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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%.

Quantitative Detection of NADH Using a Novel Enzyme-Assisted Method Based on Surface-Enhanced Raman Scattering