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

Carboxyl esterases show limited use as catalytic labels in bioassays because of slow enzymatic reaction. We report that DT-diaphorase from Bacillus stearothermophilus (DT-D, EC 1.6.99.-) shows high carboxyl esterase-like activity in the presence of reduced β-nicotinamide adenine dinucleotide (NADH) and may be used as a better catalytic label than carboxyl esterases. DT-D is a redox enzyme and can participate in signal-amplifying redox cycling. Thus, an electrochemical immunosensor using a DT-D label allows for triple signal amplification based on (i) hydrolysis of a carboxyl ester, (ii) electrochemical-chemical (EC) redox cycling involving an electrode, a hydrolysis product, and NADH, and (iii) electrochemical-enzymatic (EN) redox cycling involving an electrode, a hydrolysis product, DT-D, and NADH. Ester hydrolysis by DT-D is confirmed via spectrophotometric measurement of a chromogenic substrate (4-nitrophenyl acetate) and ¹H NMR spectra. Among two phenyl acetates and four naphthyl acetates considered, 4-aminonaphthalene-1-yl acetate (4-NH₂-NAc) is chosen as the best acetyl ester substrate because 4-NH₂-NAc is stable, its hydrolysis is slow in the absence of DT-D, its hydrolysis is very fast in the presence of DT-D, and EC and EN redox cycling involving the hydrolysis product (4-amino-1-naphthol) is rapid. However, hydrolysis of 4-NH₂-NAc by esterase from porcine liver (EC 3.1.1.1.) is very slow. When DT-D is applied to sandwich-type detection of thyroid-stimulating hormone in artificial serum, the detection limit is ∼2 pg/mL, indicating that the developed immunosensor is highly sensitive because of triple signal amplification. DT-D may be used as a catalytic label in sensitive and stable bioassays instead of common alkaline phosphatase and horseradish peroxidase.