of detection of hydrogen peroxide. At the optimum operating conditions -200 mV operating potential, 600 rpm stirring rate and in 0.05 M PBS buffer solution ( pH = 7.4 ) , when the carbon paste electrode was modified with ruthenium hexacyanoferrate(Ⅱ) [ruthenium hexacyanoferrate(Ⅱ) : carbon powders : carbon paste = 0.3：0.7：1 ( weight ratio )] , the detection limit was 0.02 mM H2O2 , the linear range was 0.02～2.7 mM H2O2 , R2 = 0.9996 , and the sensitivity was 661.8 µA/cm2．mM H2O2 . INTRODUCTION ： RESULTS AND DISCUSSION ： ( ) A study was conducted to use the ruthenium hexacyanoferrate(Ⅱ) to modify the carbon paste electrode which was 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. At 30℃, -200mV operating potential, and in 0.05 M phosphate buffer solution (PBS), the TB (Time Base) graphs for the carbon paste electrode modified with different pH of buffer solutions were plotted to evaluate the effect of pH of buffer solution on the responding current of detection of hydrogen peroxide. After the optimum conditions were determined, the carbon paste electrode was used to determine the sensitivity of detection of hydrogen peroxide. EXPERIMENTAL SECTION： ( ) Preparation of Ruthenium Hexacyanoferrate ： 30 mM Potassium Hexacyanoferrate 5 mL Fig 1. CV graphs for (A) carbon paste electrode modified with ruthenium hexacyanoferrate( the range of scanning potential: -0.8～+0.8 V) (B) unmodified carbon paste electrode( the range of scanning potential: -0.6～+0.6 V) Drying Centrifuging Repeat Centrifuging Fig. 2 The TB graphs of carbon paste electrodes for detection of H2O2 at different pH of buffer solutions ( ruthenium hexacyanoferrate : graphite carbon powders = 3 : 7); the pH values are [ (A) pH=4(B) pH=5(C) pH=6 (D) pH=7(E) pH=7.4(F) pH=8] Ruthenium Hexacyanoferrate Powders Three Times 3 mM Ruthenium Chloride Hydrate 50 mL Preparation of Working Electrode： 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 0.05 cm 0.5 cm 2. Then the ruthenium hexacyanoferrate(Ⅱ) powders, carbon powders and carbon paste were mixed with the appropriate ratio. Fig. 3 The calibration curves of different pH of buffer solutions for the carbon paste electrode modified with ruthenium hexacyanoferrate [ (A) pH=4(B) pH=5(C) pH=6 (D) pH=7(E) pH=7.4 (F) pH=8 ] Table 1 The sensitivities, responding currents, and R2 values of different pH of buffer solutions for the carbon paste electrode modified with ruthenium hexacyanoferrate Fig. 4 The TB graphs of carbon paste electrodes for determining the detection limit of H2O2 (A) stirring rate 600rpm (B) stirring rate 500rpm(C) unmodified carbon paste electrode with stirring rate 600rpm Fig. 5 The TB graphs of carbon paste electrodes for adding carbon paste ruthenium hexacyanoferrate ( appropriate ratio ) carbon powder A powder appropriate A powder 3. 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. 4. After the above mentioned carbon paste electrode was dried, the 3μL glucose oxidase solution( 3mg of glucose oxidase was dissolved in 200μL 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. Nafion Glucose Oxidase PVC cover determining the linear range of H2O2 (A) stirring rate 600rpm (B) stirring rate 500rpm(C) unmodified carbon paste electrode with stirring rate 600rpm CONCLUSIONS ：The results showed that the responding current for the carbon paste electrode modified with the ruthenium hexacyanoferrate(Ⅱ) was elevated significantly. The results of TB (Time Base) graphs showed that when pH= 8 the responding current was the highest. Because the pH value of human blood is about 7.4, the buffer solution of pH=7.4 was used in this research. The optimum weight ratio for ruthenium hexacyanoferrate(Ⅱ) : carbon powders was 3 : 7 . For the hydrogen peroxide sensor, at 30℃ , -200mV operating potential, and in 0.05 M PBS buffer solution( pH = 7.4 ), when the carbon paste electrode was modified with the ruthenium hexacyanoferrate(Ⅱ) [ruthenium hexacyanoferrate(Ⅱ) : carbon powders = 3 : 7 ( weight ratio )] , the detection limit was 0.02 mM H2O2 , the linear range was 0.02～2.7 mM H2O2 , R2 = 0.9996 , and the sensitivity was 661.8 µA/cm2．mM H2O2 . REFERENCES ： 1.Garjonyte, R. ; Malinauskas, A., Sensors and Actuaors B, 2000, 63, 122-128. 2. Pandey, P. C. ; Upadhyay, S., Sensors and Actuators B, 2001, 76, 193-198. 3. Uang, Y.-M. ; Chou, T.-C., Biosensors and Bioelectronics, 2003, 19, 141-147. 4. Singh, S. ; Chaubey, A. ; Malhotra, B. D., Analytica Chimica Acta, 2004, 502, 229-234. 5. Liu, Y. ; Lei, J. ; Ju, H., Talanta, 2008, 74, 965-970.