Single-Particle Tracking via Direct Labeling of the Viral Lipid Envelope with Cholesterol-DNA-Functionalized Quantum Dots

Quantum dots (QDs) provide rapid, continuous, and prolonged imaging of individual molecules and viruses, offering more comprehensive dynamic data than traditional fluorophores. However, their application in visualizing viral infections is limited, mostly due to the absence of a mild, efficient, and applicable labeling technique. This study presents a simple and gentle technique for labeling enveloped viruses with QDs, requiring only a single brief incubation phase. This method relies on the inherent attraction of the viral lipid envelope to cholesterol (chol), which is shown on Cadmium telluride (CdTe) QDs via Chol-DNA functionalization. The QDs were synthesized in a single step by the coordination of Cd2+ ions with phosphorothioate-modified DNA strands, maintaining high fluorescence efficiency and small particle size while enabling DNA modification. This labeling technique attains great efficiency in about one hour while maintaining viral structural integrity and infectivity. Employing this lipid-specific technique, this study established dual-color fluorescent SARS-CoV-2 spike-pseudotyped virions by labeling the envelope with QDs and the nucleic acid with Ru[(phen)2(dppz)]2+, facilitating real-time monitoring and analysis of infection dynamics. The established lipid-specific QD labeling technique provides an efficient and versatile tool for visually monitoring viruses and analyzing their infection paths, thus promoting broader utilization of single-virus identification and improving comprehension of complex infection mechanisms.