Research On The Key Technologies And System Development Of Detection Device For Self-priming And Discretization Digital PCR Chip

Development of digital PCR chip fluorescence detection device is an interdisciplinary hotspot, which covers biology, optics, mechanism, electronic and computer technologies. In this thesis, a performance prototype of detection system for Self-priming and compartmentalization (SPC) chip has been successfully developed with deep research. Meanwhile, A549 cells were used to implement digital PCR reaction and uncertainty analysis has been made to verify the effectiveness of the detection device.The specific work and main achievements in this thesis are listed as follows:1. The photoelectric system with a stable and efficient fluorescence excitation and acquisition function was designed and built. Specific as:A high power LED driving circuit with constant current output, including schematic diagram and PCB layout, was designed based on TK5401. Secondly, the wave-front recombination method was used to design the collimated light path with multi-lens combination, and a simulation was done by the Tracepro software. Thirdly, the fluorescence acquisition optical path without shortwave shift was built using a CCD of good performance and matching filters.2. An fluorescence detection and analysis system for digital PCR was developed, which has functions of image acquisition image processing、positive chambers counting, results output data management and so on. Specific as:The fluorescence image tilt correction effect was verified based on Characteristic Point method, PC A principal component analysis% Gray Projection method. In addition, according to the defects of conventional filtering algorithms and watershed algorithm, a segmentation algorithm for chip chambers based on automatic locating method was proposed. At last, the OTSU algorithm was applied to confirm the threshold and count the positive chambers. The results indicated this method was very fast and accurate.3. A study on the uncertainty of fluorescence detection system was fully developed. And the uncertainty of digital PCR detection result was evaluated.4. To realize the fluorescence sub-image acquisition and reproduction of microfluidic chip with ten thousands of chambers. Combined with a precise displacement platform, an image stitching technology based on Harris algorithm with RANSAC correction method was realized. And then, a fast and smooth splicing method based on chambers’ row-column information was proposed for regular array microfluidic chip.The successful development of the fluorescence detection equipment indicates the good integration of the detection technology and the detection equipment. And it promoted the development and the application of dPCR technology.