Department of Biomedical Engineering, Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, P.O. Box 513, Eindhoven5600 MB, Netherlands.
Laboratory of Chemical Biology and Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, Eindhoven5600 MB, The Netherlands.
ACS Sens. 2023 Jan 27;8(1):80-93. doi: 10.1021/acssensors.2c01736. Epub 2023 Jan 19.
Characterization of the number and distribution of biological molecules on 2D surfaces is of foremost importance in biology and biomedicine. Synthetic surfaces bearing recognition motifs are a cornerstone of biosensors, while receptors on the cell surface are critical/vital targets for the treatment of diseases. However, the techniques used to quantify their abundance are qualitative or semi-quantitative and usually lack sensitivity, accuracy, or precision. Detailed herein a simple and versatile workflow based on super-resolution microscopy (DNA-PAINT) was standardized to improve the quantification of the density and distribution of molecules on synthetic substrates and cell membranes. A detailed analysis of accuracy and precision of receptor quantification is presented, based on simulated and experimental data. We demonstrate enhanced accuracy and sensitivity by filtering out non-specific interactions and artifacts. While optimizing the workflow to provide faithful counting over a broad range of receptor densities. We validated the workflow by specifically quantifying the density of docking strands on a synthetic sensor surface and the densities of PD1 and EGF receptors (EGFR) on two cellular models.
在生物学和生物医学中，对 2D 表面上生物分子的数量和分布进行特征描述至关重要。带有识别基序的合成表面是生物传感器的基石，而细胞膜上的受体则是疾病治疗的关键目标。然而，用于定量其丰度的技术是定性或半定量的，通常缺乏灵敏度、准确性或精密度。本文详细介绍了一种基于超分辨率显微镜（DNA-PAINT）的简单而通用的工作流程，该流程已标准化，可提高合成底物和细胞膜上分子密度和分布的定量。基于模拟和实验数据，对受体定量的准确性和精密度进行了详细分析。通过过滤非特异性相互作用和伪影，我们证明了增强的准确性和灵敏度。在优化工作流程以在广泛的受体密度范围内提供真实计数的同时。我们通过特异性地量化合成传感器表面上的对接链的密度以及两种细胞模型上 PD1 和 EGF 受体（EGFR）的密度来验证该工作流程。
