Molecular Foundry, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
ACS Nano. 2017 Jul 25;11(7):6773-6781. doi: 10.1021/acsnano.7b01470. Epub 2017 Jun 21.
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.
半导体量子点 (QDs) 已被证明比有机或遗传编码荧光团更适合用于单分子成像，但它们受到蛋白质靶向困难、尺寸较大以及开-关闪烁等限制。在这里，我们报告了具有改进的生物缀合效率和优越的单分子光学性质的紧凑型水性 CdSe/CdS QDs。我们已经合成了对纳米颗粒使用进行了优化的共价蛋白质标记配体（即 SNAP 标签），并用这些配体功能化的 QDs 标记 SNAP 标记的蛋白质的效率比现有的 SNAP 配体高约 10 倍。这些 QDs 的单分子分析表明，荧光开态的时间占 99%，量子效率比标准 CdSe/ZnS QDs 高约 4 倍，在光漂白之前检测到 3.5 亿个光子。这些 QDs 的明亮信号使我们能够在体外跟踪驱动蛋白的步进运动，并且改进的标记效率使我们能够在活细胞中跟踪单个驱动蛋白。
