A Facile Strategy for Multiplex Protein Detection by a Fluorescent Microsphere-Based Digital Immunoassay

化学 多路复用 检出限 微球 免疫分析 荧光 链霉亲和素 色谱法 生物素 抗体 生物化学 化学工程 物理 工程类 生物 量子力学 免疫学 生物信息学
作者
Feng Gong,Zhiyou Tan,Xiaoyun Shan,Yixia Yang,Songbai Tian,Fuxiang Zhou,Xinghu Ji,Zhike He
出处
期刊:Analytical Chemistry [American Chemical Society]
卷期号:96 (8): 3517-3524 被引量:11
标识
DOI:10.1021/acs.analchem.3c05336
摘要

The digital immunoassay is a highly sensitive detection technique based on single-molecule counting and is widely used in the ultrasensitive detection of biomarkers. Herein, we developed a fluorescent microsphere-based digital immunoassay (FMDIA) by employing fluorescent microspheres as both the carriers for immunoreaction and fluorescent reports for imaging. In this approach, the target protein in the sample was captured by fluorescent microspheres to form a biotin-labeled sandwich immunocomplex, and then, the fluorescent microspheres containing the target protein molecules were captured by adding streptavidin-coated magnetic beads (SA-MBs). By counting the proportion of fluorescence-positive magnetic beads, the concentration of the target protein can be precisely quantified. As a proof of concept, α fetoprotein (AFP) and human interleukin-6 (IL-6) were used to assess the analytical performance of the proposed FMDIA, and limit of detection (LOD) values of 21 pg/mL (0.30 pM) and 0.19 pg/mL (7.3 fM) were achieved, respectively. The results of AFP detection in serum samples of patients and healthy people were consistent with the reference values given by the hospital. Furthermore, by adding fluorescent microspheres of various colors for encoding, the proposed FMDIA can easily realize the simultaneous detection of multiple proteins without the need to introduce multiple modified magnetic beads. This multiplex protein detection strategy, in which the reactions are first carried out on the fluorescent microspheres and then magnetic beads are used to capture the fluorescent reporters containing the target molecules, provides a new idea for digital assays.
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