In this study, we report an electrochemical microbial biosensor that was made by immobilizing a bacterial laccase on the surface of Escherichia coli cells followed by adsorption of modified live cells onto a glassy-carbon electrode. Expression and surface localization of laccase on target cells were confirmed by Western blotting, flow cytometry assays and immunofluorescence microscopy observation. Increased tandem-aligned anchors with three repeats of the N-terminal domain of an ice nucleation protein were used to construct a highly active E. coli whole cell laccase-based catalytic system. When the proposed biosensor was used to detect catechol, the electrochemical response under optimized pH conditions was linear within a concentration range of 0.5 μM-300.0 μM catechol. Metal ions (Mn2+, Fe3+, Cu2+, Mg2+, Al3+ and Zn2+) at concentrations from 1 to 10 mg L-1, bovine serum albumin and glucose at concentrations from 0.1 to 10 g L-1, and ascorbic acid at concentrations from 0.01 to 0.1 g L-1 did not cause a noticeable interference effect. The detection limit of 0.1 μM catechol was comparable to those of other biosensors based on purified chemically modified laccases. When used to detect catechol in real red wine and tea samples, the biosensor offered a considerable level of accuracy comparable to the HPLC method as well as high recovery rates (98.2%-103.8%) towards all of the tested samples. Moreover, the developed system also exhibited high stability and reproducibility.
Keywords: Adsorption; Bacterial laccase; Biosensor; Catechol; Cell surface display.
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