| Infectious diseases caused by pathogens are the main factors threatening human health and causing social panic.To monitor and prevent infectious diseases caused by pathogens is of great significance.When facing a sudden outbreak of unknown pathogens,how to quickly and accurately screen,identify and track the source of infection to strive for valuable time for the development of corresponding vaccines,medicines and the recovery of patients' lives is an important public health issue.In recent years,polymerase chain reaction(PCR)technology has been widely used in the diagnosis of pathogenic biology,it can achieve specific DNA replication by enzymatic synthesis of specific DNA in vitro.This technology,which can replicate a large amount of DNA in a short time,has the advantages of specificity,sensitivity,high yield,rapidity,convenience and good repeatability.Therefore,PCR technology plays an important role in various fields,and is widely used in medical detection,biological research,food safety,agricultural products detection and so on.The traditional nucleic acid detection process generally includes several steps such as sampling,samples transportation,nucleic acid extraction in laboratory,detection reagent preparation,sample mixing,amplification,product detection.The whole process is time-consuming,and the experimental operation is complex,which has high requirements for the environment and personnel,so it is difficult to meet the needs of fast detection of pathogens.Therefore,the development of small volume and low flux integrated PCR microdevice can not only prevent cross contamination,but also easy to carry out experimental operation,which enables non-professionals to carry out detection,not limited to specialized laboratories and well-trained laboratory staff.In this thesis,a pressure-driven gas-diffusion/permeation micropump,a chamber-type PCR amplification module and a continuous-flow PCR amplification module were designed for the detection of bacteria and other pathogens.Besides,this thesis proposed a novel PCR amplification microdevice which integrated the extraction of pathogen DNA,PCR amplification and product detection into a program-controlled automation system,this method reduced the detection time,eliminated the complex operation process,saved manpower and material resources,and improved the efficiency.This thesis mainly carried out the following research work:The design and experimental study of the pressure-driven gas-diffusion/permeation micropump for self-activated sample transport were carried out,including three terminal modes,namely,the end-blocked gas-permeable materials,the fluid resistance of a quartz capillary tube and the fluid resistance of a four-times stretched Teflon tube.By considering the relationship between the resistance of the fluid pipeline,the permeability of the chip and the geometry of the channel,the mathematical model of the pressure-driven gas-diffusion/permeation micropump was established based on Fick's law.It provided a new control mechanism to realize the long-time continuous and stable flow inside the microchannel at high temperature and stable transmission control of multiphase fluid.The PCR amplification microdevice was studied.It was mainly divided into chamber-type PCR microreactor and continuous-flow PCR(CF-PCR)microreactor.Based on the three basic steps of polymerase chain reaction,which were denaturation,annealing,and extension,an oil-bath chamber-type PCR microreactor,on-chip and off-chip continuous-flow PCR microreactor with constant temperature and single heat source suitable for multi-scenario PCR application were designed.This laid a foundation for miniaturization and high integration of PCR instrument.Besides,a prototype of CF-PCR microdevice was designed and built based on the Peltier effect with constant temperature and single heat source.The prototype used a pressure-driven gas-diffusion/permeation micropump to realize the steady flow of single-phase fluid in the microchannel for long time transport.This microdevice was designed by using the Peltier effect of the TEC and the thermal conductivity of ceramics,which realized the goal of small size and low power consumption.A PCR microdevice based on automatic extraction,amplification and detection of nucleic acid was designed.The microdevice was small in size,low in cost and simple in operation,can realize automatic,highly specific and highly sensitive detection of pathogens.At the same time,a DNA extraction method based on Chelex-100 and proteinase K was studied and designed.Combined with high temperature to shorten the operation time,the optimal distribution scheme of each component ratio and high temperature time was finally determined.In order to meet the needs of different use scenarios,combined with microfluidic chip technology,we studied on-chip and off-chip pathogen detection methods,carried out a series of pathogen detection experiments,and evaluated the performance of extracting nucleic acids from bacteria and human hair follicle,achieving the detection goal of "sample-in-answer-out".In addition,the micro gel electrophoresis product detection module in the microdevice can be applied not only to the detection of products after the nucleic acid amplification of pathogens,but also to the analysis of epidemic sequences,tracing the sources of infection,and sequencing of genes.In conclusion,the research of the pressure-driven gas-diffusion/permeation micropump module,PCR amplification module and nucleic acid pretreatment,amplification and detection integrated PCR microdevice for direct detection of pathogens were studied,these researches could reduce the sample detection time,provide a new analysis method for the research of life science. |
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