title: Carbon felt-based biocatalytic enzymatic flow-through detectors: chemical modification of tyrosinase onto amino-functionalized carbon felt using various coupling reagents.
abstract: Tyrosinase (TYR) was covalently immobilized onto amino-functionalized carbon felt (CF) surface via eight different coupling reagents. Prior to the TYR-immobilization, primary amino group was introduced to the CF surface by the treatment with 3-aminopropyltriethoxysilane (APTES). The APTES modification of the CF surface was confirmed by XPS and SEM measurements. The terminal amino groups on the CF surface were cross-linked with protein lysine group (or cysteine group) using various coupling reagents. The resulting TYR-immobilized CF (TYR-CF) was utilized as a working electrode unit of a biocatalytic enzymatic flow-through detector. Catechol and 4-chlorophenol (4-CP) were used as model analytes for the evaluation of catecholase activity and phenolase activity, respectively, and flow injection peaks based on the electro-reduction of the enzymatically produced o-quinone species were monitored at -0.05 V vs. Ag/AgCl. Among eight coupling reagents, glutaraldehyde (GA) exhibited the best results on the sensitivity, the operational stability and the storage stability. The detection limits of catechol and 4-CP obtained by the GA-coupling method were found to be 6.0 x 10(-9)M and 1.5 x 10(-8)M, respectively with the sample through-put of 36 samples/h. No serious degradation of the peak current was observed over 30 consecutive samples injections on the GA-coupling method, while gradual decrease in the peak currents was observed on other seven coupling reagents. The GA-coupling method showed the best results on the storage stability, and 85% of original activity for catechol oxidation remained after 25 days storage.
authors.affiliation: Department of Materials Science and Technology, Graduate School of Engineering, Saitama Institute of Technology, 1690 Fusaiji, Fukaya, Saitama 369-0293, Japan.
authors.forename: Yue
authors.initials: Y
authors.lastname: Wang
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Hasebe
mesh_terms: D000363:Agaricales / Q000201:enzymology; D055162:Biocatalysis; D002244:Carbon / Q000737:chemistry*; D000077482:Carbon Fiber; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism*; D007202:Indicators and Reagents / Q000737:chemistry; D008855:Microscopy, Electron, Scanning; D014442:Monophenol Monooxygenase / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction; D011437:Propylamines; D012821:Silanes / Q000737:chemistry*; D013057:Spectrum Analysis; D014965:X-Rays
chemical_list: D000077482:Carbon Fiber; D004800:Enzymes, Immobilized; D007202:Indicators and Reagents; D011437:Propylamines; D012821:Silanes; D002244:Carbon; D014442:Monophenol Monooxygenase; C477625:amino-propyl-triethoxysilane