Research On Droplet PCR Amplification Detection Method Based On Microfluidic Technolog

Microfludics is a new interdisciplinary for researching the physical,and biochemical processes of fluids,particles,and even biomolecules in microscale environments,which is assisted by microelectromechanical,microsystems,and micro/nano technologies.For the large differences of physical and chemical properties of the multiphase fluid in the microchannel of microfluidic chip,the whole process of generating,flowing,reacting,analyzing,screening and detecting of the microdroplet unit for reaction can be realized.The microdroplets are separated and incompatible,which provides a microcosmic reaction and detection mode from macrography.Based on droplet PCR(Polymerase chain reaction)amplification detection of microfluidics,the nucleic acid molecule is encapsulated in an independent droplet reaction unit,which can realize high-throughput detection by PCR amplification with the advantages of high detection accuracy,low reagent consumption and short detection time.Microfluidic droplet PCR has been widely conceerned as one of the important technical means for gene analysis.In this paper,the microfluidic chip droplet generation technology was studied.Based on the cross-water-in-oil principle,a droplet generation chip capable for generating uniform and stable droplets with a great quantity in a short time was designed and fabricated.A droplet generation platform was established on a computer in this research for fluid driving and controlling.The critical factors such as physical structure of the chip and fluid drive parameters were analyzed and discussed,and the droplet generation chip for PCR amplification was optimized.The microfluidic chip was researched and implemented for reliable and stable droplet amplification,and the stability of the thermal cycle in the amplification chip was tested and verified.A microfluidic droplet PCR amplification detection system was also designed and programmed to realize the multifunction PC software for the microfluidic droplet chip,which contains of UI(User Interface)interaction,PCR thermal cycle temperature control and image detection.Then,the temperature control algorithm and software of the slave machine were designed to ensure the accuracy and effectiveness of the temperature control in thermal cycle.Finally,the system integration and function verification of the microfluidic droplet PCR amplification detection device were carried out.With the microfluidic chip and clinical biological samples,the microfluidic droplets were generated in this platform which contains clinical samples,and then transferred to the droplet amplification chip.The droplet amplification chip was placed on the droplet PCR amplification detection device to perform PCR thermal cycle.When the amplification was completed,the fluorescence signal of the droplet was collected by a fluorescence camera,and the droplet was identified and interpreted with an image analysis method.In this paper,the key technologies in droplet PCR were systematically studied,especially the droplet generation,PCR amplification,and detection,and constructed a highly integrated detection system to realize droplet PCR amplification and detection.This work established a good foundation for the next step to achieve an absolutely quantitative detection of droplet digital PCR.