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A Facile Strategy for Multiplex Protein Detection by a Fluorescent Microsphere-Based Digital Immunoassay
Feng Gong1, Zhiyou Tan1, Xiaoyun Shan1
1College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China.
Analytical Chemistry
|February 15, 2024
Summary
We developed a novel fluorescent microsphere-based digital immunoassay (FMDIA) for ultrasensitive biomarker detection. This method precisely quantifies proteins like AFP and IL-6, achieving excellent sensitivity and enabling multiplexing.
Area of Science:
- Biomedical Engineering
- Analytical Chemistry
- Biotechnology
Background:
- Digital immunoassays offer high sensitivity through single-molecule counting for biomarker detection.
- Current methods face challenges in multiplexing and sensitivity for certain analytes.
Purpose of the Study:
- To develop a fluorescent microsphere-based digital immunoassay (FMDIA) for ultrasensitive and multiplexed protein detection.
- To validate the FMDIA's performance using alpha fetoprotein (AFP) and human interleukin-6 (IL-6) as model analytes.
Main Methods:
- Fluorescent microspheres were used as carriers for immunoreactions and fluorescent reporters.
- Target proteins formed sandwich immunocomplexes captured by streptavidin-coated magnetic beads (SA-MBs).
- Quantification was achieved by counting fluorescence-positive magnetic beads.
Main Results:
- Achieved limits of detection (LOD) of 21 pg/mL for AFP and 0.19 pg/mL for IL-6.
- Demonstrated high accuracy by correlating AFP detection in patient serum with hospital reference values.
- Successfully demonstrated multiplexed detection capability using color-encoded fluorescent microspheres.
Conclusions:
- The FMDIA provides a sensitive, precise, and multiplexable platform for digital biomarker quantification.
- This approach offers a new strategy for digital assays by integrating fluorescent microspheres with magnetic bead capture.
- The FMDIA has potential applications in clinical diagnostics and biomedical research requiring ultrasensitive protein analysis.
