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Rapid and Sensitive Detection of NADH and Lactate Dehydrogenase Using Thermostable DT‐Diaphorase Immobilized on Electrode
Author(s) -
Kang Juyeon,
Shin Jeonghwa,
Yang Haesik
Publication year - 2018
Publication title -
electroanalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.574
H-Index - 128
eISSN - 1521-4109
pISSN - 1040-0397
DOI - 10.1002/elan.201800119
Subject(s) - redox , chemistry , lactate dehydrogenase , diaphorase , detection limit , dehydrogenase , electrode , electrochemistry , nad+ kinase , indium tin oxide , enzyme , chromatography , nuclear chemistry , inorganic chemistry , biochemistry
Rapid and sensitive electrochemical detection of NADH, NADH‐dependent enzymes, and relevant metabolites requires a redox enzyme along with an electron mediator. Preferably, the redox enzyme should be immobilized on electrode rather than dissolved in solution. In this study, to simply immobilize a redox enzyme on electrode and maintain its enzymatic activity for long time, thermostable DT‐diaphorase (DT‐D) is immobilized on an avidin‐modified indium tin oxide (ITO) electrode. Electrochemical‐enzymatic (EN) redox cycling involving ITO electrode, ferrocenemethanol (FcMeOH), DT‐D, and NADH is employed for NADH detection. Electrochemical‐enzymatic‐enzymatic (ENN) redox cycling involving ITO electrode, FcMeOH, DT‐D, NAD + , lactate dehydrogenase (LDH), and lactate is employed for LDH detection. In both cases, a new combination of a redox enzyme and an electron mediator (DT‐D and FcMeOH) is used. The detection limits for NADH and LDH in artificial serum obtained without an incubation period are approximately 0.2 μM and 8 ng/mL, respectively. When an incubation period of 10 min is employed, the detection limit for LDH is approximately 5 ng/mL. Because the ENN redox cycling is very fast, the two detection limits are similar irrespective of incubation period. The enzymatic activity of DT‐D on ITO electrode is maintained for one month without deactivation.