title: Analysis of thiols with tyrosinase-modified carbon paste electrodes based on blocking of substrate recycling.
abstract: 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 microM 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 microM catechol), a linear range of 1-8 microM is found for GSH as compared with 5-30 microM at a higher substrate concentration of 20 microM 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.
authors.affiliation: Department of Chemistry, The University of Kansas, Lawrence, KS 66044, USA.
authors.forename: Tina H
authors.initials: TH
authors.lastname: Huang
authors.forename: Theodore
authors.initials: T
authors.lastname: Kuwana
authors.forename: Axel
authors.initials: A
authors.lastname: Warsinke
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D002244:Carbon; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods; D004566:Electrodes; D004800:Enzymes, Immobilized / Q000737:chemistry; D017022:Flow Injection Analysis / Q000295:instrumentation / Q000379:methods; D005978:Glutathione / Q000032:analysis / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D014442:Monophenol Monooxygenase / Q000737:chemistry*; D009824:Ointments; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015219:Substrate Cycling; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000737:chemistry
chemical_list: D004800:Enzymes, Immobilized; D009824:Ointments; D013438:Sulfhydryl Compounds; D002244:Carbon; D014442:Monophenol Monooxygenase; D005978:Glutathione