微流体分析中常用的荧光检测是一种模拟方式的测量，并且通常情况下测量信号的强度会随着分析物采样量的减少而减弱。本文构建了一种共焦光学系统的激光诱发荧光单分子检测仪器，可以进行单个脱氧核糖核酸分子的数字化测量，采用这种仪器检测可不依赖于样品的多少；对诱发激光的功率大小进行了优化，当诱发激光的功率为1 mW时信噪比最高，可达30；对用于制造微流体芯片的几种不同高分子基片材料也做了检验和比较，其中环烯烃共聚物具有极低的自发荧光背景，这对光子簇的识别非常有利，实验结果表明环烯烃共聚物是制造单分子检测微流体芯片的首选材料；这种单分子检测仪器能够对流经微流体通道的单个脱氧核糖核酸分子进行数字式甄别，其动态检测范围上至25 pmol/L，下至单个分子水平。本研究中对单分子检测实验所涉及的一些方面进行了考虑，这为单分子测量在更大范围内生物分析上更多的实际应用提供了依据。%Fluorescence detection commonly used in microfluidic analysis is an analog measurement,and the signal intensity is usually weakened by reduced sample size.This report presents a laser-induced fluorescence single-molecule detection instrument with confocal setup for digital counting of single DNA molecules,which does not depend on sample size.The instrument setup is thoroughly described.The applied laser power was optimized and the highest signal-to-background ratio of 30 was achieved at the optimal laser power of 1 mW. Different polymer substrates were examined and compared,and COC was demonstrated to be the substrate of choice to make microfluidic chip for single-molecule detection attributed to its superb autofluorecence background, which favors photon burst identification.This single-molecule detector is able to digitally count the number of individual DNA molecules that flow through microfluidic channels,and its dynamic range spans up to 25 pM and down to the "single molecule" level.The considerations on various aspects of the single-molecule detection experiment in this study pave a way to performing more practical single-molecule measurements in a broader range of bioanalytical applications.
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