Use of a Phosphatase-Like DT-Diaphorase Label for the Detection of Outer Membrane Vesicles.

Abstract

DT-diaphorase (DT-D) is known to mainly catalyze the two-electron reduction of quinones and nitro(so) compounds. Detection of Gram-negative bacterial outer membrane vesicles (OMVs) that contain pyrogenic lipopolysaccharides (LPSs, also called endotoxins) is required for evaluating the toxic effects of analytical samples. Here, we report that DT-D has a high dephosphorylation activity: DT-D catalyzes reductive dephosphorylation of a phosphate-containing substrate in the presence of NADH. We also report that sensitive and simple OMV detection is possible with a sandwich-type electrochemical immunosensor using DT-D and two identical LPS-binding antibodies as a catalytic label and two sandwich probes, respectively. The absorbance change in a solution containing 4-nitrophenyl phosphate indicates that dephosphorylation occurs in the presence of both DT-D and NADH. Among the three phosphate-containing substrates [4-aminophenyl phosphate, ascorbic acid phosphate, and 1-amino-2-naphthyl phosphate (ANP)] that can be converted into electrochemically active products after dephosphorylation, ANP shows the highest electrochemical signal-to-background ratio, because (i) the dephosphorylation of ANP by DT-D is fast, (ii) the electrochemical oxidation of the dephosphorylated product (1-amino-2-naphthol, AN) is rapid, even at a bare indium-tin oxide electrode, and (iii) two redox cycling processes significantly increase the electrochemical signal. The two redox cycling processes include an electrochemical-enzymatic redox cycling and an electrochemical-chemical redox cycling. The electrochemical signal in a neutral buffer (tris buffer, pH 7.5) is comparable to that in a basic buffer (tris buffer, pH 9.5). When the immunosensor is applied to the detection of OMV from Escherichia coli, the detection limit is found to be 8 ng/mL. This detection strategy is highly promising for the detection of biomaterials, including other extracellular vesicles.