DT-Diaphorase As a Promising Catalytic Label for Rapid and Sensitive Immunosensors

Abstract

The most common enzyme labels in biosensors are alkaline phosphatase (ALP) and horseradish peroxidase (HRP), which, however, have some limitations for use in electrochemical immunosensors. DT-diaphorase (DT-D) is a redox enzyme that catalyzes a two-electron reduction of quinone in the presence of NADH or NADPH. Importantly, DT-D from Bacillus stearothermophilus (EC 1.6.99.-) has a low molecular weight (30 kDa) and high thermal and long-term stability. Nevertheless, DT-D has never been employed as a catalytic label in biosensors. In this presentation, we report that DT-D can be used as a bifunctional enzyme label for rapid and sensitive electrochemical immunosensors: DT-D can convert an electrochemically inactive substrate into an electrochemically active product and DT-D can participate in electrochemical-chemical (EC) and electrochemical-enzymatic (EN) redox cycling induced by the product. We found that DT-D has high catalytic activities for nitroso reduction, phosphoester hydrolysis, and carboxyl ester hydrolysis as well as quinone reduction in the presence of NADH. 4-Nitroso-1-naphthol, 1-amino-2-naphthyl phosphate, and 4-aminonaphthalene-1-yl acetate are used as electrochemically inactive substrates for corresponding enzymatic reactions, which are converted into electrochemically active aminohydroxy-naphthalenes. This combination of DT-D and the substrates allows for high electrochemical signal-to-background ratios, because (i) the catalytic reaction by DT-D is fast, (ii) the electrochemical oxidation of the aminohydroxy-naphthalenes is rapid, even at an indium-tin oxide electrode, and (iii) two redox cycling processes significantly increase the electrochemical signal. The electrochemical immunosensor using DT-D and 4-nitroso-1-naphthol detects parathyroid hormone with a low detection limit of 2 pg/mL, the immunosensor using DT-D and 1-amino-2-naphthyl phosphate detects outer membrane vesicles from Escherichia coli with a detection limit of 8 ng/mL, and the immunosensor using DT-D and 4-aminonaphthalene-1-yl acetate detects thyroid-stimulating hormone with a detection limit of ~2 pg/mL. DT-D may be used as a catalytic label in sensitive and stable bioassays instead of common ALP and HRP.