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

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 x 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 x 10(-7) M. Two NADH samples with concentrations of 2 x 10(-4) and 2 x 10(-5) M were used to validate the linear relationship, and the logarithmic deviations were less than 3%.