Electrochemical Interface Using a New Double Mediator System for Intracellular NADH Sensing and Acute Cytotoxicity Test
Authors: Alim Mohammad Abdul , Shinohara Hiroaki , Suga Minoru 
Published: May 2020

Introduction

Nicotinamide Adenine Dinucleotide (reduced form), NADH is a very important redox compound that is essential for metabolic reactions and ATP production in all living cells. Intracellular NADH metabolism is also important to consider the cancer treatment. Intracellular NADH has been conventionally measured by WST assay using a cell membrane-permeable redox mediator, 1-methoxy PMS (1-Methoxy-5-methylphenazinium methylsulfate) and a water soluble tetrazolium to form formazan. This colorimetric method is very easy and useful for cell counting and cytotoxicity test. However, WST assay is time-consuming and then it is not able to apply for evaluation of acute cytotoxicity by chemical compounds including drugs and pollutants. On the other hand, some research groups have already shown that electrochemical method with double mediator system is useful to monitor rapidly intracellular NADH level corresponding to the cell viability [1-5].

In this paper, we would like to report the usefulness of the electrochemical interface using a small screen printed carbon electrode (SPCE) and a new double mediator system combining 1-methoxy PMS and ferricyanide. It was demonstrated that our electrochemical interface was available for faster and wide range of mammalian cell counting as compared with WST assay. Furthermore our method could be applied to acute cytotoxicity test on mammalian cells. The optimization of measurement condition is mentioned in the presentation and the advantages of our new double mediator system against other double mediator system are also discussed in the presentation.

Method

We used PC12 cell (Rat adrenal pheochromocytoma cell line) as a mammalian cell sample. The cells were grown in culture flasks containing DMEM with 5% FBS and 10%HS at 37℃ with 5% CO2 atmosphere. For the cell counting experiment, the medium was exchanged to HBSS before experiments. Cells were removed from the bottom of culture flask by trypsin treatment and suspended in HBSS. After measurement of cell density of the original cell suspension with a hemocytometer, cell suspension was exactly diluted to prepare various density of cell suspension. Each diluted cell suspension was taken in a well of 96 well plate and final concentration of 500 μM potassium ferricyanide and 10 μM 1-metoxy PMS solution were added in a drop wise to the cell suspension. After incubation for 10 minutes, a SPCE (working electrode area is 1 mm2) was immersed into the cell suspension and chronoamperometric measurement was immediately performed by potential application at +0.5V vs. Ag/AgCl reference electrode on the SPCE. The electrochemical measurement was carried out using a potentiostat (Multi-Autolab Cabinet, Metrohm Autolab).

We further tried to test the acute cytotoxicity of oxamic acid that is a famous inhibitor of anaerobic glycolysis. Acute cytotoxicity test was carried out with the same electrochemical instrument by following procedure. Each concentration of oxamic acid/HBSS was prepared and added into PC12 cell/HBSS suspension (5.5 x 105 cells /well) in a well (final concentration of oxamic acid: 0 to 20 mM) and the cell suspension was incubated for 1hour. And then potassium ferricyanide (final 500 mM) and 1-metoxy PMS (final 10 μM) solutions were added into the cell suspension. After incubation for more 10 minutes, a SPCE was immersed into the cell suspension and chronoamperometric measurement was immediately performed by potential application at +0.5V.

Results and Conclusions

In the first place, it was demonstrated that our electrochemical interface with a new combination of double mediator system was very useful for very wide range of cell counting. The amperometric oxidation current showed very good linearity vs. the number of viable cells in the range from 7500 to 964000 cells/well (Data is not shown). The result strongly supported the combination of 1-methoxy PMS and ferricyanide is effective to monitor intracellular NADH level and to speculate the cell viability.

Therefore, in the second place, we applied our electrochemical interface to test the acute toxicity by oxamic acid. The inset of Figure 1 shows the decrease of oxidation current of the double mediator system after 1 hour oxamic acid treatment. The decrease of oxidation current was clearly dependent on the concentration of oxamic acid. It indicated that intracellular NADH level corresponding to the cell viability was rapidly decreased by oxamic acid treatment. This result suggested that our electrochemical interface with a new double mediator system might be useful to evaluate acute cytotoxicity of drugs.

References

[1] A. Heiskanen, J. Yakovleva, C. Spégel, R. Taboryski, M. Koudelka-Hep, J. Emnéus, T. Ruzgas, Amperometric monitoring of redox activity in living yeast cells: comparison of menadione and menadione sodium bisulfite as electron transfer mediator, Electrochem Commun, 6 (2004) 219-224.

[2] A. Heiskanen, C. Spégel, N. Kostesha, S. Lindahl, T. Ruzgas J. Emnéus, Mediator-assisted simultaneous probing of cytosolic and mitochondrial redox activity in living cells, Anal Biochem, 384 (2009) 11-19.

[3] R.Y.A. Hassan, U. Bilitewski, A viability assay for Candida albicans based on the electron transfer mediator 2,6-dichlorophenolindophenol, Anal Biochem, 419 (2011) 26-32.

[4] M. Rahimi, H.Y. Youn, D.J. McCanna, J.G. Sivak, S.R. Mikkelsen, Application of cyclic biamperometry to viability and cytotoxicity assessment in human corneal epithelial cells, Anal Bioanal Chem, 405 (2013) 4975-4979.

[5] Y. Matsumae, Y. Takahashi, K. Ino, H. Shiku, T. Matsue, Electrochemical monitoring of intracellular enzyme activity of single living mammalian cells by using a double-mediator system, Anal Chim Acta, 842 (2014) 20-26.

Figure 1