Division of Medical Physics, Department of Radiation Oncology, Stanford University , 300 Pasteur Drive, Palo Alto, California 94305, United States.
University of California-Merced , Department of Bioengineering, 5200 North Lake Road, Merced, California 95343, United States.
Anal Chem. 2017 Jun 20;89(12):6472-6481. doi: 10.1021/acs.analchem.7b00414. Epub 2017 Jun 9.
Radiotracers are widely used to track molecular processes, both in vitro and in vivo, with high sensitivity and specificity. However, most radionuclide detection methods have spatial resolution inadequate for single-cell analysis. A few existing methods can extract single-cell information from radioactive decays, but the stochastic nature of the process precludes high-throughput measurement (and sorting) of single cells. In this work, we introduce a new concept for translating radioactive decays occurring stochastically within radiolabeled single-cells into an integrated, long-lasting fluorescence signal. Single cells are encapsulated in radiofluorogenic droplets containing molecular probes sensitive to byproducts of ionizing radiation (primarily reactive oxygen species, or ROS). Different probes were examined in bulk solutions, and dihydrorhodamine 123 (DHRh 123) was selected as the lead candidate due to its sensitivity and reproducibility. Fluorescence intensity of DHRh 123 in bulk increased at a rate of 54% per Gy of X-ray radiation and 15% per MBq/ml of 2-deoxy-2-[F]-fluoro-d-glucose ([F]FDG). Fluorescence imaging of microfluidic droplets showed the same linear response, but droplets were less sensitive overall than the bulk ROS sensor (detection limit of 3 Gy per droplet). Finally, droplets encapsulating radiolabeled cancer cells allowed, for the first time, the detection of [F]FDG radiotracer uptake in single cells through fluorescence activation. With further improvements, we expect this technology to enable quantitative measurement and selective sorting of single cells based on the uptake of radiolabeled small molecules.
放射性示踪剂广泛用于体外和体内的分子过程追踪，具有高灵敏度和特异性。然而，大多数放射性核素检测方法的空间分辨率不足以进行单细胞分析。一些现有的方法可以从放射性衰变中提取单细胞信息，但该过程的随机性排除了对单细胞的高通量测量（和分选）。在这项工作中，我们引入了一种新概念，可将放射性标记的单细胞内随机发生的放射性衰变转换为集成的、持久的荧光信号。单细胞被封装在含有分子探针的放射性荧光液滴中，这些探针对电离辐射的副产物（主要是活性氧物种或 ROS）敏感。在 bulk 溶液中检查了不同的探针，并且由于其灵敏度和重现性，二氢罗丹明 123（DHRh 123）被选为首选候选物。DHRh 123 在 bulk 中的荧光强度以每 Gy X 射线辐射 54%和每 MBq/ml 2-脱氧-2-[F]-氟-D-葡萄糖（[F]FDG）增加 15%的速率增加。微流控液滴的荧光成像显示出相同的线性响应，但与 bulk ROS 传感器相比，液滴的总体灵敏度较低（每个液滴的检测限为 3 Gy）。最后，封装放射性标记癌细胞的液滴首次允许通过荧光激活检测单细胞中 [F]FDG 放射性示踪剂的摄取。通过进一步改进，我们期望这项技术能够实现基于放射性标记小分子摄取的单细胞的定量测量和选择性分选。
