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

Area of Science:

• Analytical Chemistry
• Biochemistry
• Spectroscopy

Background:

• Accurate quantification of nicotinamide-adenine dinucleotide (NADH) is crucial in biochemical assays.
• Traditional methods for NADH detection can be limited by sensitivity or complexity.
• Surface-enhanced Raman scattering (SERS) offers high sensitivity for molecular detection.

Purpose of the Study:

• To develop a novel enzymatic method for the quantitative detection of NADH.
• To utilize surface-enhanced Raman scattering (SERS) for enhanced sensitivity in NADH detection.
• To establish a reliable and sensitive analytical method for NADH concentration determination.

Main Methods:

• An enzymatic reaction cascade involving NADH oxidase and horseradish peroxidase was employed.
• Hydrogen peroxide (H₂O₂) generated from NADH oxidation was used to oxidize a chromogen.
• o-tolidine (OT) was selected as the chromogen due to its distinct SERS spectral signature.
• Quantitative analysis was performed by measuring the integrated SERS intensity of pigment peaks.

Main Results:

• The optimal concentration of o-tolidine (OT) was determined to be 2 × 10⁻³ M.
• A wide linear range and excellent linearity were achieved between logarithmic H₂O₂ concentration and logarithmic SERS intensity at 1448 cm⁻¹.
• The developed method demonstrated a low limit of detection for NADH as low as 4 × 10⁻⁷ M.
• Validation studies showed logarithmic deviations less than 3% for known NADH concentrations.

Conclusions:

• A sensitive and quantitative enzymatic SERS method for NADH detection was successfully developed.
• The method offers a low detection limit and high accuracy, suitable for biochemical analysis.
• The use of o-tolidine as a chromogen provides a unique and reliable SERS signal for NADH quantification.