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.

为助力生理与病理机制的研究分析，亟需开发针对小型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（2-mercaptobenzimidazole）修饰的金纳米粒子电极（gold nanoparticle electrode），用于检测经培养细胞表面表达的转运蛋白排泄的尿毒症相关物质，例如尿酸（UA）与吲哚硫酸酯（IS）。当抗坏血酸、UA、多巴胺及IS的浓度以1 µg/mL为梯度线性递增时，该电极可检测磷酸盐缓冲液中不同电位下的电流变化。本研究通过安培检测法（amperometric measurement），在跨细胞转运模型中实现了对经ATP结合盒转运蛋白G2（ABCG2）表达介导的IS排泄的实时检测。即便在无血清培养基中，该电极对IS引发的电流变化仍具备极高灵敏度。我们观测到，IS浓度每升高1 µg/mL，多晶电极（polycrystalline electrode）表面每平方毫米的电流增幅约为0.10 µA。当电极置于组织培养插入式装置（tissue culture insert）中、与转染了ABCG2质粒的细胞共培养时，电流随时间推移逐渐升高，这表明IS是通过ABCG2编码的转运蛋白完成排泄的。综上，该电极可成功用于实时检测培养细胞表面ABCG2表达介导的IS排泄过程。