Potential-Resolved Multicolor Electrochemiluminescence for Multiplex Immunoassay in a Single Sample

电化学发光 化学 免疫分析 多路复用 发光体 联吡啶 分析化学(期刊) 光化学 发光 色谱法 检出限 光电子学 催化作用 有机化学 生物信息学 材料科学 免疫学 抗体 晶体结构 生物
作者
Weiliang Guo,Hao Ding,Chaoyue Gu,Yanhuan Liu,Xuecheng Jiang,Bin Su,Yuanhua Shao
出处
期刊:Journal of the American Chemical Society [American Chemical Society]
卷期号:140 (46): 15904-15915 被引量:298
标识
DOI:10.1021/jacs.8b09422
摘要

Electrochemiluminescence (ECL) is a highly successful technique used in commercial immunoassays for clinical diagnosis. Developing an ECL-based multiplex immunoassay, with the potential to enable high-throughput detection of multiple biomarkers simultaneously, remains a current research interest yet is limited by a narrow choice of ECL luminophores. Herein we report the synthesis, photophysics, electrochemistry, and ECL of several new ruthenium(II) and iridium(III) complexes, three of which are eventually used as signal reporters for multiplex immunoassay. The ECL behaviors of individual luminophores and their mixtures were investigated in multiple modes, including light intensity, spectrum, and image measurements. The spectral peak separation between Ru(bpy)2(dvbpy)2+ (bpy = 2,2′-bipyridine, dvbpy = 4,4′-bis(4-vinylphenyl)-2,2′-bipyridine), and Ir(dFCF3ppy)2(dtbbpy)+ (dFCF3ppy = 3,5-difluoro-2-[5-(trifluoromethyl)-2-pyridinyl]phenyl, dtbbpy = 4,4′-bis(tert-butyl)-2,2′-bipyridine) was up to 145 nm, thus providing the spectrum-resolved possibility of identifying light signals. The potential-resolved ECL signals were achieved for the mixtures of Ir(ppy)3 (ppy = 2-phenylpyridine) with either Ru(bpy)2(dvbpy)2+ or Ir(dFCF3ppy)2(dtbbpy)+, due to the self-annihilation ECL of Ir(ppy)3 at higher potentials, as confirmed by electrochemistry-coupled mass spectrometry. A multiplex immunoassay free of spatial spotting antibodies on plates or substrates was ultimately devised by combining luminophore-loaded polymer beads with the homogeneous sandwich immunoreaction. Using potential and spectrum dual-resolved ECL as the readout signal, simultaneous recognition of three antigens, namely, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), and beta-human chorionic gonadotropin (β-HCG), was demonstrated in a single run for a sample volume of 300 μL. These results contribute to the understanding of ECL generation by multiple luminophores and devising spot-free multiplex immunoassays with less sample consumption.
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