Sensitive electrochemical microbial biosensor for p-nitrophenylorganophosphates based on electrode modified with cell surface-displayed organophosphorus hydrolase and ordered mesopore carbons.

A novel electrochemical microbial biosensor for the rapid monitoring of p-nitrophenyl-substituted organophosphates (OPs) compounds based on glass carbon electrode (GCE) modified with both ordered mesopore carbons (OMCs) and cell surface-expressed organophosphorus hydrolase (OPH) (OPH-bacteria/OMCs/GCE) was described in this paper. The genetically engineered Escherichia coli strain surface displayed mutant OPH (S5) with improved enzyme activity and favorable stability was constructed using a newly identified N-terminal of ice nucleation protein as an anchoring motif, which can be used directly without further time-consuming enzyme-extraction and purification, thereafter greatly improved the stability of the enzyme. Compared to OPH-bacteria modified GCE (OPH-bacteria/GCE), the OPH-bacteria/OMCs/GCE not only significantly enhanced the current response but also reduced the oxidation overpotential towards oxidizable p-nitrophenol (p-NP), which was the hydrolysate of p-nitrophenyl-substituted OPs. Under the optimized experimental conditions, at +0.84 V (vs. SCE), the current-time curve was performed with varying OPs concentration. The current response was linear with paraoxon concentration within 0.05-25 μM. Similarly, linear range of 0.05-25 μM was found for parathion, and 0.08-30 μM for methyl parathion. The low limits of detection were evaluated to be 9.0 nM for paraoxon, 10nM for parathion and 15 nM for methyl parathion (S/N=3). Thus, a highly specific, sensitive and rapid microbial biosensor was established, which holds great promise for on-site detection of trace p-nitrophenyl-substituted OPs.