Carbon Felt-Based Bioelectrocatalytic Flow Detectors: Covalent Modification of Tyrosinase onto a Cyanuric Chloride-Activated Carbon Felt

Tyrosinase was covalently immobilized on the carbon felt surface by using cyanuric chloride as a linking reagent. The resulting tyrosinase-modified carbon felt was used as a working electrode unit of electrochemical flow detector, and the activity of the immobilized TYR was evaluated as a cathodic peak current response using catechol as a standard substrate. X-ray photoelectron spectroscopy, cyclic voltammetry, electrochemical impedance spectroscopy were employed to disclose the modification mechanism. The peak current responses of the immobilized tyrosinase on cyanuric chloride-activated carbon felt were much higher than those on cyanuric chloride-untreated CF. Furthermore, when the cyanuric chloride-activated carbon felt was treated with aniline or ethanolamine to block the covalent binding site of cyanuric chloride, the peak current response to catechol was much suppressed as compared with the case without blockings. These results strongly suggest that the observed peak response is originated from the covalently attached tyrosinase via the cyanuric chloride linkage. When air-saturated 0.1 M phosphate buffer (pH 7.0) was used as a carrier at 3.0 mL/min, the calibration curve of catechol exhibited linear portion over the concentration range from 0.1 to 10 M, with the sensitivity of 1.97 A/M. The detection limit was found to be 4.6 nM (S/N = 3, baseline noise level is 30 nA). The tyrosinase-immobilized carbon felt remained 85% of its original activity after one month storage in 0.1 M phosphate buffer (pH 7.0) at 4 °C. The present enzyme immobilization method would be applied for other enzyme as a simple and easy protocol of protein immobilization onto the carbon felt surface.