Diffusion-based single particle sizing method provides precise measurement of nanoparticle individual size variation and formation of bioconjugates

We present diffusion‑based single particle sizing (DBSPS), a method for determining hydrodynamic size distributions of fluorescent quantum dots (QDs) and QD bioconjugates. Using fluorescence video microscopy, individual particle trajectories were recorded and analyzed via mean square displacement to calculate diffusion coefficients, which were then converted to hydrodynamic radii using the Stokes–Einstein equation. Unlike ensemble measurements, DBSPS characterizes single particles within heterogeneous samples, enabling detection of QD–biomolecule conjugation efficiency and exclusion of aggregated QD complexes. Results confirm that DBSPS accurately measures hydrodynamic dimensions of QDs and nanospheres, offering a powerful tool for optimizing QD bioconjugate design in aqueous environments.