Combination of Double-Mediator System with Large-Scale Integration-Based Amperometric Devices for Detecting NAD(P)H:quinone Oxidoreductase 1 Activity of Cancer Cell Aggregates.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a key enzyme providing cytoprotection from quinone species. In addition, it is expressed at high levels in many human tumors, such as breast cancer. Therefore, it is considered to be a potential target in cancer treatment. In order to detect intracellular NQO1 activity in MCF-7 aggregates as a cancer model, we present, in this study, a double-mediator system combined with large-scale integration (LSI)-based amperometric devices. This LSI device contained 20 × 20 Pt working electrodes with a 250 µm pitch for electrochemical imaging. In the detection system, menadione (MD) and [Fe(CN)6]3- were used. Since MD can diffuse into cells due to its hydrophobicity, it is reduced into menadiol by intracellular NQO1. The menadiol diffuses out of the cells and reduces [Fe(CN)6]3- of a hydrophilic mediator into [Fe(CN)6]4-. The accumulated [Fe(CN)6]4- outside the cells is electrochemically detected at 0.5 V in the LSI device. Using this strategy, the intracellular NQO1 activity of MCF-7 aggregates was successfully detected. The effect of rotenone, which is an inhibitor for Complex I, on NQO1 activity was also investigated. In addition, NQO1 and respiration activities were simultaneously imaged using the detection system that was further combined with electrochemicolor imaging. Thus, the double-mediator system was proven to be useful for evaluating intracellular redox activity of cell aggregates.

Electrochemicolor Imaging Using an LSI-Based Device for Multiplexed Cell Assays.

Multiplexed bioimaging systems have triggered the development of effective assays, contributing new biological information. Although electrochemical imaging is beneficial for quantitative analysis in real time, monitoring multiple cell functions is difficult. We have developed a novel electrochemical imaging system, herein, using a large-scale integration (LSI)-based amperometric device for detecting multiple biomolecules simultaneously. This system is designated as an electrochemicolor imaging system in which the current signals from two different types of biomolecules are depicted as a multicolor electrochemical image. The mode-selectable function of the 400-electrode device enables the imaging system and two different potentials can be independently applied to the selected electrodes. The imaging system is successfully applied for detecting multiple cell functions of the embryonic stem (ES) cell and the rat pheochromocytoma (PC12) cell aggregates. To the best of our knowledge, this is the first time that a real-time electrochemical mapping technique for multiple electroactive species, simultaneously, has been reported. The imaging system is a promising bioanalytical method for exploring complex biological phenomena.