Spatial Molecular Heterogeneity on Biofunctionalized Particles Quantified by Three-Dimensional Single-Molecule DNA-PAINT

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-g-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.