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.

Detection of Gram-negative bacterial outer membrane vesicles using DNA aptamers

“…Detecting living bacteria in blood is difficult due to the immune regulation of host cells and the serum bactericidal activity 20,21 . This suggests that the detection of bacterial OMVs is more effective than the detection of bacterial cells in clinical blood samples 22 .…”

Carboxyl Esterase-Like Activity of DT-Diaphorase and Its Use for Signal Amplification

“…Enzyme labels used in combination with redox cycling are classified into 3 types: enzymes that produce redox-active species (type 1 enzymes), 9−12 enzymes that participate in redox cycling (type 2 enzymes), 6 and bifunctional enzymes (type 3 enzymes). 5,8,13 Type 1 enzyme labels, such as ALP and β-galactosidase, convert redox-inactive substrates into redox-active products, which then participate in electrochemical−chemical (EC), 11 electrochemical−chemical−chemical 9,10 and electrochemical−enzymatic (EN) redox cycling 12 (Figure 1a). Type 1 enzyme labels do not participate in redox cycling.…”

“…6 Bifunctional (type 3) enzyme labels, such as DT-diaphorase (DT-D, EC 1.6.99.2, now EC 1.6.5.2) and tyrosinase, act as both type 1 and 2 enzymes. 5,8,13 Bifunctional enzyme labels convert redox-inactive substrates to redox-active products, which participate in EC redox cycling as well as in EN redox cycling (Figure 1c). As a result, bifunctional enzyme labels allow for triple signal amplification, providing an opportunity to achieve ultrahigh signal amplification.…”

“…8 The authors also showed that DT-D catalyzes dephosphorylation of a redox-inactive phosphate-containing substrate to a redox-active dephosphorylated product in the presence of NADH, and that the dephosphorylated product participates in fast EC and EN redox cycling. 5 Both the triple amplification schemes allowed for highly sensitive detection of biomaterials, although the catalytic conversion of the nitroso compound and the phosphate-containing substrate by DT-D is not as rapid as the common catalytic reactions by ALP and HRP. Hydrolase enzymes include carboxyl esterases, phosphatases, and proteases/peptidases that break a carboxyl ester bond, a phosphoester bond, and an amide bond, respectively.…”

“…26,29 Importantly, the authors have demonstrated that DT-D [NAD(P)H dehydrogenase (quinone)] from Bacillus stearothermophilus (EC 1.6.99.-) also shows high phosphatase-like activity. 5 Because DT-D is a bifunctional (type 3) enzyme, triple signal amplification is readily achieved. Moreover, DT-D has high thermal and long-term stability.…”