State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering , Nanjing University , Nanjing 210023 , P. R. China.
Anal Chem. 2018 Jul 3;90(13):8028-8034. doi: 10.1021/acs.analchem.8b00933. Epub 2018 Jun 12.
Electrochemical immunoassay amplified with redox cycling has become a challenging topic in highly sensitive analysis of biomarkers. Here a ferritin-triggered redox cycling is reported by using a highly outersphere reaction-philic (OSR-philic) redox mediator ruthenium hexamine (Ru(NH)) to perform the OSR-philic/innersphere reaction-philic (ISR-philic) controlled signal amplification. The screened mediator can meet the needs of lower E' than ferritin, low reactivity with ISR-philic species, and quick electron exchange with ferritin redox couple. The ferritin-labeled antibody is first bonded to immunosensor surface by recognizing the target antigen capured by the immobilized primary antibody. The ferritin then mediates OSR-philic/ISR-philic transfer from Ru(NH)/immunosensor to ferritin-HO redox system. The fast mediation and excellent resistant of highly OSR-philic Ru(NH) against radical oxygen species lead to highly sensitive electrochemical readout and high signal-to-background ratio. The proposed redox cycling greatly enhances the readout signal and the sensitivity of traditional ferritin-labeled sandwich immunoassay. Using Enteropathogenic coli ( E. coli) antigen as a model analyte, the developed method shows excellent linearity over the concentration range from 10.0 pg/mL to 0.1 μg/mL and a detection limit of 10.0 fg/mL. The acceptable accuracy, good reproducibility, and selectivity of the proposed immunoassay method in real samples indicate the superior practicability of the ferritin-triggered redox cycling.
电化学免疫分析通过氧化还原循环得到了极大的发展，已成为生物标志物高灵敏分析极具挑战性的课题。本工作报道了一种基于过氧体外球体亲合（OSR-philic）氧化还原介体钌六胺（Ru(NH)）的铁蛋白触发的氧化还原循环反应，用于执行过氧体外球体亲合/内球体亲合（ISR-philic）控制的信号放大。筛选出的介体需要满足比铁蛋白更低的 E'、与 ISR-philic 物质低反应性以及与铁蛋白氧化还原偶快速电子交换的条件。铁蛋白标记的抗体首先通过识别固定化的一级抗体捕获的靶抗原结合到免疫传感器表面。铁蛋白随后介导从 Ru(NH)/免疫传感器到铁蛋白-HO 氧化还原体系的过氧体外球体亲合/内球体亲合转移。高过氧体外球体亲合的 Ru(NH)快速介导和对自由基氧物种的良好抗性导致了高灵敏度的电化学读出和高信号与背景比。所提出的氧化还原循环极大地增强了传统铁蛋白标记三明治免疫分析的读出信号和灵敏度。以肠致病性大肠杆菌（E. coli）抗原为模型分析物，所开发的方法在 10.0 pg/mL 至 0.1 μg/mL 的浓度范围内表现出优异的线性关系，检测限为 10.0 fg/mL。该免疫分析方法在实际样品中具有可接受的准确性、良好的重现性和选择性，表明铁蛋白触发的氧化还原循环具有优异的实用性。
