Key Technologies Of Space Microfluidic Chip For Gene Amplification And Related Devices Development

With the rapid development of the China's manned space career in recent ten years,the Chinese space station,which will become the important research bases of space science and new technology,will be built completely in 2022.Space life sciences is one of the important research direction in field of space science.Space equipment is the indispensable research platform of space life sciences and also immediate impacts its research capacity.Due to the speciality of space environment and the limitation of the payload capacity,the conventional experimental methods and equipment do not meet the demand of the space life science and space loading conditions.Therefore,not only the research method innovation in concept,principle and technology,but also the space environment adaptability of experimental equipment will be needed.With the supports of 973 program,863 program and the National Special R&D Program for Key Scientific Instruments and Equipment,the research works of space life sciences equipment with miniaturization,low power consumption,high integration and automatization were being carried out.The mature research results of these programs will be loaded and applied in space station in the future.Space gene amplification is the important mean of the space life sciences.The study on the equipment of space gene amplification will not only build a platform for the organisms injured,the genetic variation mechanism and its protection which caused by the microgravity and radiation in space,but also benefit the health-related research of astronauts in long-term space flight.With the special requirements of space station loading and the advantage of microfluidic chip,we developed a microfluidic gene amplification device aimed at applying in the field of space life sciences in our study.We choos variable PCR technology for gene amplification in consideration of high fidelity requirement for gene amplification in space.We also make a breakthrough of key techniques in DNA extraction,cell direct-PCR,BSA passivation and temperature improvement according to the gene amplification characteristics of microfluidic chip in space.In addition,we also developed another microfluidic miniaturizing gene amplification device based the study on the equipment of space gene amplification which civilianizes space technology applying in the field of life science instrument and medical apparatus.This paper achieved following innovative research results:(1)We introduce three standard microfluidic chip fabricating methods for glass,PMMA and NOA81 based on the existing fabricating method in our laboratory.This standard fabricating method for three different method avoid the experimental error caused by the differentiation in chip fabricating.(2)Designed and fabricated a glass microfluidic chip with the structure of straight channel and multiple chamber in both side.We extracted the genomic DNA in 200?l human blood sample in 25 min with the liquid-liquid extraction method.Using these genomic DNA as gene template,the PCR products of HLA-DRB1 was half of the conventional DNA extraction method.This kind of microfluidic DNA extraction chip has the advantages of continuously and rapidly which could simple the DNA extraction steps greatly,reduced the device volume,improve the DNA extraction ability and increase the automation.If the microfluidic chip for DNA extraction is integrated with the microfluidic device of gene amplification in space,the integrated device is right for DNA extraction and PCR analysis of biological sample in space.(3)We designed and fabricated a NOA81 microfluidic chip for cell direct-PCR and introduced a temperature gradient cycle method for cell disruption on the PCR chip.With this method,we successfully achieved the integrated operation for HEK cell disruption and direct-PCR on the chip.This technology of direct-PCR,which greatly reduces the device volume and the complex pipeline connection,has the advantages of simply and rapidly without any extra DNA extraction steps.If this cell direct-PCR microfluidic chip is integrated with the microfluidic device of gene amplification in space,the automated analysis ability for biological sample in space will be greatly improved.In addition,this integrated device with the advantages of miniaturization,low power consumption and automation is suitable for space loading and carrying out life science research in space.(4)We developped a BSA coated method on NOA81 microfluidic chip for reducing the inhibition effect on PCR caused by the NOA81 surface adsorption to the Taq enzyme.With the BSA coated method,the PCR efficiency of HLA-DRB1 on NOA81 microfluidic chip has been increased for 7 times and the contrast PCR of AEB-HV was successful investigated under the cobalt radiation.This BSA coated method with the advantages of easy to use,practical and good biological compatibility makes a good foundation for NOA81 microfluidic chip applying in space.(5)We improved a temperature improvement method for NOA81 microfluidic chip.This method improved the temperature property of Peltier base NOA81 microfluidic chip by copper-sputtering on the surface of Peltier.With this method,the temperature integrity,the cooling speed and the PCR efficiency of NOA81 microfluidic gene amplification chip was significantly improved.This method with good effect in temperature improvement greatly increased the accuracy and reliability of the NOA81 microfluidic gene amplification chip and made this chip potential using in the space in the future.(6)We developed a microfluidic device for space gene amplification and designed a matching NOA81 microfluidic chip.This device integrated four main unit: sample controlling unit,microfluidic chip unit,circuit controlling and detection unit.The device could successful achieve simultaneous PCR of IL-6,TNF-?,HMGB-1and BDNF gene.In addition,this device could also realize the gene probe hybridization detection of cy3-IL-6 on the chip.This device with the advantages of miniaturization,low power consumption and automation is suitable for space loading and carrying out gene amplification of space biological sample.(7)We developed a miniaturizing gene amplification microfluidic device and designed a pluggable microfluidic chip for gene amplification.With this miniaturizing device,we achieved rapid PCR,STR-PCR and LAMP on the microfluidic chip.In addition,this miniaturizing device was used in the field of forensic identification and the rapid detection of salmonella.In the future,this miniaturizing device with the advantages of miniaturization in volume,rapid in PCR,low-cost in chip fabricated and flexible in functions will be used widely in the field of POCT,forensic identification and food safety testing if we make further optimizing in structure and appearance.