A selective and sensitive d-xylose electrochemical biosensor based on xylose dehydrogenase displayed on the surface of bacteria and multi-walled carbon nanotubes modified electrode

Abstract

A novel Nafion/bacteria-displaying xylose dehydrogenase (XDH)/multi-walled carbon nanotubes (MWNTs) composite film-modified electrode was fabricated and applied for the sensitive and selective determination of d-xylose (INS 967), where the XDH-displayed bacteria (XDH-bacteria) was prepared using a newly identified ice nucleation protein from Pseudomonas borealis DL7 as an anchoring motif. The XDH-displayed bacteria can be used directly, eliminating further enzyme-extraction and purification, thus greatly improved the stability of the enzyme. The optimal conditions for the construction of biosensor were established: homogeneous Nafion–MWNTs composite dispersion (10μL) was cast onto the inverted glassy carbon electrode, followed by casting 10-μL of XDH-bacteria aqueous solution to stand overnight to dry, then a 5-μL of Nafion solution (0.05wt%) is syringed to the electrode surface. The bacteria-displaying XDH could catalyze the oxidization of xylose to xylonolactone with coenzyme NAD+ in 0.1M PBS buffer (pH7.4), where NAD+ (nicotinamide adenine dinucleotide) is reduced to NADH (the reduced form of nicotinamide adenine dinucleotide). The resultant NADH is further electrocatalytically oxidized by MWNTs on the electrode, resulting in an obvious oxidation peak around 0.50V (vs. Ag/AgCl). In contrast, the bacteria-XDH-only modified electrode showed oxidation peak at higher potential of 0.7V and less sensitivity. Therefore, the electrode/MWNTs/bacteria-XDH/Nafion exhibited good analytical performance such as long-term stability, a wide dynamic range of 0.6–100μM and a low detection limit of 0.5μM d-xylose (S/N=3). No interference was observed in the presence of 300-fold excess of other saccharides including d-glucose, d-fructose, d-maltose, d-galactose, d-mannose, d-sucrose, and d-cellbiose as well as 60-fold excess of l-arabinose. The proposed microbial biosensor is stable, specific, sensitive, reproducible, simple, rapid and cost-effective, which holds great potential in real applications.