Applied Research and Technology, Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, IL 60064.
Applied Research and Technology, Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, IL 60064
Proc Natl Acad Sci U S A. 2021 Apr 6;118(14). doi: 10.1073/pnas.2025033118.
Every year, over 100 million units of donated blood undergo mandatory screening for HIV, hepatitis B, hepatitis C, and syphilis worldwide. Often, donated blood is also screened for human T cell leukemia-lymphoma virus, Chagas, dengue, Babesia, cytomegalovirus, malaria, and other infections. Several billion diagnostic tests are performed annually around the world to measure more than 400 biomarkers for cardiac, cancer, infectious, and other diseases. Considering such volumes, every improvement in assay performance and/or throughput has a major impact. Here, we show that medically relevant assay sensitivities and specificities can be fundamentally improved by direct single-molecule imaging using regular epifluorescence microscopes. In current microparticle-based assays, an ensemble of bound signal-generating molecules is measured as a whole. By contrast, we acquire intensity profiles to identify and then count individual fluorescent complexes bound to targets on antibody-coated microparticles. This increases the signal-to-noise ratio and provides better discrimination over nonspecific effects. It brings the detection sensitivity down to the attomolar (10 M) for model assay systems and to the low femtomolar (10 M) for measuring analyte in human plasma. Transitioning from counting single-molecule peaks to averaging pixel intensities at higher analyte concentrations enables a continuous linear response from 10 to 10 M. Additionally, our assays are insensitive to microparticle number and volume variations during the binding reaction, eliminating the main source of uncertainties in standard assays. Altogether, these features allow for increased assay sensitivity, wide linear detection ranges, shorter incubation times, simpler assay protocols, and minimal reagent consumption.
每年，全世界有超过 1 亿单位的捐献血液接受强制性的 HIV、乙型肝炎、丙型肝炎和梅毒筛查。通常，捐献的血液也会筛查人类 T 细胞白血病-淋巴瘤病毒、恰加斯病、登革热、巴贝西虫病、巨细胞病毒、疟疾和其他感染。全世界每年进行数十亿次诊断测试，以测量 400 多种与心脏、癌症、传染病和其他疾病相关的生物标志物。考虑到如此庞大的数量，每次检测性能和/或通量的提高都会产生重大影响。在这里，我们展示了通过使用常规的明场荧光显微镜直接进行单分子成像，可以从根本上提高与医学相关的检测灵敏度和特异性。在当前基于微球的检测中，作为一个整体来测量结合的信号产生分子的集合。相比之下，我们获取强度分布来识别并然后对结合到抗体包被的微球上的目标的单个荧光复合物进行计数。这增加了信号与噪声的比率，并提供了对非特异性影响的更好区分。它将检测灵敏度降低到模型检测系统的皮摩尔级（10-18M），并降低到检测人血浆中分析物的飞摩尔级（10-19M）。从计数单分子峰过渡到在较高分析物浓度下平均像素强度，使得从 10 到 1010M 的连续线性响应成为可能。此外，我们的检测对结合反应期间微球数量和体积的变化不敏感，消除了标准检测中不确定性的主要来源。总的来说，这些特性提高了检测灵敏度、宽线性检测范围、缩短了孵育时间、简化了检测方案和最小化了试剂消耗。
