Amperometric H2O2 biosensors based on horse radish peroxidase and catalase immobilized at nanomaterial modified electrodes

• Main Authors: Shan-Wei Ting, 丁山圍
• Other Authors: 陳生明
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/4cp5v8

碩士 === 國立臺北科技大學 === 化學工程研究所 === 99 === Part I：In this work, Ruthenium oxide nanoparticles were electrochemically deposited on a glassy carbon electrode (GCE) by 30 repeated cyclic voltammetry (CV) potential scans in the potential range between -0.3 and 1.2 V using RuCl3. nH2O aqueous solution as a precursor. The model redox enzyme, horseradish peroxidase (HRP) was covalently immobilized at the electrochemically deposited RuNPs film surface using Chitosan-glutaraldehyde (Chi-GAD) cross-linking. The surface morphology of RuNPs, HRP and HRP/Chi-GAD/RuNPs films were investigated using scanning electron microscopy (SEM) and atomic force microscopy (AFM) studies. The SEM and AFM results revealed that the average sizes of the RuNPs were 60 – 160 nm. Furthermore, UV-visible absorption spectroscopy studies reveal that both HRP and HRP/Chi-GAD/RuNPs films exhibit soret band at 406 nm which confirms that HRP retains its native structure at the composite film. Electrochemical impedance spectroscopy (EIS) results reveal that rapid electron transfer process occurs at the composite film surface. Furthermore, the electrochemical impedimetric catalysis results obtained at the HRP/Chi-GAD/RuNPs composite film for H2O2 quantification authenticate show that the electron transfer resistance (Ret) values vary linearly with H2O2 concentrations between 131 µM – 794 µM. The prepared HRP/Chi-GAD/RuNPs film was also successfully employed for the amperometric determination of H2O2 in both lab and real samples containing H2O2 and we achieved satisfactory results with good repeatability and reproducibility. The proposed HRP/C-G/RuNPs biosensor exhibits excellent amperometric i-t response towards H2O2 in both lab and real samples in the linear concentration range between 5.09 mM to 16.15 mM. Furthermore, the HRP/Chi-GAD/RuNPs biosensor exhibits good selectivity towards H2O2 without any common interferences. PartⅡ：In our present work, we have synthesized graphene oxide (GO) by adapting Staudenmaier method via oxidative treatment of graphite. The prepared GO was modified on a glassy carbon electrode (GCE) and electrochemically reduced to graphene by recording 15 continuous potential scans in N2 saturated pH 5.0 PBS in the potential range between 0 and -1.5 V. The electrochemically reduced graphene oxide (ERGO) modified GCE surface was utilized as an immobilization matrix for exploring the direct electrochemistry of catalase (CAT). In order to improve the stability and selectivity of the CAT biosensor, a thin nafion (NF) layer was used as an outer protecting layer. The prepared GO, ERGO, CAT and NF/CAT/ERGO films have been characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM) studies. The electrochemical behavior of the modified electrodes has also been probed via electrochemical impedance spectroscopy (EIS) technique. The prepared NF/CAT/ERGO film modified GCE has been employed for insulin quantification at physiological pH conditions. The NF/CAT/ERGO film showed excellent amperometric i–t response towards H2O2 in the linear concentration range 0.05 mM to 1.91 mM. The developed NF/CAT/ERGO film biosensor is highly selective towards H2O2 and it can be used to deteced H2O2 present in the contact lens cleaning solution.