Single Cell Nucleic Acid Analysis Based On Microfluidic Digital Isothermal Amplification

Molecular diagnostic technology based on nucleic acid amplification plays an important role in the prevention and control of major infectious epidemics,cancer screening,and genetic disease diagnosis.The conventional nucleic acid detection method is polymerase chain reaction,which has long detection time,precise temperature control,and requires special instruments and professional technical personnel to operate.In order to solve the shortcomings and problems existing in traditional amplification detection,this paper,based on microfluidic technology,uses looped mediated isothermal amplification technology to detect the common mutation sites in non-small cell lung cancer cells specifically.The main research contents are as follows:A dLAMP-based microfluidic digital nucleic acid detection platform was established and successfully applied to the mutation screening of non-small cell lung cancer(NCI-H1975)cells.First,based on the soft lithography technology,multiple parallel array chips were designed.By PDMS modification and negative pressure treatment,100%sample injection volume and fast loading of samples and reagents are realized.In addition,the design of sandwich structure reduces the evaporation of reagents during the amplification process.Secondly,the factors that affect LAMP reaction were optimized,such as betaine,primer concentration,dye concentration,etc.,and the mutation detection of NCI-H1975 was successfully realized under the optimal reaction conditions,and the detection limit was single cell copy number.Finally,the microarray and q PCR technology were used to test the unknown samples and compare the results,the detection sensitivity was higher.A dLAMP-based digital single-cell array nucleic acid analysis platform was established.A digital single-cell array was constructed using a hydrophobic microarray through restricted dilution,and the formed single-cell array was verified and evaluated.The factors affecting the amplification of LAMP were further optimized and the specific screening of EGFR L858 R mutation gene in MC38 and NCI-H1975 cell samples with different mixed proportions was successfully realized.In order to further improve the stability and reproducibility of this method,this paper further proposes two methods for constructing single cell arrays: one was the SU-8 microwell array based on cell size,and the single cell capture efficiency of this method was as high as 62.15%.The repeatability was good,but the amplification system was too small for subsequent amplification reactions;the other was a double-layer capture array based on droplet size,which captured single-cell through the lower microwell structure,and the upper "inverted eight" structure captured the large droplets,which expanded the amplification system and the droplet capture rate was close to 100%.This article compared and analyzed three methods for forming digital single-cell arrays,laying the foundation for further single-cell nucleic acid detection.