A Self-digitization Chip Integrated With Hydration Layer For Low-evaporation And Robust Digital PCR

Due to the properties of strong specificity,high sensitivity,absolute quantification,simplicity and rapidity,digital polymerase chain reaction(dPCR)technology,has become an important detection method in the field of life sciences.Digital PCR not only enables absolute quantification of target nucleic acid,but also has potential applications for gene sequencing,gene mutation,early disease diagnosis,clinical tracking and evaluation.However,the existing commercial dPCR platforms require either expensive instruments or complicated operation,which limit their extensive application.Although the current polydimethylsiloxane(PDMS)-based dPCR chips,reduce the cost and difficulty of production,the permeability and hydrophobicity of PDMS also brings some problems,such as excessive evaporation of water,protein adsorption and other issues,which limit the applications of dPCR chip.To overcome the limitation of the existing dPCR chips,this thesis presents a self-powered and zero-water-loss microfluidic digital PCR device to enable low-cost and robust quantitative nucleic acid assays.In this device,a thin cavity is integrated beneath the reaction chamber array.By utilizing the permeability of polydimethylsiloxane(PDMS),the integrated cavity serves a dual function:vacuum“battery”and hydration“reservoir”.The combination of pre-stored pumping energy and water compensation for evaporation loss enables simple,robust single-DNA-molecule amplification and unambiguous detection in nanoliter-volume reactors in this device.The main contents of this thesis are summarized as follows:(1)After analyzing and summarizing the research status and development trend of the existing dPCR technologies,we intend to develop a self-digitalization and low-water-loss dPCR chip.(2)We present a novel strategy for building a self-powered and zero-water-loss microfluidic digital PCR device,and develop a feasible fabrication process.(3)After pre-degassing,the integrated cavity layer of the chip can store the vacuum energy,which enables the power-free sample partitioning,acavitying to use the complicated and expensive pumps,and reducing the application costs.The power-free sample partitioning capacity of the prepared dPCR chip was characterized.The results showed that the injection speed of this chip is faster and the pre-degassing time used is also greatly reduced.(4)Due to the high permeability of PDMS,PDMS-base dPCR chips have severe water evaporation.To solve this problem,water or buffer was filled into the integrated cavity to compensate for the water loss of each reaction micro-chamber.The anti-evaporation capacity of the prepared dPCR chip was characterized.The results showed that this chip configuration can perfectly prevent water evaporation from the whole micro-chamber array of the chip.(5)Finally,we demonstrated the absolute quantitation capability of the prepared self-digitalization and low-water-loss dPCR chip.The target DNA solution was diluted successively by 1×10~1 copies/?l,1×10~2 copies/?l,1×10~3 copies/?l,1×10~4 copies/?l,1×10~5 copies/?l five series to amplification separately,and the results showed that there had a good linear relationship between the target copy number detected by the chip and the expected target copy number,and a very good absolute quantitative detection could be achieved.