Dynamic Polymer Cross-linking Limits the Homogeneity of Compact Quantum Dots for Single-Particle Tracking.

Quantum dots (QDs) are semiconductor nanocrystals with stable and bright fluorescence, attributes particularly valued for single-molecule imaging in the life sciences. For these applications, QDs must be compact and homogeneously dispersed as single colloids, attributes enabled by multidentate polymer coatings. However, high-resolution analyses show evidence of clusters of two or more QDs (multimers) that may dominate measurements at the single-particle level. Here, we study the mechanisms of multimer formation using chromatographic separation, microscopy, spectroscopy, and affinity measurements. We find that multimers derive from dynamic polymer cross-linking and exist in a reversible state that is concentration-dependent and influenced by free polymers. Compared with monomers, QD multimers exhibit heterogeneous brightness, protein-induced aggregation, and enhanced nonspecific binding to cells, effects that bias single-particle measurements in live cells. Multimers can be depleted by purification, blocking desorbed binding groups on polymers, or increasing the net electrostatic charge. These findings provide solutions for improving nanocrystal quality for life science applications and point toward reporting standardizations of nanoparticle concentration and sample purity during characterization and application.