Revealing Spatial Molecular Heterogeneity of High-Density Biofunctionalized Surfaces Using DNA-PAINT

The quantification and control of molecular densities and distributions on biofunctionalized surfaces are key for enabling reproducible functions in biosciences. Here, we describe an analysis methodology for quantifying the density and spatial distribution of high-density biofunctionalized surfaces, with densities in the order of 10²–10⁵ biomolecules per μm² area, in a short measurement time. The methodology is based on single-molecule DNA-PAINT imaging combined with simulation models that compensate for lifetime and spatial undersampling effects, resulting in three distinct molecule counting methods and a statistical test for spatial distribution. The analysis methodology is exemplified for a surface with ssDNA affinity binder molecules coupled to a PLL-g-PEG antifouling coating. The results provide insights into the biofunctionalization efficiency, yield, and homogeneity. Furthermore, the data reveal that heterogeneity is inherent to the biofunctionalization process and shed light on the underlying molecular mechanisms. We envision that DNA-PAINT imaging with the developed analysis framework will become a versatile tool to study spatial heterogeneity of densely biofunctionalized surfaces for a wide range of applications.