用于单细胞内/表面超痕量蛋白实时、精准检测的超高灵敏度激光诱导荧光检测器（LIF）研究

Single cell analysis can reveal differences between cells. Detection of ultra-trace proteins (zmol-fmol) in/on a single cell can reveal life phenomena at the molecular level, which has important biological and medical significance. Laser induced fluorescence detection (LIF) with super high sensitivity and selectivity is the best choice for ultra-trace detection. In this project, ultra-high sensitive LIF in confocal arrangement will be developed. Study includes 1) adding spherically negative GRIN in front of the detection cell, the GRIN and the objective lens work together to focus and collect, to improve the collection efficiency; refractive index matching fluid (RIMF) is used between GRIN and the detection cell to reduce the scattered light; 2) LIF with long Stokes Shift is designed to avoid the scattering of solvent and silicon, reducing the scattered light; 3) developing appropriative filter technology of bandpass combined with polarization for ultra-high sensitive LIF, to improve the signal-to-noise ratio (SNR); 4) adding “the interference fringe” and “the center spot” adjusting method, to make the alignment better and more repetitive. The developed LIF is combined with capillary electrophoresis or microfluidic chip system. The lowest detection number is close to 20 molecules for active caspase3 proteins in a single leukemia cell, about 0.15 molecular per μm2.

单细胞分析能揭示细胞间的差异性。检测单细胞内/表面的超痕量蛋白（zmol-fmol）能在分子水平上揭示生命现象，有重要的生物学和医学意义。激光诱导荧光检测（LIF）有极高的灵敏度和选择性，是超痕量检测的最佳选择。本项目拟研制超高灵敏度共聚焦LIF。研究包括1）检测池前增加消球差的梯度折射率透镜（GRIN），GRIN与物镜组合聚焦和收集，提高收集效率；GRIN和检测池之间填充折光补偿液体，降低杂散光；2）设计长斯托克斯位移LIF，避开溶剂和硅基底的散射光谱，降低杂散光；3）研制超高灵敏度LIF专用的带通、偏振组合滤光技术，提高信噪比；4）增加“干涉条纹”和“中心光斑”调光路方法，LIF对准更佳、更重复。研制的LIF与毛细管电泳或微流控芯片结合，对单个白血病细胞内的active caspase3蛋白的最低检测分子数接近20个，约0.15个分子/μm2。

单细胞分析能揭示细胞间的差异性。检测单细胞内/表面的超痕量蛋白（zmol-fmol）能在分子水平上揭示生命现象，有重要的生物学和医学意义。激光诱导荧光检测（LIF）有极高的灵敏度和选择性，是超痕量检测的最佳选择。本项目拟研制超高灵敏度共聚焦LIF。研究包括：1）检测池前增加消球差的梯度折射率透镜（GRIN），GRIN与物镜组合聚焦和收集，提高收集效率；GRIN和检测池之间填充折光补偿液体，降低杂散光；2）设计长斯托克斯位移LIF，避开溶剂和硅基底的散射光谱，降低杂散光；3）研制超高灵敏度LIF专用的带通、偏振组合滤光技术，提高信噪比；4）增加“干涉条纹”和“中心光斑”调光路方法，LIF对准更佳、更重复。. 经过研究团队共同努力，本项目凝练出单分子级超高灵敏度小型激光诱导荧光检测器（mLIF）设计的创新关键科学原理和方法。1）研制出超高灵敏共聚焦mLIF检测器，检测灵敏度比进口名牌产品指标提高10倍以上；同时实现漂移≤0.12 mV/h；2）将所研制的mLIF检测器与毛细管电泳（CE）结合，实现对单个白血病细胞内的active caspase3蛋白的实时、精准定量检测，最低检测分子数7个，约0.06个分子/μm2；3）设计了球面二向色反光镜（SDR），mLIF的检测限达到了流动注射（FIA）评测的最高灵敏度，对荧光素钠检测限0.15 pM（LOD，3σ方法），与之前的工作相比，灵敏度提高了30倍；mLIF定量检测三种氨基酸，检测限介于pM至fM之间，是报道的检测氨基酸的最高灵敏度；4）研制了1套近红外LIF检测器，对CF790染料检测限5.1×10-14 mol/L，优于文献报道的光纤式LIF数百倍；将检测器用于检测离体病变的PG样本，准确率为83.3%，符合临床PG检测要求。