Research On Active-passive Multi-stage Integrated Chip For Accurate Sorting Of Circulating Tumor Cells

After circulating tumor cells are detached from the original tumor lesion,they will enter the blood circulation and migrate with the blood to other organs of the body to form new tumor lesions,which will eventually lead to the deterioration of cancer disease.Therefore,the study of circulating tumor cells in blood is of great significance to the early diagnosis of cancer,the evaluation of subsequent treatment effects and the development of related drugs.In fact,the number of circulating tumor cells in blood is scarce,and one milliliter of adult blood contains only about 10 tumor cells.Therefore,how to quickly and precisely extract the rare circulating tumor cells from the huge amount of background blood cells is the main research of this thesis.Based on the summary of various microfluidic cell sorting techniques and the existing results in the laboratory,the magnetic sorting technique among the active sorting methods was explored in depth and integrated with the trapezoidal spiral sorting technique to further improve the sorting accuracy while retaining the advantages of high throughput and high recovery rate of the trapezoidal spiral sorting technique.Finally,the combination of the micromixing technique realized rapid in-sheet magnetic bead labeling and improved the efficiency of cell pre-processing.The specific research results achieved in this paper are as follows:(1)A magnetically enhanced microfluidic chip was developed to accomplish the separation of high-purity tumor cells from lysed red blood cells as well as clinical samples(pleural and abdominal fluid).In this chapter,a magnetically enhanced microfluidic chip was proposed,in which the magnetic field strength and magnetic field gradient in the sorting flow channel were enhanced by adding Permalloy strips between adjacent magnets and high permeability flow channels on both sides of the sorting flow channel,thus improving the sorting accuracy.The magnetic polystyrene particles were used to simulate the magnetic bead-labeled leukocytes in blood and the ordinary polystyrene particles to simulate tumor cells to verify the sorting performance of the chip and investigate the optimal sorting flow rate of the chip.The magnetic sorting chip was tested to achieve perfect sorting of polystyrene particles,with 100%recovery and 100% purity of 15 μm particles.The optimal flow rate band was applied to cell sorting experiments in lysate blood doped with higher/lower concentrations of multiple tumor cell lines and clinical samples(pleural and abdominal fluid).The test showed that average recovery of the magnetic sorting chip for the three tumor cell lines was 88.37% and the average purity was 81.73% at the higher tumor cell doping levels.At a lower tumor cell doping level,the average recovery of the three tumor cell lines was 92.40% and the average purity was reduced to 65.56%.For clinical samples(pleural and abdominal fluid),the detection rate of tumor cell was 100%,and the average purity was 50.88%.(2)To address the problems of insufficient throughput and recovery rate of magnetic sorting chip,a multi-stage sorting chip with integrated trapezoidal spiral flow channel and magnetic sorting technology was developed to separate tumor cells from lysed red blood cells and clinical samples(thoracic and abdominal fluid)with higher throughput and accuracy.In view of the excellent effect of single-stage magnetic sorting chip in sorting purity,in order to further improve the throughput and sorting accuracy,the trapezoidal spiral sorting structure was integrated with the magnetic sorting chip.The multi-stage integrated sorting method was used to further improve the sorting effect.The performance of the integrated chip was first verified by polystyrene particles,and the chip was tested to achieve perfect sorting of polystyrene particles with 100% recovery and 100% purity of 15 μm particles.To further improve the cell sorting accuracy,two new antibodies,CD66 b and CD16,were introduced to label the leukocytes.The average recovery of the magnetic sorting chip for the three tumor cell lines was improved to 94.37% and the average purity was improved to 94.58% at higher tumor cell doping levels.At a lower tumor cell doping level,the average recovery of the three tumor cell lines was improved to 96.83% and the average purity was improved to 78.14%.For clinical samples(pleural and abdominal fluid),the detection rate of tumor cell was 100%,and the average purity was 70.52% with a maximum purity close to 90%.(3)In order to further improve the experimental efficiency and reduce the sample preprocessing time,a microfluidic chip with integrating micromixing and sorting functions was developed,which accomplished the on-chip magnetic bead labeling as well as high-throughput and high-precision separation of tumor cells from lysed red blood cells and clinical samples(thoracic and abdominal fluid).A micromixing microfluidic chip was designed to introduce Dean flow and vortex flow by setting up microstructures and vortex flow to achieve fast and efficient mixing of the solution on the chip.The optimal flow rate of the chip was investigated by numerical simulations and experimental tests.The throughput was increased to the required value for sorting by multi-layer stacking of thin film chips.The average recovery of the multifunctional integrated chip for sorting three tumor cell lines was 92.86% and the average purity was 91.26% at higher tumor cell doping.Under the lower tumor cell doping amount,the average recovery of the three tumor cell lines was 96.37%,and the average purity was 82.87%.For clinical samples(pleural and abdominal fluid),the detection rate of tumor cell was 100%,and the average purity was 67.67%.