Research On Key Technologies For Portable Nucleic Acid Amplification And Detection Based On Static Chamber Chip With Multi-surface Variable Temperature Heat Source

Conventional nucleic acid amplification and testing approach requires sample collection,transportation,extraction,amplification,and detection in the professional molecular diagnostic laboratories.However,the testing process is complicated and the sample is easily contaminated during sample transfer.The current integrated nucleic acid amplification and detection systems are typically large in size and is not applicable for onsite testing.Microfluidic chips have the advantages of small size and low reagent consumption,which makes microfluidics suitable for on-site testing.Many researchers have proposed micro-PCR chips for nucleic acid amplification and detection.However,most of these approaches have the drawbacks of uneven heating,temperature instability,slower heat transfer and integration,which limits the microfluidics’ application in the field of onsite testing.In this study,multi-surface variable temperature heat transfer model combines with static chamber microfluidic chip is proposed for the first time,which can significantly improve the control accuracy of liquid temperature and the heat transfer efficiency of same volume of liquid in a chip.Meanwhile,the key problems of microfluidic technology application in nucleic acid amplification and detection were also studied,including chip design,chip preparation,portable nucleic acid amplification and detection system construction and on-chip experimental research,which expected to lay the foundation for future advances of microfluidic technology for the pathogen nucleic acids in on-site testing.The contents of this study are as follows.(1)Numerical simulation study of heat transfer model with multisurface variable temperature heat source using COMSOL Multiphysics.Firstly,we analyzed the heat transfer process according to the heat transfer theory and built the heat transfer models;then,the heating and cooling saturation speed were discussed for four metal materials used in this study,and it was observed that the silver,aluminum,copper and nickel decrease in turn on heating and cooling speed.Aluminum is the best material for the heat transfer medium as a result of its thermal conductivity,thermal mass,and low price.Finally,the steady state and transient simulation results show that the static chamber chip with multi-surface variable temperature heat source has a maximum temperature increase of 10℃ when the steady state temperature change from 30°C to 100°C and heat transfer time also cut in half compared with the heat transfer model with single-surface variable temperature heat source.(2)Design and preparation of static chamber chip with multi-surface variable temperature heat source for on-chip asymmetric and parallel fluorescent nucleic acid amplification and detection.Firstly,the structure of static chamber chip for three-step amplification and nucleic acid molecular hybridization assays based on the polypropylene was designed,meanwhile,the multilayer polypropylene chip bonding method was proposed using a hot press machine;then,a parallel fluorescence detection chip was designed and prepared,which consists of a static chamber chip with multi-surface variable temperature heat source for two-step amplification and fluorescence detection,as well as a liquid distribution chip that can improve the dispensing efficiency of the parallel detection process,and is capable of realizing single sample loading and four detections.(3)A portable and integrated system was built for nucleic acid amplification and detection was built based on static chamber chip.Firstly,the temperature control system for nucleic acid amplification was established,and the ramp rate of temperature rise is 3℃/s and the temperature control accuracy is ±0.5℃.Then,we develop a smartphonebased fluorescence detector for the detection of SYBR Green I fluorescent dye.The entire system has a weigh of 1.5kg and can be used to up to 16 samples in parallel.(4)Two amplification detection mechanisms are carried out to verify the validity of the portable system based on static chamber chip for immediate on-site detection.Firstly,we proposed a method to obtain the probe and single stranded DNA of Escherichia coli by asymmetric nucleic acid amplification,then the single stranded DNA hybridized with the probe that was labeled and immobilized on the chip surface,and the detection of target sequence was realized by molecular hybridization techniques of nucleic acids.Secondly,using the human β-actin gene as the objective for a single sample loading and four experiments of parallel fluorescent nucleic acid amplification and detection experiments.The detection of target sequence examined by fluorescence signal.The results of amplification and detection from both types of experiments were consistent with the results of commercial experiments.