DT-Diaphorase as a Bifunctional Enzyme Label That Allows Rapid Enzymatic Amplification and Electrochemical Redox Cycling

5 years ago

DT-Diaphorase as a Bifunctional Enzyme Label That Allows Rapid Enzymatic Amplification and Electrochemical Redox Cycling

Nam-Sihk Lee, Juyeon Kang, Cheolho Kang, Haesik Yang, Young Ho Yoon

The most common enzyme labels in enzyme-linked immunosorbent assays are alkaline phosphatase and horseradish peroxidase, which, however, have some limitations for use in electrochemical immunosensors. This Article reports that the small and thermostable DT-diaphorase (DT-D) and electrochemically inactive 4-nitroso-1-naphthol (4-NO-1-N) can be used as a bifunctional enzyme label and a rapidly reacting substrate, respectively, for electrochemical immunosensors. This enzyme–substrate combination allows high signal amplification via rapid enzymatic amplification and electrochemical redox cycling. DT-D can convert an electrochemically inactive nitroso or nitro compound into an electrochemically active amine compound, which can then be involved in electrochemical–chemical (EC) and electrochemical–enzymatic (EN) redox cycling. Six nitroso and nitro compounds are tested in terms of signal-to-background ratio. Among them, 4-NO-1-N exhibits the highest signal-to-background ratio. The electrochemical immunosensor using DT-D and 4-NO-1-N detects parathyroid hormone (PTH) in phosphate-buffered saline containing bovine serum albumin over a wide range of concentrations with a low detection limit of 2 pg/mL. When the PTH concentration in clinical serum samples is measured using the developed immunosensor, the calculated concentrations are in good agreement with the concentrations obtained using a commercial instrument. Thus, the use of DT-D as an enzyme label is highly promising for sensitive electrochemical detection and point-of-care testing.

Publisher URL: http://dx.doi.org/10.1021/acs.analchem.7b01223

DOI: 10.1021/acs.analchem.7b01223