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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-1 8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。
To achieve simultaneous detection of multi-target DNA fragments on a microfluidic chip, a multi-color fluorescence detection system based on a microfluidic chip was developed. According to the characteristics of polymer chain reaction (PCR) reaction chamber in microfluidic chip, an orthogonal fluorescent detection system based on ultra-bright white LED module, light stick, dichroic mirror and CCD was designed. The CCD can collect the fluorescence signals of all the PCR reaction chambers on the microfluidic chip at a time. By the combination of the filter wheel and the wheel assembly, the simultaneous detection of various fluorescent labels can be realized. Fluorescein sodium solution was used to test the homogeneity of the excitation light. The uniform fluorescence of the fluorescence image was 93.99%, which could meet the requirement of simultaneous detection of multiple reaction chambers on microfluidic chip. At the same time, pUC-18 artificial plasmid DNA as a standard, carried out microfluidic fluorescence PCR biological experiments to verify the system performance. The experimental results showed that the change of DNA concentration in microfluidic PCR reaction chamber was consistent with the change of fluorescence signal. The detection limit of pUC-18 was 0.05 pg / uL and the amplification efficiency was 97.28%. The melting curve showed no primer dimerization Body production, specificity, and reached the level of commercial equipment.