| Circulating tumor cells(CTCs),nomadic cells which are shed into peripheral blood from a primary tumor,play an important role in the metastasis of tumor cells.Therefore,the measurement of the number of CTCs in blood plays a critical role in early cancer diagnosis and treatment monitoring since it allows early cancer detection and treatment monitoring.Due to the extremely low number of CTCs in blood(only 1-100 CTCs in one milliliter of blood cells),separation with high purity and recovery rate has been a challenging but essential pretreatment step for the efficient detection of CTCs.Based on the latest research progress of circulating tumor cells sorting by microfluidic methods and the preliminary research of our group,this dissertation systematically studied the technique of inertial spiral sorting with the advantage of high throughput,and based on which,two kinds of integrated chip combined with the deterministic lateral displacement(DLD)or magnetic sorting technologies were proposed.Significant breakthrough in rapid and precise separation of tumor cells was made with the sequence of coarse sorting and precise sorting.The innovative achievements are mainly described as follows:(1)A simple spiral channel device for rapid separation of tumor cells from blood was devised.The device was first characterized with diluted whole blood at different concentrations and flow rates to obtain the optimal operational parameters.Then,at a flow rate of 400 m L/min,we continuously separated the human breast cancer cells MCF-7 from the 100× diluted whole blood.The experimental results showed that 91.97±0.57% of blood cells were removed and 85.06±9.27% of MCF-7 cells were collected after the first separation.After the second separation,the remove rate of blood cells increased to about 99.07%.Meanwhile the purity of captured MCF-7 cells were increased to 8.10±2.63%,which means an 81-fold increase in the purity of MCF-7 cells was observed within the sample collected from the middle outlet with respect to the original sample.The separation results proved its excellent capability of detecting rare CTCs in blood.(2)An integrated device combining inertial and deterministic lateral displacement methods for rapid and precise separation of tumor cells was devised.Aiming at the relative shortcoming of inertial spiral technology in sorting precision,a size-sensitive deterministic lateral displacement method was introduced and integrated in series to thoroughly remove residual blood cells with small size.With the two stage of highthrought coarse separation by spiral channel and precisely purified by DLD,highly efficient capture of tumor cells was achieved.In the design of integrated chip,the particle-free flow at the outer wall of the spiral channel was ingeniously introduced into the secondary DLD channel,to replace the sheath flow which is additionally introduced by the conventional DLD chip.According to this design,the final integrated chip has a single inlet and three outlets,which greatly facilitates the operation of the separation process.Based on the optimization of spiral and DLD unit respectively,the flow rate and flow resistance of the integrated chip were matched.The 100% separation of polystyrene particles by the integrated device demonstrates its enormous advantages and great potential in high-throughput,high-purity separation.The experiment of MCF-7 sorting from blood was carried out,and obvious advantages on separation performance were observed compared to single-stage spiral structure.The purity of captured MCF-7 cells was even as high as 93.59% under the condition of lower concentration of initial sample,with the 99.94% removal rate of blood cells.We also spiked an extremely low number of MCF-7 cells(500 cells per m L)into the diluted blood,and detectable tumor cells were collected from the DLD upper outlet.Moreover,a more efficient separation of MCF-7 cells from a lysed buffy coat was also demonstrated,with the about 98.63% remove rate of similar size WBCs.In addition,re-culture of the captured tumor cells indicated that they still maintained great viability.(3)An integrated device combined inertial and magnetic methods for rapid and precise separation of tumor cells was devised.Because the sorting techniques of inertial spiral and DLD,which were employed in the integrated chip as above mentioned,are size-based,they have significant limitations in terms of accurate separation of white blood cells(about 10-25 ?m in diameter)and tumor cells(about 15-40 ?m in diameter).In order to overcome this deficiency,a size-independent magnetic separation method was introduced.With the accurate depletion of red blood cells and magnetically-labeled white blood cells by inertial spiral and magnetic unit respectively,a high-purity,high-recovery rate,and high-throughput capture of tumor cells was achieved.The integrated chip includes three sorting stages: dual-inlet spiral channel for RBCs depletion,asymmetric sinusoidal channels for focusing,and magnetic sorting channel for magnetically-labeled WBCs depletion.In order to fabricate low-cost and disposable microchip,a flexible process of laser cutting membrane chip was deployed.Optimization of the spiral channel unit and the focusing/magnetic sorting units were respectively carried out,then flow rate through three-stages was matched by one layer of spiral chip with a 3-layer focusing/magnetic chips.Based on the flow resistance compensation of each outlet,a final integrated membrane chip containing a 19-layer structure was devised.Using the integrated chip for MCF-7 sorting from blood,the results showed that 93.84±9.01% of cancel cells can be captured with the purity of 51.47±6.27%.By decreasing the cell concentration of initial sample,the purity of MCF-7 cells can increase significantly up to 93.60±5.97%.The integrated chip also has an excellent performance in the sorting of ultra-low concentration tumor cells(500 cells/m L).Benefiting from the magnetic beads were tagged on white blood cells,the captured MCF-7 cells still had great viability and physical biological properties of their own. |
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