Precision Molecular Diagnosis Of Lung Cancer Based On Microfluidic Chip And Isothermal Amplification Technology

Currently,the diagnosis and staging of lung cancer is based on pathological findings as the gold standard,which usually requires surgical procedures to obtain samples,which is difficult,time-consuming,and puts patients at surgical risk.In order to establish a minimally invasive,precise,efficient and relatively low cost molecular diagnosis of lung cancer,we selected a total of 22 lung cancer tumor-related markers(carcinoembryonic antigen CEA,cytokeratin fragment 19 CYFRA21,etc.)and autoantibodies(tumor-related gene protein p53,sex-determining gene family SOX2,etc.)through literature research.Sequence fragments were selected as targets for primer design and optimization,and performance validation was performed for the specificity and sensitivity of the above indicators to finally screen a set of combined assays for lung cancer diagnosis.A total of seven cell lines(NCI-H69,NCI-H226,NCI-H1975,BEAS-2B,NCI-H460,NCI-H1299and HCC827)were cultured for several common lung cancer subtypes(including small cell lung cancer,lung squamous cell carcinoma,lung adenocarcinoma,large cell lung cancer and other non-small cell lung cancers)and one human normal lung epithelial cell.The mRNA of the cells was extracted at a density of about 10~6 cells/m L,and the combined assay was performed by isothermal amplification technique using the screened set of indicators,and the results of each indicator were calculated by Ct value to calculate the corresponding concentrations,and compared with the results of the internal reference genes and normal lung epithelial cells longitudinally and horizontally,respectively,and the results showed that the correspondence between the detection indicators and lung cancer subtypes in the literature research was generally The results showed a general agreement with the correspondence between the assay indexes and lung cancer subtypes in the literature study,and tentatively demonstrated the correlation between mRNA expression and protein expression.In order to further optimize the detection process,this study combined traditional biology experiments with microfluidic chip technology,using the characteristics of microfluidic chip miniaturization,microminiaturization,integration and high throughput,transferring the experimental platform from a multi-well plate to a disk chip with a radius of only 31 mm,integrating all 22 assays and one internal reference gene GAPDH on a single chip for simultaneous detection,and the experimental system The experimental system was reduced from 25μL in the test tube to 0.94μL in the reaction chamber of the chip,which greatly reduced the reagent consumption.In order to simplify the instrumentation requirements and reduce the operational difficulties,the loop-media isothermal amplification(LAMP)technique is used instead of RT-PCR for mRNA detection,and the instrumentation requirements are simplified from a precise real-time fluorescence quantitative PCR instrument to a small isothermal amplification instrument.The amplification experiment can even be carried out using a constant temperature water bath;and the reaction time is shortened to about 40 min,and the detection time can be about 15 min for indicators with high sensitivity.It was initially verified in the microfluidic chip that the independent reactions between reaction chambers can basically be satisfied without cross-reactivity,and comparing with the results obtained from traditional biological experiments,it can be found that the used multi-indicator,microsystem and highly efficient combined detection platform is feasible.In this study,an innovative microfluidic device was applied to develop a combined multi-indicator assay platform for lung cancer with a microsystem of less than 1μL,which combined with LAMP technology effectively improves the assay efficiency and provides a reliable basis for clinical diagnosis and staging of lung cancer without surgical sampling.Due to the simplicity of the test,it is possible to achieve widespread multi-regional screening of tumors in the future,so that more patients can be diagnosed and treated accordingly at an early stage,thus effectively improving the quality of life of a large group of society.