Abstract
An enzymatic method for quantitative detection of the reduced form of nicotinamide-adenine dinucleotide (NADH) using surface-enhanced Raman scattering was developed. Under the action of NADH oxidase and horseradish peroxidase, NADH can generate hydrogen peroxide (H2O2) in a 1:1 molar ratio, and the H2O2 can oxidize a chromogen into pigment with a 1:1 molar ratio. Therefore, the concentration of NADH can be determined by detecting the generated pigment. In our experiments, eight chromogens were studied, and o-tolidine (OT) was selected because of the unique Raman peaks displayed by its corresponding pigment. The optimal OT concentration was 2 × 10−3 M, and this gave the best linear relationship and the widest linear range between the logarithmic H2O2 concentration and the logarithmic integrated SERS intensity of the peak centered at 1448 cm−1. Under this condition, the limit of detection for NADH was as low as 4 × 10−7 M. Two NADH samples with concentrations of 2 × 10−4 and 2 × 10−5 M were used to validate the linear relationship, and the logarithmic deviations were less than 3%.
Highlights
Nicotinamide adenine dinucleotide (NAD+ ) and its reduced form (NADH) are ubiquitous biomolecules found in eukaryotic and prokaryotic organisms
Quantitative detection of NADH allows for understanding of the overall cellular energy metabolism and monitoring of fermentation processes
We found the characteristic peak of NADH
Summary
Nicotinamide adenine dinucleotide (NAD+ ) and its reduced form (NADH) are ubiquitous biomolecules found in eukaryotic and prokaryotic organisms. Both these compounds are key central charge carriers in living cells and are essential in energy metabolism, reductive biosynthesis, and antioxidation [1,2,3]. Several methods have been utilized to determine NADH, such as fluorescence spectra and electrochemical methods [8,9,10,11,12]. These methods are invasive, time consuming, and complex. Spectroscopic techniques may overcome these problems, and surface-enhanced Raman scattering (SERS) is attractive
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