Cheng Yuanhua, Nguyen Alya, Nelson Wyatt, Fujimoto Bryant S, Zhao Mengxia, Chiu Daniel T
Department of Chemistry, University of Washington, Seattle, Washington 98195, United States.
Pangnostics, Inc., Bothell, Washington 98021, United States.
ACS Nano. 2025 Jul 1;19(25):23442-23449. doi: 10.1021/acsnano.5c07028. Epub 2025 Jun 17.
Digital assays such as digital PCR for nucleic acids and digital ELISA for proteins provide absolute quantitation and greater accuracy, sensitivity, and reproducibility than their analogue counterparts (real-time PCR and standard ELISA), but current digital assays involve amplification (e.g., DNA amplification in digital PCR and signal amplification in digital ELISA), which makes high multiplexing difficult, often requires complex and expensive sample compartmentalization, and adds reaction steps. We have developed a single-molecule sensitive flow cytometer, which we termed a digital flow cytometer (dFC). dFC optimizes the sensitivity and efficiency of single-molecule detection by using smaller, planar microfluidic channels, a smaller probe volume, and a shorter working distance/higher numerical aperture objective than used in current commercial high-sensitivity flow cytometers, allowing digital assays via direct single-molecule counting. This paper describes our characterization of the analytical performance of this system when detecting antibody-dye conjugates and demonstrates absolute concentration measurements of commercial antibody-dye conjugates. The dFC exhibited a single-molecule detection efficiency with which over 98% for antibodies conjugated with 18 different small-molecule, phycobiliprotein, and semiconducting polymer dyes were separated from noise, a low false-positive rate, a stable baseline signal, and accurate concentration measurements with a dynamic range spanning 4 orders of magnitude. This system can be used for authenticating antibody-dye conjugates used in flow cytometry and tissue imaging studies and in the development of multiplexed, amplification-free digital assays for nucleic acids and proteins.
诸如用于核酸的数字PCR和用于蛋白质的数字ELISA等数字检测方法，相较于其类似物（实时PCR和标准ELISA），可提供绝对定量以及更高的准确性、灵敏度和可重复性。但目前的数字检测方法涉及扩增（例如数字PCR中的DNA扩增和数字ELISA中的信号扩增），这使得高多重分析变得困难，通常需要复杂且昂贵的样品分区，并且增加了反应步骤。我们开发了一种单分子敏感流式细胞仪，我们将其称为数字流式细胞仪（dFC）。dFC通过使用比当前商用高灵敏度流式细胞仪更小的平面微流控通道、更小的探针体积以及更短的工作距离/更高数值孔径的物镜，优化了单分子检测的灵敏度和效率，从而能够通过直接单分子计数进行数字检测。本文描述了我们在检测抗体 - 染料偶联物时对该系统分析性能的表征，并展示了对商用抗体 - 染料偶联物的绝对浓度测量。dFC表现出单分子检测效率，与18种不同的小分子、藻胆蛋白和半导体聚合物染料偶联的抗体中，超过98%能够与噪声分离，假阳性率低，基线信号稳定，并且在跨越4个数量级的动态范围内能够进行准确的浓度测量。该系统可用于鉴定流式细胞术和组织成像研究中使用的抗体 - 染料偶联物，以及用于开发针对核酸和蛋白质的多重、无扩增数字检测方法。
