Real-time Quantitative Detection Of Nucleic Acid Based On Water-cooling-based PCR Chip

Polymerase chain reaction(PCR)chip is a kind of microfluidic chip for the amplification and detection of nucleic acid.With the advantages of integration,miniaturization,and high portability of microfluidic chips,PCR chips have obvious advantages in terms of cost,period and integration level than traditional detection methods of nucleic acid,which makes PCR chips one of the important applications of microfluidic chips.Static chamber PCR chips,an important branch of PCR chips,have shown good prospects in many fields such as disease diagnosis and environmental monitoring due to their simple chip structure and system composition.Currently,a variety of nucleic acid detection technologies based on static chamber PCR chips have been proposed.Based on the requirements of static chamber PCR chips in terms of detection cycle,cost,and processing difficulty,this thesis applied water-cooling technology to PCR chips and proposed a water-cooling-based PCR chip,which can quickly achieve PCR reactions with the advantages of easy fabrication,parallel detection and so on.Then the technology of real-time quantitative detection of nucleic acid based on the water-cooling-based PCR chip was proposed.The contents of this thesis are as follows:(1)Design and study of heat transfer performance of the water-coolingbased PCR chipThe design and heat transfer performance of the PCR chip are significant to the detection cycle and detection performance due to the chip is the realization platform of nucleic acid real-time quantitative detection.In this thesis,three kinds of water-cooling-based PCR chips were proposed,which were named PCR chips with exposed chambers,semi-embedded chambers,and embedded chambers,respectively.By analyzing the heat transfer mechanism of the PCR chip and combining the theory of fluid flow and heat transfer,the theoretical model of the chip was established.Then numerical simulations were performed to investigate their heat transfer performances to choose the PCR chip with better performance.Based on the simulation,three kinds of PCR chips were prepared by 3D printing technology to verify their heat transfer performances.In addition,the tests of the influence of the flow rate of cooling water,the thickness of the water layer,and the thickness of the polymer wall on the heat transfer performances of PCR chips were implemented,which provided a basis for the subsequent experiments and the cycle optimization of real-time quantitative detection.(2)Development of the real-time quantitative detection technology of nucleic acid based on water-cooling-based PCR chipsAfter confirming the structure of the water-cooling-based PCR chip,the real-time quantitative detection technology of nucleic acid based on the PCR chip was developed,which can be divided into three steps.Amplification module: To realize the amplification of nucleic acid,the water-cooling-based PCR chip was fabricated by CNC processing technology with polypropylene material,followed by the performance test of the chip.Then a corresponding amplification module based on the chip was designed and developed to realize the nucleic acid amplification;Temperature control module: To meet the temperature requirements of nucleic acid amplification,a temperature control module was developed to accurately control the temperature of PCR reagents in the chip,followed by the test of temperature control performance of the module;Fluorescence detection module: To achieve real-time quantitative detection of amplification products,a fluorescence detection module was designed,and the important performance parameters such as light trajectory and light uniformity of the module were analyzed by optical simulation software.Then,the performance of the fluorescence detection module was evaluated.Based on the above three modules,the real-time quantitative detection technology of nucleic acid was developed,and a corresponding real-time quantitative detection device was proposed.(3)Experimental study on the real-time quantitative detection of nucleic acid based on water-cooling-based PCR chipsTo verify the validity of the developed real-time quantitative detection technology,the above-mentioned real-time quantitative detection device was used to detect the c DNA samples of the vesicular stomatitis virus.By analyzing the "real-time" process of nucleic acid amplification and the mechanism of "quantitative" detection in the experiment,the validity of the proposed real-time quantitative detection technology was verified.Then,the amplification efficiency of the proposed device and a commercial instrument were compared to evaluate the performance of the proposed device.In addition,parallel detection based on the chip was performed to improve the detection efficiency of nucleic acid real-time quantitative detection.Finally,agarose gel electrophoresis was performed to test the specificity of products,which proved the reliability of nucleic acid real-time quantitative detection technology.(4)Cycle optimization of the real-time quantitative detection of nucleic acid based on water-cooling-based PCR chipsTo further shorten the cycle of the proposed real-time quantitative detection technology,the water-cooling-based PCR chip and PCR parameters were optimized.Optimization of water-cooling-based PCR chips: According to the heat transfer mechanism of the chip and the above-mentioned research results,the water-cooling-based PCR chip made of PDMS material was prepared in combination with the reverse molding and plasma bonding process to improve the heat transfer efficiency of the chip,followed with important parameters such as the preparation performance,bonding performance and heat transfer performance of the chip were tested.PCR parameter optimization:Adjust PCR reaction parameters to further shorten the cycle of the real-time quantitative detection technology.After the above optimization,the c DNA samples of the vesicular stomatitis virus were used to verify the proposed technology,and then the specificity of the product was verified.