Supporting Information for "Development of a Molecular Recognition Electrode and Investigation of a Biomolecular Application in Non-Aqueous Media -Electrochemical Detection of Uremia-Related Substances Excreted via ATP-Binding Cassette Transporter G2-"

Detection methods for small biological molecules are needed to facilitate analysis of physiological and pathological mechanisms. We aimed to construct a 2-mercaptobenzimidazole modified gold nanoparticle electrode for detection of uremia-related substances, e.g. uric acid (UA) and indoxyl sulfate (IS), excreted via transporters expressed on cultured cells. This electrode detected the current changes in phosphate buffer at different potentials as the concentrations of ascorbic acid, UA, dopamine, and IS were linearly increased in 1 µg/mL increments. Real-time detection of IS excretion via ATP-binding cassette transporter G2 (ABCG2) expression was performed in a transcellular transport model with amperometric measurement. The electrode was highly sensitive to the current changes with IS even in a serum-free culture medium. We observed an increase in current of approximately 0.10 µA per mm2 of polycrystalline electrode surface area with each 1 µg/mL increase in IS concentration. The current increased with time when the electrode was exposed to cells transfected with ABCG2 plasmid in tissue culture insert, indicating that IS excretion occurred via the transporter encoded by ABCG2. In conclusion, the electrode could be successfully used for the real-time detection of IS excreted via ABCG2 expressed on cultured cells.

为促进生理与病理机制的分析，亟待开发针对小生物分子的检测方法。本研究旨在构建一种经2-巯基苯并咪唑修饰的金纳米粒子电极，以实现对尿毒症相关物质，如尿酸（UA）和吲哚硫酸盐（IS），通过培养细胞上表达转运蛋白分泌的检测。该电极能够检测磷酸盐缓冲液中不同电位下的电流变化，当抗坏血酸、UA、多巴胺和IS的浓度以1 µg/mL的增量线性增加时。在一种跨细胞转运模型中，通过ATP结合 cassette转运蛋白G2（ABCG2）表达实现了IS排出的实时检测。即使在无血清的培养介质中，该电极对IS引起的电流变化亦表现出高度敏感性。我们观察到，随着IS浓度的每1 µg/mL增加，多晶电极表面面积每平方毫米的电流增加约为0.10 µA。当电极暴露于转染了ABCG2质粒的组织培养插片中细胞时，电流随时间增加，这表明IS的排出是通过ABCG2编码的转运蛋白实现的。综上所述，该电极可成功用于通过在培养细胞上表达的ABCG2实时检测IS的排出。