The detection of proteins is of central importance to biomolecular analysis and diagnostics, yet fundamental limitations due to the surface-based nature of most sensing approaches persist, and limited improvements have been designed to integrate multimodal information beyond concentration measurements. Here we present a single-molecule microfluidic sensing platform for digital protein biomarker detection in solution, termed digital immunosensor assay (DigitISA). DigitISA is based on microchip electrophoretic separation combined with single-molecule detection and enables absolute number–concentrations quantification of proteins in a single, solution-phase step. Applying DigitISA to a range of targets including amyloid aggregates, exosomes, and biomolecular condensates, we demonstrate that the assay provides information beyond stoichiometric interactions, and enables characterization of immunochemistry, binding affinity, and protein biomarker abundance. Together, DigitISA constitutes a new experimental paradigm for the digital sensing of protein biomarkers, and enables analyses of targets that would otherwise be hard or impossible to address by conventional immuno-sensing techniques.

中文翻译：

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溶液中蛋白质生物标志物的直接数字传感

蛋白质的检测对于生物分子分析和诊断至关重要，但由于大多数传感方法基于表面的性质，其基本局限性仍然存在，并且已经设计了有限的改进来整合浓度测量之外的多模态信息。在这里，我们提出了一种用于溶液中数字蛋白质生物标志物检测的单分子微流体传感平台，称为数字免疫传感器测定 (DigitISA)。DigitISA 基于微芯片电泳分离与单分子检测相结合，能够在单个溶液相步骤中对蛋白质进行绝对数量-浓度定量。将 DigitISA 应用于一系列目标，包括淀粉样蛋白聚集体、外泌体和生物分子凝聚物，我们证明该测定提供了化学计量相互作用以外的信息，并能够表征免疫化学、结合亲和力和蛋白质生物标志物丰度。DigitISA 共同构成了蛋白质生物标志物数字传感的新实验范例，并能够分析传统免疫传感技术难以或不可能解决的目标。