自吸离散式数字PCR芯片检测装置的关键技术研究与系统开发

发布时间：2019-01-05 08:18

【摘要】：数字PCR微流控芯片荧光检测装置的研发是生物学、光学、机械及电子等多学科交叉的重要热点领域。本文基于本研究组研制的自吸离散式微流控芯片,研究了荧光检测分析系统中的部分关键技术,并开发了相应的性能样机,同时,通过肺癌细胞扩增实验与不确定度理论分析进行了检测系统评价与有效性验证。本文的具体工作和成果主要包括：1、设计和搭建了具有稳定有效的荧光激发与采集功能的光电系统。具体包括：设计了基于TK5401的大功率LED激发光源恒流驱动电路(含原理图和PCB版图)；利用波前重组技术设计了多透镜组合准直均匀光路,并利用Tracepro软件对其进行了光路仿真；采用CCD及配套光学膜片搭建了无短波移荧光采集光路。2、开发了具备图像采集与处理、亮点计数、结果输出及数据管理等功能,且人机交互性能较好的数字PCR荧光检测分析系统。具体包括：验证了主成分分析法、投影法的倾斜校正效果；针对常规滤波和分水岭算法的不足,提出了自动定位小室分割方法；采用大津法进行阈值计算及阳性小室计数,计数快速而准确。3、开展了荧光检测系统不确定性与不确定度的研究,对数字PCR检测结果进行了不确定度评价。4、为了实现万级高密度芯片荧光图像采集与还原,结合位移平台,实现了结合Harris算法和RANSAC修正的荧光图像拼接。进而针对规则检测阵列,提出了基于小室行列信息的快速拼接方法。本装置的成功开发,标志着高精度定量检测技术和检测仪器的成功集成,并将促进数字PCR技术发展及应用推广。
[Abstract]:The research and development of digital PCR microfluidic chip fluorescence detector is an important and hot field in biology, optics, mechanics and electronics. Based on the self-priming discrete microfluidic chip developed by this research group, this paper studies some key technologies of the fluorescence detection and analysis system, and develops the corresponding performance prototype, at the same time, The system evaluation and validity of lung cancer cell expansion test and uncertainty analysis were carried out. The main work and achievements of this paper are as follows: 1. A photoelectric system with stable and effective fluorescence excitation and acquisition function is designed and built. The main contents are as follows: the constant current driving circuit (including schematic diagram and PCB layout) of high power LED excitation light source based on TK5401 is designed, the multi-lens combination collimation uniform optical circuit is designed by using wavefront recombination technology, and the optical path simulation is carried out by Tracepro software. CCD and supporting optical diaphragm are used to construct the light path of fluorescence acquisition without shortwave shift. 2. The functions of image acquisition and processing, bright spot counting, result output and data management are developed. And the digital PCR fluorescence detection and analysis system with good human-computer interaction performance. The main contents are as follows: the principal component analysis (PCA) method and the tilt correction effect of projection method are verified, and the automatic location chamber segmentation method is proposed to overcome the shortcomings of conventional filtering and watershed algorithm. The threshold value and positive chamber count are calculated by using the method of Dajin. 3. The uncertainty and uncertainty of fluorescence detection system are studied, and the uncertainty of digital PCR detection results is evaluated. In order to realize the fluorescence image acquisition and restoration of the high density chip, combining with the displacement platform, the fluorescence image mosaic based on Harris algorithm and RANSAC correction is realized. Then, a fast stitching method based on column and column information is proposed for rule detection array. The successful development of this device marks the successful integration of high precision quantitative detection technology and testing instrument, and will promote the development and application of digital PCR technology.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：TP391.41

【参考文献】