Detection system based on a substrate specific enzyme such as oxygenase and dehydrogenase is used for a measurement of a substrate such as glucose in a cell culture or a blood. In particular, the measurement system based on a dehydrogenase has highly valuable as it is hardly affected by oxygen in the atmosphere. Upon reacting the dehydrogenase with the substrate, a co-enzyme such as nicotine amide nucleotide (NAD) and nicotine amide dinucleotide phosphate (NADP) is reduced (NAD+→NADH, NADP+→NADPH). Concentration of the substrate can be determined from electric current or color intensity by transferring electron from the reduced form of the co-enzyme thus formed to an electrode or a reductive chromogenic dye via an electron mediator such as PMS (phenazine methosulfate), 1-Methoxy PMS (1-methoxy phenazine methosulfate) and PES (phenazine ethosulfate). Also, the concentration of the dehydrogenase can be determined using similar system by adding the substrate externally.
On the other hand, the electron mediator as mentioned above has a property to accept the electron from living cells. Various methods for measuring activities of living cells via the electron mediators that carry out electron transfer such as 1-Methoxy PMS and PES have been employed in a cell proliferation assay and cytotoxicity assay used in a drug screening in drug discovery and toxicity analysis for various substances. The method for evaluating the cytotoxicity by measuring the activity of the enzyme leaked out of the part of the dead cells into a culture medium via the electro mediators. However, the sample for the measurement has to be prepared by separating the living cells from the culture medium because the electron mediators as mentioned above also carries out the electron transfer with living cells (see F. K.-M. Chan et al., Methods Mol. Biol., 2013, 979, 65-70.). The method in which the living cells are not separated is also proposed, however, the influence of the living cells cannot be avoided, which requires to lower the reaction temperature and shorten the reaction time (see JPA 2005-523022).
In the cytotoxicity assay, the method in which the dehydrogenase from dead cells is assayed using diaphorase, an enzyme mediating electron transfer, instead of low molecular electron mediator to suppress the electron transfer from the living cells is proposed. However, a reagent solution is of poor solubility and not easy for handling because of the use of the enzyme.