Study On Capture Of Circulating Tumor Cells By Using Microfluidic Techonology

Major:Clinical Laboratory DiagnosticsObjective:Conventional methods based on antibody capture of circulating tumor cells(CTCs)have limitations with lower capture efficiency and impairation of cell viability.In this study,the flexible design of microfluidic chip and effective chip surface modification were both used to innovatively combine with the method of antibody-based capture CTCs.The microfluidic capture system was comprehensively optimized to achieve high CTCs capture efficiency,which can be combined with clinical testing and reduced physiological damage to cells.Methods:This experiment belongs to the methodological study.The establishment method and the flow cytometry are used to analyze and compare the cell capture efficiency,and the reliability of the established method is determined.In this study,the capture efficiency is highly improved by the internal chemical modification of the chip,and a DNA fragment is modified inside the chip channel by biotin and streptavidin binding system,and then combined with the epithelial cell adhesion factor(EpCAM).It makes the antibody completely exposed to the flow region of the chip,and through this extended tentacle of"DNA fragment",which can efficiently capture tumor cells expressing EpCAM on the surface.The paper is divided into three parts to optimize and demonstrate its feasibility:1.Through a series of parameter optimization,the chip capture system can stably capture CTCs.2.Compare with the capture efficacy by using flow cytometry to demonstrate the feasibility of the chip.3.Use this method to detect the simulated samples and compare the results with the flow cytometry to elaborate the clinical utility.Results:(1)A series of parameter optimizations was obtained for the stability and high efficiency of the capture system,including PBS buffer rinsing rate of 3μL/min,chip channel width of 300μm,DNA fragment length of 482bp,DNA fragment concentration of 150ng/mL and anti-EpCAM concentration of 40ng/mL.(2)The optimized chip capture efficiency of CTCs was 80.8±4.7%,which is significantly superior to flow cytometry labeling efficiency(67.3±2.0%).(3)In the simulated blood sample tests,the concentration of tumor cells mixed into the peripheral blood of normal people was 0,8,16,24,32 and 40×10~4per/mL,the chip capture efficiencies were4.4±0.7%,54.2±4.1%,60.0±4.0%,69.0±4.5%,71.3±3.3%and 74.2±2.1%,respectively,which were superior to flow cytometry labeling efficiencies(-0.5±0.2%,36.7±5.0%,29.7±4.0%,42.7±4.4%,44.5±3.2%and 37.1±2.5%).Conclusion:The capture efficiency was optimal when the parameters were setted as:PBS buffer washing rate of 3μL/min,chip channel width of 300μm,DNA fragment length of 482bp,DNA fragment concentration of 150ng/mL and anti-EpCAM concentration of 40ng/mL.The developed chip capture efficiency has superior performance compared with that of flow cytometry.In the simulated clinical sample tests,the improved chip also performs superior to flow cytometry.