DNA分析仪的灵敏度分析

Detection Limit of DNA Analyzer

设计了DNA分析仪,采用激光诱导荧光对样品的毛细管电泳结果进行检测,对仪器的灵敏度进行了理论分析和实验测试.分析表明:荧光的探测灵敏度取决于入射到光探测器(PMT或CCD)上的荧光强度及光探测器自身的探测能力.结合设计参量,计算得到DNA分析仪针对染料TO(thiazole orange)作为荧光标记的探测灵敏度为0.127fluor/μm3个荧光分子.和实验结果相比,双链DNA样品pGEM-3Zf(+)/Hae Ⅲ Markers加TO进行毛细管电泳实验,得到实际的探测灵敏度为0.1454fluor/μm3荧光分子.研究表明:提高探测灵敏度的措施是制备性能良好的毛细管涂层和合理使用筛分介质,合理增大激光功率、尽可能增大物镜的数值孔径、提高光电倍增管的探测下限.

The sensitivity of the DNA analyzer, which was designed by authors group to detect the samples of capillary electrophoresis （CE） with Laser induced fluorescence （LIF）, was analyzed in theory and experiment. The detection sensitivity of fluorescence depends on both the fluorescent intensity entering the sensors （PMT or CCD） and the performance of sensors. Given an actual DNA analyzer, the calculated detection limit of TO（thiazole orange） fluorophore was 0. 127 1 - 0. 127 2 fluors per cubic micron. The experimental result of CE about double-stranded （ds） TO probes （the standard sample pGEM-3Zf（＋）/ Hae Ⅲ Markers） is 0. 145 4 fluors per cubic micron. The study shows that the approaches for improving detection limit are to make the good coating on inner walls of capillary, choose the suitable sieving matrix, enlarge laser power, increase numerical aperture of objective as much as possible and raise the detection lower limit of photomultiplier.