| Citation: | HU Peng-tao, GAO Ruo-qian, GE Ming-feng, DONG Wen-fei, LI Li, SU Xin-ran. Design of flow-phase single-molecule immunoassay detection system[J]. Chinese Optics, 2025, 18(5): 1055-1065. doi: 10.37188/CO.2025-0045
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We develop a single-molecule immunoassay detection system for flow-phase samples to enable early diagnosis of tumor biomarkers. The system includes an optical fluorescence imaging platform and an image processing algorithm. First, we developed a flow-phase single-molecule immunoassay detection method suitable for real-time detection of low-concentration, high-throughput samples. Second, we designed a set of fluorescence imaging detection platform compatible with microfluidic chips. Through the rational configuration of optical filters and beam splitters, high-resolution fluorescence imaging was achieved by utilizing multi-module integration. Finally, based on the image detection and counting algorithm for out-of-focus particles, we optimized a feature-matching algorithm to effectively detect out-of-focus fluorescent particles. Experimental results demonstrate a detection limit of 0.001 pg/mL within a linear range of 0.001−1 pg/mL, with coefficient of variation below 10%. The system can process up to 10 samples per hour. These findings indicate that our system meets the requirements for stable, sensitive, and high-throughput single-molecule immunoassay detection, showing promising potential for early cancer screening.
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