1. An Analysis of Variance of Detection of Hydrogen Peroxide for Carbon Paste Electrode Modified with Copper Hexacyanoferrate Chia-Cheng Hsiao ( 蕭佳政 ), Chih-Ying Wu ( 巫致穎 ), 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 Due to hydrogen peroxide is widely used in the industry and food preservation, developing a hydrogen peroxide sensor which can detect the hydrogen peroxide rapidly and conveniently 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 rapid and convenient glucose biosensor also has become an important research subject. Because the copper( Ⅱ ) hexacyanoferrate possesses the excellent catalytic characteristic it can be used with the carbon paste and graphite carbon powders which possess the excellent conductivity to make the carbon paste electrode and elevate the responding current of the hydrogen peroxide. The carbon paste electrode is used to detect the responding current of hydrogen peroxide in phosphate buffer solution (PBS) and the concentration of hydrogen peroxide can be determined from the responding current of hydrogen peroxide. The glucose and oxygen can be catalyzed by the glucose oxidase to produce the gluconic acid and hydrogen peroxide, and therefore, as the concentration of hydrogen peroxide can be determined, the concentration of glucose can also be determined. A study of factorial design involving three factors (operating potential, stirring rate, and pH value of PBS) and two levels was performed to analyze the effect of reaction parameters on the sensitivity of detection of hydrogen peroxide for the carbon paste electrode modified with Copper( Ⅱ ) Hexacyanoferrate (Cu( Ⅱ )HCF). The results showed that the main effect of operating potential ( F = 31.755, p value ＜ 0.05 ) and the main effect of pH value ( F = 27.798, p value ＜ 0.05 ) were significant on the sensitivity of detection of hydrogen peroxide. At 30 ℃, -200 mV operating potential, 500 rpm stirring rate and in 0.05 M PBS buffer solution ( pH = 7.4 ), when the carbon paste electrode was modified with Copper( Ⅱ ) Hexacyanoferrate (Cu( Ⅱ )HCF) [Cu( Ⅱ )HCF : graphite carbon powders : carbon paste = 0.3 ： 0.7 ： 1 ( weight ratio )], the detection limit was 0.02 mM H 2 O 2, the linear range was 0.02 ～ 2.6 mM H 2 O 2, R 2 = 0.9945, and the sensitivity was 121.606 µA/cm 2 ． mM H 2 O 2. Fig 1. The theory of detection of glucose for the biosensor CuSO 4 ． 5H 2 O (aq) (MERCK ） 3(CuSO 4 ． 5H 2 O)+ 2 〔 K 3 [Fe(CN) 6 ] 〕 Cu 3 [Fe(CN) 6 ] 2 +3K 2 SO 4 +15H 2 O Coprecipitation method ： K 3 Fe(CN) 6(aq) （ MERCK ） Cu 3 [Fe(CN) 6 ] 2 (aq) Separation of side product Drying at 60 ℃ (48 hours) copper( Ⅱ ) hexacyanoferrate Stoichiometry 3 ： 2 (mole ratio) INTRODUCTIO N EXPERIMENTAL RESULTS AND DISCUSSION Fig 2. CV graphs for (A) carbon paste electrode modified with copper 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) CONCLUSIONS The results showed that the responding current for the carbon paste electrode modified with Copper( Ⅱ ) Hexacyanoferrate was elevated significantly. For the factorial design, the results showed that the main effect of operating potential ( F = 31.755, p value ＜ 0.05 ) and the main effect of pH value ( F = 27.798, p value ＜ 0.05 ) were significant on the sensitivity. Because the pH of human blood is about 7.4, pH = 7.4 was used in this research. The results showed that the optimum operating conditions for this research were operating potential = –200mV, stirring rate = 500 rpm, and pH = 7.4. Also the results showed that at –200mV operating potential, 500 rpm stirring rate and in 0.05M PBS buffer solution (pH=7.4), the detection limit was 0.02 mM H 2 O 2, the linear range was 0.02~2.6 mM H 2 O 2, R 2 =0.9945 and the sensitivity was 121.606 μA/cm 2 ּmM H 2 O 2. 1. D. C. Montgomery, “ Design and Analysis of Experiments,” 5th Edition, 170 (2001). 2. S. Timur, U. Anik, D. Odaci and L. Gorton, “Development of a Microbial Biosensor Based on Carbon Nanotube (CNT) Modified Electrodes,” Electrochemistry Communications, 9, 1810 (2007). 3. Y. Liu, J. Lei and H. Ju, “Amperometric Sensor for Hydrogen Peroxide Based on Electric Wire Composed of Horseradish Peroxidase and Toluidine Blue-Multiwalled Carbon Nanotubes Nanocomposite,” Talanta, 74, 965 (2008). REFERENCES Preparation of Copper( Ⅱ ) Hexacyanoferrate(Cu( Ⅱ )HCF) ： N2N2 Temperature at 30 ℃ CounterElectrode PtPt Working Electrode ReferenceElectrode Ag/AgCl pH 7.4 之 0.05 M PBS Buffer Solution ※ Three Electrodes System ： Preparation of the working electrode : 1. 7 cm 0.5 cm 0.05 cm Surface area of electrode=0.0805cm 2 The mixing of Cu( Ⅱ )HCF and carbon powders was even. Mixing with equal amount of carbon paste 銅 芯 電 線 2. Then the Copper( Ⅱ ) Hexacyanoferrate powders, graphite carbon powders and carbon paste were mixed with the appropriate ratio (Copper( Ⅱ ) Hexacyanoferrate : 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. Table 1 Three factors and two levels of the factorial design Table 3 The results of analysis of variance for the sensitivity of detection of hydrogen peroxide for the carbon paste electrode modified with Copper( Ⅱ ) Hexacyanoferrate Table 4 The TB graphs of carbon paste electrode electrodes for detection of the detection limit of H 2 O 2 (Copper( Ⅱ ) Hexacyanoferrate: graphite carbon powders = 3 : 7, pH= 7.4 ) Table 5 The TB graphs of carbon paste electrodes for detection of the linear range of H 2 O 2 (Copper( Ⅱ ) Hexacyanoferrate : graphite carbon powders = 3 : 7, pH= 7.4 ). Table 2 The results of the sixteen experiments of the factorial design ( A: Operating Potential(mV), B: Stirring Rate(rpm), C: pH valus )