Abstract：Reduced nicotinamide adenine dinucleotide (NADH) plays a crucial role in many biochemical reactions in human metabolism. Noninvasive and in vivo monitoring of the NADH level in skin tissue is of great interest. In this paper, the Raman scattering experiment and density functional theory (DFT) calculation have been applied to investigate the vibrational properties of NADH in the spectral range of 200～3 300 cm-1. The DFT calculation was performed with hybrid exchange functional using B3LYP functions with a polarized 6-311+G(d,p) basis. To achieve accurate analytical vibrational frequency calculation, the ground-state geometry of NADH molecule was first optimized at B3LYP/6-311+G(d,p) level of theory without any symmetry restrain, and the bond lengths and bond angles of NADH molecule were calculated. Then, the calculated wavenumbers were normally scaled with a necessary wavenumber linear scaling procedure by accounting for anharmonicity in DFT calculation. The DFT calculated spectrum of NADH is in good agreement with the Raman experimental spectrum: a good linear correlation between calculated and experimental wavenumbers has been obtained in the spectral range of 200～3 300 cm-1, and the deviations are smaller than 5 cm-1. In addition, the characteristic vibrational modes of the three parts adenine, nicotinamide, and dinucleotide of NADH molecule have been assigned and discussed, which would be helpful for the noninvasive and in vivo analyses of NADH. The characteristic mode of adenine at 732 cm-1 can be chosen as the most representative model for analyzing NADH. The characteristic mode of nicotinamide at 1 690 cm-1 can be chosen as another representative mode for further analyzing NADH. The characteristic modes of dinucleotide at 1 086 and 1 339 cm-1 can be chosen as a combination for further more accurately analyzing NADH. Therefore, when applying the Raman method for noninvasive and in vivo monitoring of the NADH level in skin tissue, first, the most representative mode at 732 cm-1 can be used for quick analyses, then the mode at 1 690 cm-1 and/or the combination modes of 1 086 and 1 339 cm-1 can be used for further accurate analyses.
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