Rapid and Highly Sensitive Electrochemical Technique for Cell Viability Assay via Monitoring of Intracellular NADH with New Double Mediator System

抄録

It is very important to assess cell viability rapidly and sensitively for the cell biology research, medical and pharmaceutical application. Compared to conventional methods, we have established a new rapid and sensitive bio-electrochemical system using small screen-printed carbon electrode (SPCE) and 1-methoxy-5-methylphenazinium methyl sulfate (mPMS)/[Fe(CN)₆]³⁻(FeCN) as double electron mediators for monitoring cell viability through the measurement of intracellular NADH. Combination of 10 µmol L⁻¹ (µM) mPMS and 500 µM FeCN was the optimum concentration and 10 minutes incubation was enough to monitor intracellular NADH by chronoamperometry at +0.5 V applications. This mPMS/FeCN system works as useful as previously reported menadione (Mena)/FeCN system. We confirmed that the electron transfer from intracellular NADH to mPMS occurred non-enzymatically, though the electron transfer from intracellular NADH to Mena was catalyzed by cytosolic enzyme. We applied our system to count the three kinds of mammalian cells. The oxidation current in chronoamperometry after 10 minutes incubation showed a good linear relationship in two times wider of cell concentration as compared to the cell concentration detected with WST-1 assay. The results indicated that the metabolically active mammalian cells could be quickly quantified by our method. Furthermore, we have applied this method to evaluate the acute cytotoxicity of oxamic acid on cytoma cells. Only 10 minutes incubation and high sensitivity embellished this method. These results strongly supported that our electrochemical method might be potent to alternate to WST assay for cell viability and acute cytotoxicity test.