Tan Wei Shan, de Jong Arthur M, Prins Menno W J
Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven 5612 AZ, The Netherlands.
Institute for Complex Molecular Systems (ICMS), Eindhoven University of Technology, Eindhoven 5612 AZ, The Netherlands.
Langmuir. 2025 Aug 26;41(33):22181-22192. doi: 10.1021/acs.langmuir.5c02403. Epub 2025 Aug 13.
Quantifying and controlling the spatial molecular heterogeneity on biofunctionalized particles is essential for understanding and improving their functionality in bioscience applications. Here, we describe an analysis framework based on single-molecule localization microscopy that can quantitatively assess the spatial molecular properties of affinity molecules conjugated to particles. We performed 3D DNA-PAINT imaging on biofunctionalized particles and established analysis methods to correlate single-molecule data to the particle outer surfaces, count the number of conjugated molecules, and quantify the spatial distributions of the conjugated molecules. We show that imaging data combined with simulation-based molecular counting gives access to high densities of conjugated molecules and enables quantification of their spatial distributions. The analysis is exemplified for particles with a diameter of 1 μm functionalized with single-stranded DNA molecules via two bioconjugation methods, namely, streptavidin-biotin coupling and PLL--PEG-based click chemistry. The data reveal interparticle and intraparticle spatial heterogeneities that are dependent on the bioconjugation methods and conditions. With the analysis framework, 3D DNA-PAINT imaging becomes a versatile characterization technique to study biofunctionalized particles and guide future biofunctionalization strategies for a wide range of applications.
量化和控制生物功能化颗粒上的空间分子异质性对于理解和改善其在生物科学应用中的功能至关重要。在此，我们描述了一种基于单分子定位显微镜的分析框架，该框架可定量评估与颗粒共轭的亲和分子的空间分子特性。我们对生物功能化颗粒进行了三维DNA-PAINT成像，并建立了分析方法，以将单分子数据与颗粒外表面相关联、计数共轭分子的数量并量化共轭分子的空间分布。我们表明，成像数据与基于模拟的分子计数相结合，可以获得高密度的共轭分子，并能够量化它们的空间分布。通过两种生物共轭方法，即链霉亲和素-生物素偶联和基于聚赖氨酸-聚乙二醇的点击化学，对直径为1μm的单链DNA分子功能化的颗粒进行了分析示例。数据揭示了颗粒间和颗粒内的空间异质性，这些异质性取决于生物共轭方法和条件。借助该分析框架，三维DNA-PAINT成像成为一种通用的表征技术，可用于研究生物功能化颗粒并指导未来广泛应用的生物功能化策略。
