微流控聚合物链式反应芯片的多色荧光检测

为了实现微流控芯片上多目标DNA片段的同时检 测,建立了基于光棒匀光结构的微流控芯片多色 荧光检测系统。根据微流控芯片中的聚合物链式反应(PCR)反应腔的特点,设计了基于超亮 白光LED模组、光棒、二向色镜 和CCD的正交型荧光检测系统。CCD可一次采集微流控芯片上所有PCR反应腔中的荧光信号 ,通过滤光 片轮组合的变换,可实现多种荧光标记物的同时检测。采用荧光素钠溶液对激发光的均匀性 进行了测试, 激发产生的荧光图像均匀度达到93.99%,可满足微流控芯片上多反应 腔同时检测的需求。同时,以pUC-18人工质粒DNA作为标准品,开展了微流控荧光PCR生物实验,对系统性能进行验证。实验结 果表明:微 流控PCR反应腔的 DNA浓度变化与荧光信号的变化相一致,pUC-18样品的检测限达到0.05pg/uL,扩增 效率为97.28%,熔解曲线显示无引物二聚体产生,特异性好,达到了 商业化仪器的水平。

Multicolor fluorescence detection for microfluidic PCR chip

In order to achieve multitarget DNA test in microfluidic chip at th e same time,this paper develops a multicolor fluorescence detection system for microfluidic chip based on a uniform illumination structure of light pipe homogenizer.According to the characteristi cs of the polymerase chain reaction (PCR) chamber in microfluidic chip,an orthog onal type fluorescent detection system is designed using super bright white LED modules,light pipe homogenizer,dichroic mirror and CCD.The f luorescent signals of all PCR reaction chambers in microfluidic chip can be collected at the same time,and also a variety of fluorescent markers can be detected simultaneously through the transformation of the optical filter. Fluorescein sodium solution was carried out to test the uniformity of excitation light,the fluorescence image uniformity is 93.99%,and it can meet the design requirements.To evalu ate the performance of the developed fluorescence detection system,we successfully performed PCR analysis of pUC-18.The experimental results show that the DNA concentration change of PCR reaction cha mber is consistent with that of fluorescent signal,and the limit of detection of pUC-18is 0.05pg/uL.The melting curves indicate that there are no primer dimers and specificity is good.The amplification efficiency is 97.28% and can achieve commercial-level.