Construction Of Microbial Point-of-care Testing Platform Based On Microfluidic Technology And Loop-mediated Isothermal Amplification Strategy

Pathogenic microorganisms have always been a major threat to human health.In recent years,infectious diseases caused by pathogenic microorganisms have occurred frequently,such as Ebola virus and avian influenza virus,which have caused a large number of deaths and economic losses worldwide.In addition,pathogenic microorganisms are the cause of many diseases,such as pneumonia,meningitis,tumors and other diseases.Therefore,the rapid and sensitive detection of pathogenic microorganisms is of great significance for controlling infectious diseases and early diagnosis and treatment of related diseases.Recently,due to superior sensitivity and specificity,nucleic acid testing(NAT)has garnered much attention for the identification of microbial species.The isothermal amplification method represented by the loop-mediated isothermal amplification technology(LMAP)can complete nucleic acid amplification under constant temperature conditions,avoiding the thermal cycling process and eliminating the need of large instruments.LAMP has the advantages of simple operation,rapid sensitivity and high specificity,and is more suitable for the immediate detection of pathogenic microorganisms.Microfluidic technology is a multi-disciplinary subject.Through micro-processing,the chemical and biological analysis systems are integrated on the chip to realize sample preparation,transportation,reaction,analysis and other operations.In this paper,the integrated microfluidic nucleic acid testing platform is constructed by combining the loop-mediated isothermal amplification method and microfluidic technology,and the nucleic acid extraction,isothermal amplification and detection are completed on the chip to realize the "sample-to-answer" detection of pathogenic microorganism.The main research work is as follows:(1)A three-dimensional electrochemical paper microdevice with simple operation and high sensitivity was developed based on paper origami technology.The steps of sample preparation,isothermal amplification and product detection can be completed by simply folding the device panel.The paper-based electrochemical unit was constructed by combining the screen printing electrodes with wax printing technology.The loop-mediated isothermal amplification was monitored by the electrochemical interrogation reaction between the electrochemically active molecule methylene blue(MB)and the LAMP double-stranded amplicon(LAMP),successfully applied the electrochemical method to the "sample-to-answer" detection of pathogenic microorganisms,and realized the rapid and highly sensitive detection of pathogenic microorganisms in blood and food samples.(2)In order to realize on-chip reagents storage and delivery,the simple programmed operation for non-professionals and rapid identification of pathogenic microorganisms without laboratory equipment,we have developed an integrated portable microfluidic platform.The platform uses an innovative atmospheric pressure valve to store the reagents needed for nucleic acid extraction,amplification and detection on the chip without the need for off-chip reagent storage and delivery,further improving device integration and portability.The platform utilizes positive temperature coefficient(PTC)combine with appropriately selected light emitting diodes(LEDs)to provides a constant temperature for loop-mediated isothermal amplification(LAMP),followed by take images by a smartphone camera.Through this platform,we have demonstrated the reproducible detection of pathogenic microorganisms in plasma and the simultaneous detection of multiple target microorganisms from a single sample.