University of Leipzig, Institute of Analytical Chemistry, Leipzig, Germany.
Electrophoresis. 2011 Nov;32(22):3108-14. doi: 10.1002/elps.201100204.
Herein we introduce deep UV fluorescence lifetime detection in microfluidics applied for label-free detection and identification of various aromatic analytes in chip electrophoresis. For this purpose, a frequency quadrupled Nd:YAG (neodymium-doped yttrium aluminum garnet) picosecond laser at 266 nm was incorporated into an inverse fluorescence microscope setup with time-correlated single photon counting detection. This allowed recording of photon timing with sub-nanosecond precision. Thereby fluorescence decay curves are gathered on-the-fly and average lifetimes can be determined for each substance in the electropherogram. The aromatic compounds serotonin, propranolol, 3-phenoxy-1,2-propanediol and tryptophan were electrophoretically separated using a fused-silica microchip. Average lifetimes were independently determined for each compound via bi-exponential tail fitting. Time-correlated single photon counting also allows the discrimination of background fluorescence in the time domain. This results in improved signal-to-noise-ratios as demonstrated for the above model analytes. Microchip electrophoretic separations with fluorescence lifetime detection were also performed with a protein mixture containing lysozyme, trypsinogen and chymotrypsinogen emphasizing the potential for biopolymer analysis.
在这里，我们介绍了应用于微流控的深紫外荧光寿命检测，用于芯片电泳中各种芳香分析物的无标记检测和识别。为此，我们将 266nm 的倍频 Nd:YAG（掺钕钇铝石榴石）皮秒激光器集成到具有时间相关单光子计数检测的反向荧光显微镜设置中。这使得能够以亚纳秒精度记录光子定时。从而可以实时采集荧光衰减曲线，并确定电泳图谱中每种物质的平均寿命。使用熔融硅微芯片对血清素、普萘洛尔、3-苯氧基-1,2-丙二醇和色氨酸等芳香族化合物进行了电泳分离。通过双指数尾部拟合，独立确定了每种化合物的平均寿命。时间相关的单光子计数还允许在时域中区分背景荧光。如上述模型分析物所示，这可提高信噪比。还使用含有溶菌酶、胰蛋白酶原和糜蛋白酶原的蛋白质混合物进行了带有荧光寿命检测的微芯片电泳分离，强调了用于生物聚合物分析的潜力。
