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%.
Keywords: NADH; enzyme-assisted; quantitative detection; surface enhanced Raman scattering.