大视场dPCR芯片荧光检测系统的设计与优化

Design and improvement of large field of view fluorescent detection system for dPCR gene chip

数字PCR(dPCR)技术作为传统PCR技术的更新迭代,有着绝对定量的特点,在新冠病毒核酸序列检测、癌细胞探测等生物学领域有着巨大的潜力。现有的dPCR荧光检测系统在检测通量、检测速度以及成本之间难以做出合理的权衡。由于成像视野小、需多次图像拼接极大影响了dPCR荧光检测系统效率和时间。文章构建了一套采用大视场荧光显微设计的多通道dPCR检测系统实验平台,依托该实验平台,运用Zemax软件优化结构,改进显微成像物镜设计,能够实现直径18mm的全视场范围FAM、HEX、ROX三种荧光通道分辨率在6μm的多通道成像。采用随机霍夫变换算法(RHT)图像处理分析方法,对常规均匀“圆孔”和新型“雪花”、“树枝”等非均匀结构的微流控荧光芯片,均能实现高效检测,得到清晰、稳定的荧光图像。

As a Digital PCR(dPCR) technology,an updated iteration of traditional PCR technology with absolute quantification,has great potential in biological fields such as neo-coronavirus nucleic acid sequence detection and cancer cell detection.Existing dPCR fluorescence detection systems are challenging to make a reasonable trade-off between detection throughput,detection speed,and cost.The small imaging field of view and multiple image stitching greatly limit the accuracy and detection time of dPCR fluorescence detection systems.Therefore,a digital PCR detection experimental platform is builded with multi-channel based on a large field-of-view fluorescence microscope system.Rely on this platform,By simulation experiments of Zemax to improve the design of microscopy objective,Relying on this experimental platform,Zemax software is used to optimize the system structure and improve the design of microscopic objectives,which can realize an 18mm full field of view and 6.3μm resolution among the FAM、HEX、ROX channels.By using the Randomized Hough transform algorithm(RHT),the microfluidics chip including the normal "holewell" structure、the new structure such as "snowflake","branch" and other Irregular structures can be obtain a clear and stable fluorescence images under the premise of a large field of view and fast detection.