| Circulating tumor cells(CTCs)are shed from cancerous tumors,enter the circulatory system,and migrate to distant organs to form metastases that ultimately lead to the death of most patients with cancer.The isolation of rare CTCs from peripheral blood has been regarded as a non-invasive liquid biopsy technique,which has great significance for clinical applications such as the evaluation of cancer treatment effects,personalized treatment,early diagnosis of cancer,and R & D cancer drug screening.However,the number of CTCs in human peripheral blood is extremely rare,with an average of one circulating tumor cell per billion blood cells.Therefore,this thesis mainly studies how to achieve high-precision,high-throughput sorting of circulating tumor cells.Based on the existing research results of our laboratory,the inertial spiral sorting technology has been deeply explored and integrated with the deterministic lateral displacement technology.While retaining the advantages of the high throughput of the spiral sorting technology,the deterministic lateral displacement technology has been added to further improve the sorting accuracy.Finally,combined with the laser cutting technology and the bonding process,the multi-core superposition of the spiral structure has been achieved,and its throughput has been further improved.The specific research results obtained in this thesis are as follows:(1)A simple dual-port inertial spiral chip with simple structure,low cost,easy processing,and high throughput was developed,which successfully completed the isolation of various tumor cells from diluted whole blood,lysed whole blood,and clinical samples(peritoneal effusion).Polystyrene microspheres with different diameters were used to simulate red blood cells,white blood cells,and tumor cells in the blood to verify the chip sorting performance and to explore the range of flow rates that can be used to sort tumor cells.According to the flow rate range determined by the microsphere sorting,the blood cells and tumor cells are sorted by the chip respectively to further verify the performance of the chip and determine the optimal flow rate of sorting.Optimal flow rates were used to sort tumor cells from diluted whole blood,lysed whole blood,and clinically authentic samples(peritoneal effusion).Tests show that at higher doping levels,the chip has a removal rate of blood cells greater than 99.60%,a recovery rate of three tumor cells greater than 99.99%,and a purity of about 16.87%-20.31%.For clinical samples(peritoneal effusion),the detection rate of tumor cells is 100%,and the purity is about 2%.(2)Aiming at the low accuracy of the DFF chip,a sorting chip integrating DFF and DLD was developed,and the tumor cells was isolated from diluted whole blood and clinical samples(peritoneal effusion)with high accuracy cell by the chip.In the view of the excellent performance of DFF chip sorting throughput and tumor cell recovery rate.In order to further improve the chip sorting performance,integrating deterministic lateral displacement with high sorting accuracy advantages and dual-entry spiral structure.Two-stage sorting is used to improve tumor cell purity.The chip sorting performance was verified by chip sorting of polystyrene microspheres close to the cell size.After testing,it was found that the integrated chip sorting microsphere experiment reached a nearly perfect sorting effect.The recovery rate of 15μm microspheres was ～ 100%,and the purity was ～ 100%.The integrated chip is used for the sorting of dilute whole blood with high / very low concentration of a variety of tumor cell samples and clinical real samples(peritoneal effusion).After testing,compared to the DFF chip,the integrated chip has a removal rate of blood cells of more than 99.9%,and the purity of tumor cells is increased to ～ 60% at a higher doping amount,and 22.23% at a very low doping amount.For the peritoneal effusion,the purity of tumor cells in the recovered fluid was increased to22.16%.(3)In order to meet the requirements for rapid sorting of circulating tumor cells and further increase the chip throughput,a multi-stage sorting chip integrated with quadcore spiral,serpentine pre-focus,and deterministic lateral displacement was developed.Taking advantage of the easy stacking of thin-film chips,the four-core superposition of the dual-entry spiral structure is used to improve the chip throughput.DFF structure superimposes four cores to increase the throughput of the chip.The serpentine flow channel is introduced to realize the regular arrangement of particles in the flow channel.Then the deterministic lateral displacement is used to achieve accurate cell sorting.The performance and throughput of the quad-core spiral sorting unit,serpentine focusing unit,and lateral displacement unit are verified respectively,so that the final structure of the integrated chip is determined as a quad-core spiral 、two-core serpentine pre-focus and deterministic lateral displacement.The integrated chip was used for the sorting of dilute whole blood with high / very low concentration of a variety of tumor cell samples and clinical real samples(peritoneal effusion).Compared with DFF + DLD chip,the integrated chip’s removal rate of blood cells reaches more than 99.99%,the throughput is increased by 4.8 times,and the purity of tumor cells is increased to 90.57% at a high doping amount,and～ 75% at extremely low doping amount.For peritoneal effusion,the purity of tumor cells is increased to ～ 60%. |
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