Experimental And Theoretical Studies On Droplet Digital PCR And Aptamer Screening Based On Microfluidic Droplet Technology

Droplet microfluidic technology has shown many outstanding advantages in miniaturization,automation,high throughput and low cross-contamination.In recent years,droplet microfluidics in digital PCR and high-throughput screening of nucleic acid aptamers research has become a very important technology platform.However,the droplet microfluidic have some shortcomings in operation performance,detection flux,droplet stability,which restrict the large-scale application of droplet microfluidic.In this paper,we develop a new method to produce droplets and a capillary integrated droplet digital PCR system(CI-ddPCR)is proposed to solve the shortcomings of the existing ddPCR system.Later,the CI-ddPCR system is applied to nucleic acid aptamer screening.This thesis first summarizes the development and current situation of droplet microfluidic technology.Then,we develop two novel droplet generators and study the droplet generation simulation.By tuning the flow rate ratio,capillary inner diameter,tip diameter and other related parameters,we are able to determine the breaking mode of droplets.At the same time,we also numerically study on droplet formation in the co-flow mode at various flow rate and material parameters based on the VOF method in FLUENT.On this basis,an integrated droplet digital PCR system with complete intellectual property rights has been successfully developed,which includes a droplet generator and a thermal cycler of our own.Increase in detectable droplets effectively reduces detection errors and improve detection sensitivity.In this study,we also conduct the research on droplet stability in the two-phase liquid flow stability and thermal cycling,resulting in optimization of the system.With the lung cancer gene LUNx as a target,we verify the system in terms of its sensitivity as well as detection limit.The observed results of the integrated droplet digital PCR system indicate a good reproducibility.The observed detection range is 5 orders of magnitude in theory,the detection limit reaches down to 1 copy/?L,and it shows extremely high reliability.This system demonstrates the advantages of microfluidic equipment integrated with high precision machine of adjustable flux and complete automation.Based on CI-ddPCR,a high throught-put droplet sorting system combined with a flow cytometer is developed.This protocol can acquiring single aptamets themselves by one step.In the strategy,one first isolates aptamers into individual reaction chambers of their own,and the fluorescence in chambers will be activated upon hybridization of their single aptamers with the target.By reading and sorting those reported chambers,people can have single aptamers of interest immediately.The digital selection is not based on sequencing,thus saving time and costs related to the repetitive sequencing,as well as the uncertainties of synthetic aptamers.The reliability of the integrated system was verified by high-throughput sequencing,and the aptamer shows high affinity(Kd=0.296 ± 0.004 ?M)and specificity to the target AFB1.Finally,we use a CI-ddPCR system to produce a monodisperse emulsion with beads in them as well as isolating amplification in droplets from outsides.After amplification,multiple copies of a single nucleic acid sequence cover each bead surface and then uses flow cytometry to straight observe the binding of one aptamer particle with the target molecules,and only beads with the highest affinity finally are sorted.In this method,we have obtained DNA aptamers of PD-1(Kd=2.7 ± 0.05 nM)with a?60 fold higher affinity than reported for this protein.