Acridine orange-induced signal enhancement effect of tyrosinase-immobilized carbon-felt-based flow biosensor for highly sensitive detection of monophenolic compounds.
Tyrosinase (TYR: EC 1.14.18.1) was covalently modified onto the surface of a cyanuric chloride-activated carbon felt (CF) from the mixed buffer solution of TYR and acridine orange (AO). The resulting TYR-immobilized CF (TYR/AO-CF) was used as a working electrode unit of an electrochemical flow-through detector for mono- and di-phenolic compounds (i.e., p-chlorophenol (p-CP), p-cresol, phenol, and catechol), which detects the reduction current of enzymatically produced o-quinones at -0.05 V (vs. Ag/AgCl). The presence of AO (0.2 mM) in TYR solution during the enzyme immobilization step was significantly effective for the signal enhancements especially for p-CP, and the cathodic peak currents of p-CP by the TYR/AO-CF-based detector were much larger than those by the TYR-CF-based detector prepared from TYR solution without AO. The oxymetry with Clark-type oxygen electrode revealed that monophenolase activity of free TYR in 1 mM phosphate buffer (pH 7.0) was greatly enhanced in the presence of AO (0.2 mM), whereas diphenolase activity was not so much influenced. Furthermore, the comparison of cyclic voltammograms of TYR/AO-CF and TYR-CF in air-saturated phosphate buffer containing each substrate revealed that the electrochemical reduction rate of p-chloro-o-benzoquinone at TYR/AO-CF was faster than that at TYR-CF. In addition, the electrochemical impedance spectroscopy revealed that the structural properties of immobilized TYR on the CF would be influenced by AO. Some kinds of interaction of AO with TYR would affect the enzymatic kinetics and the structural properties of the immobilized TYR, leading to the signal enhancement of the TYR-CF-based flow biosensor especially for monophenolic compounds.
Department of Materials Science and Engineering, Graduate School of Engineering, Saitama Institute of Technology, 1690 Fusaiji, Fukaya, Saitama 369-0293, Japan.
Yue
Y
Wang
Yasushi
Y
Hasebe
D000165:Acridine Orange / Q000494:pharmacology*; D015374:Biosensing Techniques; D002244:Carbon / Q000737:chemistry*; D000077482:Carbon Fiber; D002396:Catechols / Q000032:analysis; D002733:Chlorophenols / Q000032:analysis; D003408:Cresols / Q000032:analysis; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013499:Surface Properties; D014227:Triazines / Q000737:chemistry
D000077482:Carbon Fiber; D002396:Catechols; D002733:Chlorophenols; D003408:Cresols; D004800:Enzymes, Immobilized; D014227:Triazines; C032538:4-cresol; C029107:4-chlorophenol; C019309:cyanuric chloride; D002244:Carbon; D014442:Monophenol Monooxygenase; D000165:Acridine Orange; C034221:catechol