Paper-based Microfluidic Nucleic Acid Detection For Microbial Point-of-care Testing On Space Station

With the improvement of the condition of scientific research and the development of space resource during the long-term flight of space station,microbia of space station has a good living environment.However,microbial proliferation can destroy the equipment of space station and pollute the contamination of air,water and food,making astronaut infection.Therefore,it is necessary to monitor the contamination condition of microbia during the long-term flight of space station.In this study,we mainly focus on developing a new paper-based nucleic acid extraction technology,paper-based sensitivity enhancement technology and an integrated paper-based nucleic acid device for nucleic acid extraction,amplification and testing.Finally,we also detected the microbial sample and blood sample ofhuman in laboratory.The main results are as follows:1.As for paper-based nucleic acid extraction,we developed a new paper-based DNA extraction device by incorporated sponge-based buffer storage,a paper-based valve and channels of different length to autonomously direct the reagent and sample to the Fusion 5 disk for DNA capturing.With this device,DNA can be extracted within 2min from only 30?L samples of whole blood,serum,breast cancer cell,saliva,sputum and bacterial suspension.The performance of the device was similar to that of the commercial QIAamp DNA micro kits and the FTA card.In our perception,this simple,inexpensive,portable and disposable device holds great promise in terms of POC testing in resource-poor settings,especially in space station.2.As for the improvement sensitivity of paper-based detection,one aspect,we developed a novel device by integrating dialysis-based concentration method into LFAs.The device successfully achieved 10-fold signal enhancement in Human Immunodeficiency Virus(HIV)(as a model target),nucleic acid detection with adetection limitof 0.1nMin less than 25min.Another aspect,we also developed a low-cost,simpleand easy-to-use method to improve analytical sensitivity by integrating sponge shunt into LFA to decrease the fluid flow rate.The thickness,length and hydrophobicity of the sponge shunt were sequentially optimized,and achieved 10-fold signal enhancement in nucleic acid testing of HBV(as model target)as compared to the unmodifiedLFA.The enhancement was further confirmed by using HBV clinical samples,where we achieved the detection limit of 10~3copies/ml as compared to10~4copies/ml in unmodifiedLFA.This simple,low-cost and portable integrated device and improved LFA hold great potential for point-of-care testing in space station.3.As for the integrated paper-based sample-to-answer device for nucleic acid testing,we demonstrated for a fully disposable and integrated paper-based sample-in-answer-out device for nucleic acid testing by integrating nucleic acid extraction,helicase-dependent isothermal amplification and lateral flow assay detection into one paper device.This simple device allows on-chip dried reagent storage and equipment-free nucleic acid amplification with simple operation steps,included adding sample,pressing the button,move the insulation pad,move the copper sheets to the amplification line and move to the copper sheets to the detection line,which could be performed by untrained users in remote settings within 75min.The proposed device consists of sponge-based reservoir and paper-based valve for nucleic acid extraction,integrated battery,PTC ultrathin heater,temperature control switch and on-chip dried enzyme mix storage for isothermal amplification,and lateral flow test strip for naked-eye detection.It can sensitively detect Salmonella typhimurium(as a model target),as a model target,with a detection limit of as low as 10~2cfu/ml in wastewater and egg,and10~3cfu/ml in milk and juice in about an hour.This fully disposable and integrated paper-based device holds great potential for future POC applications in resource-limited settings,especially in space station.4.As for the microbial contamination of space station and crewed health,in this study,we used our developed paper-based devices to detect model targets such as Staphylococcus aureus and HBV target of whole blood sample in the laboratory.In the experiment,we firstly extracted DNA from different samples and then amplified DNA using an integrated paper-based sample-to-answer device.Next,nucleic acid electrophoresis,normal LFA,paper-based concentration device,sponge-based improvement LFAwere sequentially used to detect Staphylococcus aureus and HBV target of human whole blood sample.These results indicate that our developed paper-based nucleic acid devices hold great potential for future POC applications in resource-limited settings,especially in space station.