Analysis of thiols with tyrosinase-modified carbon paste electrodes based on blocking of substrate recycling

被引:46
作者
Huang, TH
Kuwana, T
Warsinke, A
机构
[1] Univ Potsdam, Inst Biochem & Biol, Dept Analyt Biochem, D-14476 Golm, Germany
[2] Univ Kansas, Dept Chem, Lawrence, KS 66044 USA
关键词
tyrosinase; biosensor; glutathione; thiol detection; carbon paste electrode; substrate recycling;
D O I
10.1016/S0956-5663(02)00105-7
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
The enzyme, tyrosinase, was immobilized inside carbon paste electrodes (CPE) for the analysis of thiol-containing compounds such as the reduced form of glutathione (GSH) and L-Cysteine. The measuring principle of this sensor is based on the blocking of the substrate recycling process between the enzyme and the electrode. The current response is monitored at -0.050 V versus Ag/AgCl. At this low potential, interferences from easily oxidizable species such as ascorbic acid and uric acid are minimized. The tyrosinase CPE is characterized both in steady state experiments and by flow injection analysis (FIA). GSH is used as the model thiol-containing compound for the study. The highest response for GSH was obtained around pH 6.5. A detection limit of 100 nM and 1 muM is achieved for GSH in steady state and in flow measurements, respectively. The analytical range for GSH is dependent on the concentration of the tyrosinase substrate (catechol). In steady state experiments, and at a lower substrate concentration (10 muM catechol), a linear range of 1-8 muM is found for GSH as compared with 5-30 muM at a higher substrate concentration of 20 muM catechol. Current response of the tyrosinase CPE is not affected by the oxidized form of GSH and L-Cysteine (glutathione disulfide, GSSG, and L-Cystine, respectively) and sulfur-containing compound such as methionine. The tyrosinase CPE can also detect coenzyme A, which makes it possible to construct biosensors based on enzymes producing or utilizing coenzyme A. (C) 2002 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:1107 / 1113
页数:7
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