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

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. A combination of 10 µmol L−1 (µM) mPMS and 500 µM FeCN was the optimum concentration and, 10 minutes (min) 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 min incubation showed a good linear relationship in two times wider of cell concentration as compared to the cell concentration detected with water soluble tetrazolium-1 (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 min 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.

在细胞生物学研究、医疗及制药应用中，对细胞活力进行迅速且敏感的评估至关重要。相较于传统方法，本研究构建了一种新型的快速灵敏生物电化学系统，该系统采用小型网印碳电极（SPCE）及1-甲氧基-5-甲基苯并嗪明甲磺酸盐（mPMS）/[Fe(CN)6]3−(FeCN)作为双电子介质，通过测量细胞内NADH水平来监测细胞活力。10 µmol L−1（µM）mPMS与500 µM FeCN的混合物为最佳浓度，且仅需10分钟（min）的孵育时间便足以通过计时电流法在+0.5 V的条件下监测细胞内NADH。该mPMS/FeCN系统在效用上与先前报道的甲萘醌（Mena）/FeCN系统相当。我们证实，细胞内NADH向mPMS的电子转移过程非酶促进行，而细胞内NADH向Mena的电子转移过程则由细胞质酶催化。我们将本系统应用于计数三种哺乳动物细胞。10分钟孵育后的计时电流法中氧化电流与细胞浓度的线性关系良好，其范围比使用水溶性四唑盐-1（WST-1）检测的细胞浓度范围宽两倍。结果表明，通过我们的方法可以迅速定量具有代谢活性的哺乳动物细胞。此外，我们还将此方法应用于评估草酰乙酸对瘤细胞的急性细胞毒性。仅需10分钟的孵育时间和高灵敏度便凸显了此方法的优势。这些结果强烈支持，我们的电化学方法有望成为WST检测细胞活力和急性细胞毒性的替代方案。