Application Research Of Microfluidic Chip Technology In Detecting Circulating Tumor Cells In Peripheral Blood Of Lung Cancer

Circulating Tumor Cells(CTCs)are directly derived from tumor tissues and have the characteristics of tumor tissue,which are closely related to the occurrence and development of tumors,especially metastasis and recurrence of tumors.The number of CTCs and the changes of gene characteristics in peripheral blood of patients with tumors are all can be used to diagnose and monitor the prognosis and development of tumors.However,the number of CTCs in peripheral blood is sparse.1mL(containing10~9 red blood cells,10~6 white blood cells)may contain only a few CTCs in the blood.The detection method with high sensitivity and specificity has important clinical application significance.Microfluidic chips are the most promising CTCs detection technology due to their small size,high integration,low reagent consumption and fast response.In this paper,based on the characteristics and detection requirements of CTCs,a CTCs-rich chip based on cell size and a multi-functional microfluidic chip integrating CTCs enrichment,cleavage and nucleic acid detection were designed.The research content mainly includes the following two parts:1.A microfluidic chip for CTCs enrichment was designed.This chip is designed based on the size of cells,which is used for CTCs enrichment,and verify the clinical utility of the chip by detecting peripheral blood CTCs in patients with lung cancer.The chip structure includes an inlet,cell pellet filtration zone,single cell filtration channel,and a sample port.The cell mass filtration zone is a row of hexagonal columns for blocking the cell mass to prevent clogging of the single cell filtration array,and the single cell filtration zone is arranged in parallel by the main pipe,the filtration pipe and the side pipe.The chip can capture more than 80%of cancer cells.CTCs are detected in peripheral blood samples of 100 patients with lung cancer and 100 healthy donors,and the CTCs were identified by staining with epithelial marker(CK8+18,CK19),mesenchymal marker(Anti-Vimentin)and immune molecule(PD-L1)of tumor cells.Characterization of CTCs revealed that the number of CTCs in patients with lung cancer was significantly higher than that in healthy donors(P<0.001).The receiver operating characteristic curve(ROC)analysis showed that the microfluidic device has high sensitivity and specificity(92%sensitivity and 91%specificity),and can also identify CTCs with different immunophenotypes,which has important reference value and clinical application significance in tumor diagnosis and prognosis monitoring.2.An integrated microfluidic chip is designed based on the chip of CTCs enrichment.The chip consists of three parts,including CTC capture area,single layer membrane valve area and nucleic acid detection and analysis region based on digital polymerase chain reaction(dPCR).The CTCs capture area of chip is coupled to the nucleic acid detection chip through a control valve,and the chip integrates the capture,cell lysis and gene detection of CTCs.The chip was used to detect the gDNA of lung cancer cell lines(H1975,A549),and the EGFR-L858R mutation was detected in H1975cells,no mutations were detected in A549,it is further verified that the chip can successfully detect the gene mutation in CTCs for reference of tumor targeted drugs and precise diagnosis and treatment.In conclusion,based on the difference in cell size,we designed a microfluidic chip for CTCs enrichment consisting of a hexagonal column that filters cell pellets and a filter channel array that filters single cells,combined with the epithelial marker(CK8+.18,CK19),the mesenchymal marker Vimentin and immune molecule PD-L1for the identification and analysis of CTCs.It has been verified in clinical samples,which proves that it has high sensitivity and specificity and can provide important reference value for clinical application.In addition,based on the requirements of CTCs gene detection,we developed a microfluidic chip that integrates CTCs capture,cleavage and gene detection on the basis of CTCs capture chip.The chip can realize the enrichment of CTCs and the detection of mutant genes,and is verified in the lung cancer cell line H1975 cells,which is expected to become a practical tool for clinical detection.