The Study Of Methodology And Applications Of Detection Of Nicotinamide Adenine Dinucleotide By Direct Electrochemisty

Objective:To establish an electrochemical method for direct detection ofreduced nicotinamide adenine dinucleotide (NADH) with high sensitivity.Based on the method established, we developed an electrochemicalmethod for indirect detection of the concentration of total bile acidwithout any modification and detecting the concentration of total bile acidin clinical serum sample，and developed an electrochemical method forindirect detection of the concentration of alcohol in human serum withoutany modification.Methods:1. The direct electrochemical detection of NADH: The NADH inphosphate buffer solution (PBS) was detected by cyclic voltammetry (CV)and differential pulse voltammetry (DPV). The electrode is a screen printed carbon electrode (SPCE): a work electrode and a counterelectrode are printed by carbon thick liquid; reference electrode is printedby Ag/AgCl thick liquid.2. The determination of total bile acid in human serum: After proteinprecipitation and dried under nitrogen stream, the serum sampleredissolved in PBS. Then, it mixed with oxidized nicotinamide adeninedinucleotide (NAD+) and catalyzed by3a-hydroxysteroid dehydrogenase(3α-HSD). The reaction media was drop on SPCE and detected by DPV.The concentration of total bile acid in35clinical serum samples weredetected by this method and enzymatic cycling method，respectively.3. The determination of alcohol in human serum: Alcoholdehydrogenase (ADH) and NAD+in PBS reacted with serum sample for10min at room temperature. The reaction media was drop on SPCE anddetected by DPV.Results:1. The linearity of NADH by SPCE was in the range of2.5×10-6~2.0×10-4mol/L. The regression equation was Y=0.125X-0.233,with the correlation coefficient of0.9992. The detection limit was1.0×10-6mol/L. The coefficient of the assay detecting NADHconcentrations of100.0μmol/L and10.0μmol/L were1.5%and4.2%,respectively. 2. The linearity of total bile acids in serum was in the range of5~400μmol/L. The regression equation was Y=0.0038X+0.2739, with thecorrelation coefficient of0.9959. The detection limit of this assay was2μmol/L. The within-and between-day coefficients were1.1%~5.2%and3.2%~6.1%, respectively. The recoveries were between75%and113%.The method was used for detection of concentration of total bile acid in35clinical serum samples. The results by this method and enzymaticcycling method showed a good correlation(r=0.9438)3. The linearity of alcohol in PBS was in the range of7.5~250μmol/L.The regression equation was Y=0.0064X+0.0747, with the correlationcoefficients of0.9951. The linearity of serum-alcohol was in the range of4~24μmol/L. The regression equation was Y=0.0918X+0.0705, with thecorrelation coefficient of0.9987. The within-and between-daycoefficients were2.0%~4.1%and3.9%~4.6%, respectively. Therecoveries were between93%and102%.Conclusions:1. The SPCE for detection of NADH has high sensitivity, goodreproducibility and fast speed. The SPCE is mass production, low costand disposable and is potential in the development ofdehydrogenase-based biosensors.2. A simple, cheap, cost-effective, and efficient method for determination total bile acids by electrochemistry was established. Thisdeveloped method is available for detecting total bile acids of specimenserum and helpful for developing portable sensor.3. Detecting serum-alcohol concentration by the electrochemistry wasestablished. The method is rapid, cheap, and convenient.