Research On The Fluorescence Detecting Technology In Microfluidic Channel

Biological analysis technology based on microfluidic channel is a focus for scientists in the field of biological detection, which covers multi-disciplines such as microfabrication, biological engineering, analytical chemistry, polymer science, optical engineering and so on. Correlative technologies and applications have developed greatly in China. Some research results have reached or have been closed to international top level. But in some key technologies such as high speed detecting and high sensitivity detecting, our research still has a large gap with the international top level. This dissertation carries on the research on fluorescence detecting technology with fast speed and high sensitivity used for suspension fluorescence microspheres' array and capillary electrophoresis system for DNA analyzing.The parallel detecting technology for suspension fluorescence microspheres' array in this dissertation adopted a pulsed Xenon lamp to expose flowing fluorescence microspheres serially, which had a 2D distributing in microfluidic channel. CCD was used to capture images of multi-microsphere at the same time. Microspheres were recognized in fluorescence images and parallel detection was achieved. Dynamic images of fluorescence microspheres with diameter of 6 microns were captured for the first time in the system. Limit of detection of the system was fewer than 10 fluorescence molecules per square micron. Detecting speed could reach 30000 fluorescence microspheres per second, which was higher than that of flowing cytometry by 1～2 orders of magnitude.DNA analyzing system by capillary electrophoresis in the dissertation adopted a laser of 488nm or 532nm to excite fluorophore labeling DNA separated by electrophoresis in a capillary. Fluorescence was collected by an f-theta lens and was detected by a PMT with confocal optical arrangement. The electrophoresis condition was optimized in experiments. Due to the system optimization, dsDNA fragments" were separated and single base pair resolution was achieved in the system. Electrophoresis analysis of Short Tandem Repeats loci in a capillary with effective length of 14cm could be completed. Resolution for all loci couldbe closed to baseline level in 23 minutes, which was shorter than half of the time used by ABI 310. Research on spatial distribution of florescence and noise indicated that compared with the noise caused by refraction and reflection from inner and outer walls of the capillary, noise reduced by Rayleigh scattering could be ignored. The optimal angle between the fluorescence collecting direction and the exciting laser beam was 75° .At last, a new architecture of fluorescence detecting for microfluidic channel in microfluidic chip was presented. In this design, a prism was utilized to couple exciting laser into the microfluidic channel and an elliptical reflector was used to focus fluorescence outside of the microfluidic chip and to reduce the divergent angle of fluorescence. Theoretical requirement for working distance of objective for this structure could be reduced to be infinitely short. Fluorescence collection efficiency was improved for about 6.3 times.