Influence of different covalent immobilization protocols on electroanalytical performance of laccase-based biosensors.

This paper presents a simple and effective flow-through electrochemical biosensor, consisting of Trametes versicolor laccase (Lac)-based mini-reactor and a tubular detector of silver solid amalgam (TD-AgSA), capable of rapid and selective detection of phenolic compounds. Amperometric detection relies on the reduction of the quinone molecule (formed during the enzymatic reaction in a mini-reactor) on TD-AgSA at -50 mV vs SCE. Since different enzyme immobilization techniques may contribute to differing biosensor performances, four covalent strategies for Lac attachment were compared: (i) through glutaraldehyde to supports -NH, (ii) via disuccinimidyl suberate to supports -NH, (iii) using EDC/NHS for Lac coupling by its -COOH groups to supports -NH, and (iv) using EDC/NHS to supports -COOH. Additionally, five supports (mesoporous silica (SBA-15, MCM-41), cellulose, carbon-based (glassy carbon, graphite) powders) were investigated. It was found that different methods of immobilization, as well as different types of supports, significantly affect the amount of immobilized Lac and, in turn, the analytical characteristics of the obtained biosensors. Thus, TD-AgSA with enzymatic mini-reactor based on Lac covalently attached via glutaraldehyde to aminated MCM-41 proved to be the most promising biosensor, providing the best detection limit (18.3 μmol L) and the long-term stability (47.9 % of the initial response/4 months/100 measurements).