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

Ichzan, Andi Muhammad, Lee, Sohee, San Fang, Chiew, Nandhakumar, Ponnusamy, Ha, Hyeri, Joo, Jung Min, Kim, Kwang-sun, Yang, Haesik
Analytical chemistry 2019 v.91 no.7 pp. 4680-4686
Escherichia coli, Gram-negative bacteria, NAD (coenzyme), NAD(P)H dehydrogenase (quinone), absorbance, antibodies, ascorbic acid, biocompatible materials, buffers, catalytic activity, dephosphorylation, detection limit, electrochemistry, electrodes, endotoxins, immunosensors, indium tin oxide, lipopolysaccharides, oxidation, pH, phosphates, quinones, toxicity
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.