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摘要:
为实现肿瘤标志物的早期诊断,本文设计了一种适用于流动相样本的单分子免疫检测系统,并对其光学荧光成像平台及图像检测计数算法进行了研究。首先,为满足低浓度、高通量样本的即时检测需求,提出了一种基于流动相的单分子免疫检测方法。其次,结合微流控芯片的规格要求,设计了一套光学荧光成像检测平台,通过滤光和分光元件的合理配置,利用多模块集成实现荧光样本的高分辨率成像。最后,在离焦粒子的图像检测计数算法的基础上,优化了特征匹配方法,以高效处理非焦面荧光粒子信号。实验结果表明,本系统在单分子免疫标志物样本检测中的下限可达到0.001 pg/mL,在0.001~1 pg/mL的理论检测范围内,变异系数均小于10%,可在一小时内完成最多十份样本的检测。本系统满足了单分子免疫检测的稳定性、高灵敏度和高通量检测需求,在癌症早期筛查领域具有重要的应用前景。
Abstract: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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表 1 a样本荧光粒子检测结果
Table 1. Detection results of fluorescent particles in sample a
稀释倍率 a1 a2 a3 平均值 1∶500 4.54 3.94 5.1 4.53 1∶ 1000 2.23 2.13 1.65 2 1∶ 3000 0.53 0.79 0.78 0.7 1∶ 4000 0.64 0.49 0.38 0.5 1∶ 5000 0.55 0.32 0.42 0.43 1∶ 10000 0.32 0.25 0.18 0.25 1∶ 50000 0.01 0.01 0 0.01 表 2 b样本荧光粒子检测结果
Table 2. Detection results of fluorescent particles in sample b
稀释倍率 b1 b2 b3 平均值 1∶500 4.41 4.14 3.93 4.16 1∶ 1000 2.09 1.37 1.56 1.67 1∶ 3000 0.98 0.77 0.64 0.79 1∶ 4000 0.46 0.43 0.4 0.43 1∶ 5000 0.57 0.56 0.26 0.46 1∶ 10000 0.24 0.31 0.17 0.24 1∶ 50000 0 0.01 0 0 表 3 本文系统与Simoa HD-X系统性能对比
Table 3. Performance comparison between the proposed system and Simoa HD-X system
性能参数 Simoa系统 本系统 取样量 1~100 μL 1~100 μL 反应量 0.048 μL 0.2~1 μL 检测限 0.01~100 pg/mL 0.001~1 pg/mL 动态范围 ≥4个数量级 ≥3个数量级 精确度 CV<10% CV<10% -
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