| The separation and analysis of circulating tumor cells is of great significance for the diagnosis of metastatic cancer,the evaluation of prognosis,the determination of individualized treatments,and the development of cancer drugs.However,circulating tumor cells is in the background of a large number of blood cells,and their physical characteristics are different but partly the same as those of blood cells.Therefore,it is necessary and also challenging to study how to achieve label-free high-purity sorting of circulating tumor cells.Active and passive methods for cell separation based on microfludic chip,which are distinguished by the presence or absence of external field force,rend high sorting purity but low throughput and high throughput but low sorting purity,respectively.And both of the them require external connecting equipment such as pressure pumps and pipelines.The system as a whole is too complicated.The centrifugal cell sorting technology relies on chip rotation to drive fluid and cell movement,and has the advantages of simple operation,low cost,simple and high integration in its equipment.However,the recovery ratio and separation purity of target cells obtained by centrifugal sorting are relatively low.And it is still challenging to precisely manipulate cells.This paper reviews the existing microfluidic chip for cell sorting and analysis methods,and starts from cell density difference and size difference according to the characteristics of centrifugal microfluidic chips that can distinguish both cell density and size.Firstly,most of the red blood cells and white blood cells with relative sizes to circulating tumor cells were removed based on density differences,and then circulating tumor cells were preliminarily enriched.Then,the white blood cells with similar density to circulating tumor cells were removed based on size difference,and the circulating tumor cells were collected and analyzed.Further study of cell sorting mechanism,chip design and preparation,sorting performance of chip and analysis system integration were conducted to solve the problems of particle trajectory deviation affected by non-steady flow in sizebased centrifugal separation mechanism,low recovery ratio and separation purity of tumor cells,and material packaging failure in chip fabrication to achieve the label-free high-purity sorting and analysis of circulating tumor cells.The detailed research contents of this paper are as follows:（1）A centrifugal cell sorting microfluidic chip based on density difference was developed.Firstly,a density gradient centrifugation method was proposed to remove red blood cells and some white blood cells of similar size to tumor cells for tumor cell preliminarily enriching,given the difference in density between tumor cells and blood cells.Secondly,considering the instability and difficulty in manually extracting cell layers in blood samples with traditional density gradient centrifugation methods,a density gradient centrifugation method based on centrifugal microfluidic chip was designed.Besides,the chip structure was designed for automatic sample addition and extraction.The factors that affecting the stability of white blood cell layers and the extraction were studied.Finally,the preliminary processing of automatic sample addition and extraction of blood samples was realized.（2）A rectification method was proposed to change the flow state of centrifugal microfluid.The research on centrifugal cell sorting based on the size difference reveal the problem that the unsteady flow prevailing in the centrifugal microfluidic chip affects the particle trajectories.By designing the rectifier structure in the chip,the unsteady flow process of centrifugal separation is modified into a locally stable continuous steady flow process,solving the problem of inconsistent trajectories of the same particles caused by the changes in unsteady flow speed,and making the manipulate of cells in centrifugal microfluidic chips controllable.In addition,the level set method was used to describe the fluid interface theoretically,and the rectifier structure was numerically simulated to analyze the change of the flow velocity at the outlet of the sample cavity.The effectiveness of the rectifier of changing the fluid flow state was proved both theoretically and experimentally.（3）A cell-sorting centrifugal microfluidic chip with a rectifier was developed.For tumor cells and leukocyte samples with similar densities but different sizes,in order to sort tumor cell out,it is necessary to analyze the manipulation of particles with different sizes.Firstly,the Lagrangian theorem was used to solve Newton’s second law,the numerical simulation of the particle trajectory was carried out,and the influence of the rotation speed on the particle trajectory was analyzed furthermore.Therefore,the sorting performance of the chip was theoretically proved.In the experiment,the separation experment of the mixed suspension of polystyrene particles with size 25 um and12um was conducted.The recovery ratio is close to 100%,and the separation purity is higher than 95%.Furthermore,the prostate cancer cells were separated from highconcentration leukocytes with recovery ratio of 90% and separation purity of 88.24%.Considering the stable sorting performance,this chip has significant potential use in biomedical and clinical applications.（4）Study on the preparation process of centrifugal microfluidic chips,including material selection,chip processing and packaging.The manufacturing and packaging process of PMMA,epoxy resin and PDMS materials were researched.Aiming at the problem that laser engraving machine cutting PMMA material will form surface recast layer and cause high adhesive bonding failure rate,a mask-cooling process was proposed to reduce the protrusion of the recast layer on the surface of the structure,thereby improving the success rate of chip bonding.In order to solve the problem that adhesive bonding of epoxy resin chip affects the surface properties of structure,a fusion bonding method with same material of epoxy resin is proposed,which provides a new method for packaging epoxy resin chip.（5）Study on the system integration of centrifugal cell sorting and imaging detection.An integrated system of centrifugal cell separation and imaging detection was designed and built to realize in situ detection of centrifugal cell separation.Firstly,the overall structure of the centrifugal cell sorting and imaging detection system was designed,and the system was then assembled completed to obtain high-resolution images of cells in the chip.Then,according to the obtained in-chip cell image,the image recognition method is further used to count the cells without manual operation.The system is of high integration,simple structure and simple operation,which proves the great potential in cancer clinical diagnosis. |
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