Time-resolved microfluidic flow cytometer for decoding luminescence lifetimes in the microsecond region
Time-resolved microfluidic flow cytometer for decoding luminescence lifetimes in the microsecond region
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DOI：
10.1039/c9lc00895k
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发表时间：
2020-02-07
期刊：
影响因子：
6.1
通讯作者：
Lu，Yiqing
中科院分区：
文献类型：
--
作者：
Wang，Yan;Sayyadi，Nima;Lu，Yiqing
使用寿命在微秒区域的长寿命探针的时间分辨发光检测在超灵敏和多重生物分析中显示出巨大的潜力。然而，在流式细胞术中，长寿命对测量提出了重大挑战，其中检测窗口通常太短而不能确定衰减特性。在这里，我们报告了一个时间分辨的微流控流式细胞仪（tr-mFCM），结合了一个声聚焦芯片，它允许减慢的流动，同时为每个目标提供相同的检测条件，实现准确的寿命测量无自体荧光干扰。通过相对于时间选通序列配置流速和检测孔径，在最大激发和检测效率下捕获每个事件的多循环发光衰减曲线。然后开发了一种自定义拟合算法，以解决铕染色的聚合物微球以及白血病细胞对丰富的荧光颗粒，实现计数效率接近100%和寿命CV（变异系数）约2- 6%。我们进一步证明了用不同的铕探针染色的前列腺癌和膀胱癌细胞的寿命多路复用检测。我们的声聚焦tr-mFCM为快速筛选含有多种细胞类型的生物流体样品提供了一种实用的技术，特别是在资源有限的环境中，如区域和/或欠发达地区以及即时应用。
Time-resolved luminescence detection using long-lived probes with lifetimes in the microsecond region have shown great potential in ultrasensitive and multiplexed bioanalysis. In flow cytometry, however, the long lifetime poses a significant challenge to measure wherein the detection window is often too short to determine the decay characteristics. Here we report a time-resolved microfluidic flow cytometer (tr-mFCM) incorporating an acoustic-focusing chip, which allows slowing down of the flow while providing the same detection conditions for every target, achieving accurate lifetime measurement free of autofluorescence interference. Through configuration of the flow velocity and detection aperture with respect to the time-gating sequence, a multi-cycle luminescence decay profile is captured for every event under maximum excitation and detection efficiency. A custom fitting algorithm is then developed to resolve europium-stained polymer microspheres as well as leukemia cells against abundant fluorescent particles, achieving counting efficiency approaching 100% and lifetime CVs (coefficient of variation) around 2–6%. We further demonstrate lifetime-multiplexed detection of prostate and bladder cancer cells stained with different europium probes. Our acoustic-focusing tr-mFCM offers a practical technique for rapid screening of biofluidic samples containing multiple cell types, especially in resource-limited environments such as regional and/or underdeveloped areas as well as for point-of-care applications.