Semiconductor quantum dots (QDs) have proven to be superior probes for single-molecule imaging compared to organic or genetically encoded fluorophores, but they are limited by difficulties in protein targeting, their larger size, and on-off blinking. Here, we report compact aqueous CdSe/CdS QDs with significantly improved bioconjugation efficiency and superior single-molecule optical properties. We have synthesized covalent protein labeling ligands (i.e., SNAP tags) that are optimized for nanoparticle use, and QDs functionalized with these ligands label SNAP-tagged proteins ∼10-fold more efficiently than existing SNAP ligands. Single-molecule analysis of these QDs shows 99% of time spent in the fluorescent on-state, ∼4-fold higher quantum efficiency than standard CdSe/ZnS QDs, and 350 million photons detected before photobleaching. Bright signals of these QDs enable us to track the stepping movement of a kinesin motor in vitro, and the improved labeling efficiency enables tracking of single kinesins in live cells.
Keywords: SNAP tag; bioconjugation; bioimaging; kinesin; motor protein; quantum dot; single-molecule biophysics.