As the need for single-molecule techniques grows, commensurate with the desire to study complex samples and the heterogeneity of systems, we present the single-molecule microfluidic diffusional sizing (sm-MDS) platform and demonstrate its capabilities at performing diffusional sizing at picomolar sensitivity. Microfluidic diffusional sizing (MDS) is a robust, in-solution method to measure hydrodynamic radii (R hyd ) of particles. The range of MDS is very much suited to measuring the size of individual proteins and higher order protein assemblies, and MDS has indeed been applied to good effect to study proteins and the strength of their interactions. However, conventional MDS is inherently an ensemble-level method—it is performed in the widefield optical regime, and it is fluorescence intensity data that is used for analysis and calculation, which ultimately gives an ensemble-averaged measurement. This limits its detection sensitivity to the nanomolar regime, and limits its capability to probe heterogenous mixtures. By combining confocal illumination with digital single-molecule counting, we establish single-molecule microfluidic diffusional sizing capable of detecting particles at concentrations as low as 1 pM. This enables direct measurement of hydrodynamic radius and dissociation constants in the picomolar range without the need for surfaces, providing a powerful new approach for quantifying high-affinity interactions. We further demonstrate the method’s ability to resolve heterogeneous mixtures at the single-molecule level by distinguishing monomeric and oligomeric α-synuclein, the proteins associated with Parkinson’s disease.
Fan et al. (Sun,) studied this question.