Visualization Detection Of Respiratory Virus Based On Microfluidic Technology

Virus-caused respiratory infections,particularly those caused by the Novel Coronavirus in late 2019,pose a significant threat to global public health.In order torealize the rapid diagnosis and timely treatment of acute respiratory diseases and effectively control the spread of infectious diseases,it is of great significance to develop a high sensitivity,high specificity,simple and rapid detection method for respiratory viruses.Aim to the needs of pre-research of commercial respiratory virus detection,combining microfluidics technology with colloidal gold visualization detection method,respiratory virus immune detection based on microfluidics technology and respiratory virus nucleic acid detection based on microfluidics and RPA-CRISPR/LbCas12a were developed.The main research contents are as follows:(1)Preparation of microfluidic chips.The 2D structure of microfluidic chip was drawn by Auto CAD.Polydimethylsiloxane(PDMS)and polymethyl methacrylate(PMMA)were used to fabricate microfluidic chips.The preparation technology and material applicability of the two kinds of chips were compared.The results showed that PDMS has stronger hydrophobicity than PMMA,and the hydrophilicity of modified PMMA chip is better than that of PDMS chip.In order to increase the hydrophilicity of the chip and reduce the non-specific adsorption of proteins,PMMA material was selected as the base material for subsequent microfluidic chip preparation.(2)Development of visual immune detection method of respiratory virus based on microfluidic technology.The injection area,coating dissolution area,mixing area and detection area were integrated on the microfluidic chip.The injection pump was used as the fluid driving force and the visual detection of inactivated influenza A(H1N1),influenza B,respiratory syncytial virus and novel Coronavirus standard antigens was realized through precise fluid control.The overall detection time was 10min,and the sample size of a single time was 80μL.The limits of detection for influenza A antigen,influenza B antigen,RSV antigen and Novel coronavirus antigen are 37.6 ng/m L,17.8μg/m L,112.5 ng/m L and0.05 ng/m L respectively.The developed immune microfluidic chip has the characteristics of high specificity,small sample size,simple operation,rapid detection and no cross-reaction,which can realize the rapid detection of respiratory pathogens,timely distinguish COVID-19 from common respiratory infections,and improve the efficiency of epidemic prevention and control.The technology can be used for point of care testing(POCT)field rapid detection.(3)Development of visual nucleic acid detection of respiratory virus based on microfluidic RPA-CRISPR/LbCas12a.RPA constant temperature amplification,CRISPR/LbCas12a detection and colloidal gold side flow chromatography strip visual detection were integrated on microfluidic chip for influenza A,influenza B and novel coronavirus nucleic acid detection.The limits of detection for influenza A,influenza B,novel coronavirus plasmids and novel coronavirus pseudovirus were2.33×10~2copies/μL,8.76×10~1copies/μL,1.38×10~2copies/μL and 8.4×10~2copies/μL respectively.The whole reaction can be carried out at 37℃,and the detection results can be obtained in 60 minutes,without expensive nucleic acid amplification equipment and complex nucleic acid amplification procedures.