| Early diagnosis and prognostic evaluation has practical significance for cancer research.As the key precursor of tumor metastasis,circulating tumor cells(CTCs)in peripheral blood can be used to predict the overall survival of patients,thus it is vital important to find effective solutions for the fast capture and enrichment of CTCs.In this work,we developed a novel microfluidic instrument for the high-throughput separation and enrichment of label-free CTCs.Our work is demonstrated as follows.(1)Research of working principle of microfluidic passive flow regulator,design,fabrication and characterization of the flow regulator.In order to control the fluid precisely,we propose a novel microfluidic passive flow regulator to regulate the fluid flowing through it automatically.Firstly,COMSOL Multiphysics software was used to build the 3D fluid-structure interaction model of the flow regulator,and simulated results show the significant nonlinear relationship between the input pressure and output flow-rate.Next,based on the simulation results,a set of prototype devices of the flow regulators with different dimensions were designed and fabricated.By connecting the device in a liquid circuit system,flow-rates of the device under varied input pressures were measured,and constant flow-rate autoregulation performance of the device was observed.Then,influence of critical structural parameters of the device on the flow-rate was investigated systematically.It is found that the flow-rate is decreased with the increase of control channel width or the decrease of cross-sectional area of through channel.The above conclusion provides useful guidance for designing the flow regulators with different flow-rates.Finally,damping performance of the flow regulator was studied by installing the device in a gas circuit system.Through the estimation of a classical resistor-capacitor(RC)low-pass filter,the damping capacity of the system was found to be increased with the increase of gas volume and input pressure frequency.(2)Investigation of particle inertial separation in gas-driven flow system,development of low-cost sample injection,transportation and separation system.Laboratory syringe pump for precise fluid infusion is expensive in price and bulky in volume,thus the pump is not suitable for miniaturized integration of microfluidic system.In order to develop lightweight and portable microfluidic instrument,a low-cost gas-driven flow system was proposed to realize the precise sample infusion,transportation and particle separation.Firstly,an inertial microfluidic chip(e.g.DFF chip)was designed for the separation of 5?m and 15?m polystyrene microbeads due to their different flow behaviors in the spiral channel of the chip.As 5?m microbeads were transported to the outer outlet due to the dominate Dean drag force and 15?m microbeads were migrated to the inner outlet due to the strong inertial lift force,two microbeads can be separated effectively,and the optimal flow-rates for cell separation were measured.Next,the gas-driven flow system was designed and constructed.In the system,low-pressure compressed air was applied as the power source,and the microfluidic flow regulators and DFF chip were integrated into the system.Finally,the microbead transportation in the spiral channel was investigated,and successful separation of microbeads was observed totally independent of the varied pressures.The results demonstrate that the gas-driven flow system integrated with the passive flow regulators can be used for the precise fluid infusion in microfluidic environment,thus the system has the good potential in constructing portable microfluidic instrument.(3)Study of fabrication process of thermoplastic polymer membrane chip,design and fabrication of cell high-throughput separation device which is integrated with passive flow regulators.In order to fabricate low-cost and disposable microchip,a novel membrane chip of thermoplastic polymer material was proposed.Firstly,the membrane chip was fast fabricated using UV laser cut and thermal lamination method,and the influence of the laser and lamination parameters on the cross-sectional dimensions of microchannel was investigated systematically,thus the optimal parameters for chip fabrication were obtained.Then,a DFF membrane chip was fabricated according to the above parameters,and the optimal flow-rate for separating microbeads of 5?m,7?m,15?m and 20?m was measured.The membrane chip was also used to separate breast adenocarcinoma cell lines MCF-7 from human blood,and the experimental results show the good separation efficiency of MCF-7 cells and blood cells.Next,a cell high-throughput separation device was designed and fabricated.Since the parallel integration of eight spiral channels,the fluid throughput of the device was over 12ml/min.After that,a fluid autoregulatory device was designed and fabricated.The fluid autoregulatory device was integrated with nine microfluidic passive flow regulators,thus it provides the precise fluid infusion for the sheath and sample fluids in cell separation device separately.Finally,a totally integrated microfluidic device was obtained through integrating the cell separation device and the flow autoregulatory device.The overall dimension of the integrated device is 51x53x2.7mm3,and the material cost of the device is only 3.5RMB,which meets for the low-cost and portable microchip requirements in practical application.(4)Design,fabrication and application of CTCs automatic separation instrument.The microfluidic instrument was designed according to the concept of the gas-driven flow system.Firstly,the application requirement of the instrument was analyzed,and the technical parameters of the microfluidic device and the control system of the instrument were determined.Based on the requirement analysis,key electric components were chosen and mechanical structures of the instrument were designed and fabricated.Next,cell separation mode,product backflow mode,cell enrichment mode,and automatic mode of the instrument was developed.By analyzing the stepping motion of the electric component at every mode,control program of the instrument was developed.After the instrument was assembled,system debugging was performed,and the optimal operation parameters were set.Then,the practical function of the instrument was tested,and the pressure and flow-rate of the system was measured.The experimental results show that sheath and sample flow-rates of the system were 10.91 ±0.34ml/min and 1.26±0.03ml/min respectfully,which meets for the flow-rate requirement of cell separation.Finally,the instrument was applied to sort cancer cell lines MCF-7 from blood cells.The removing ratio of blood cells was found to be over 99%,the capture efficiency of cancer cells was about 80%,and the purity of the captured cancer cells was about 30%.The whole automatic operation process of the instrument was about 23 min.In comparison to the previously reported cell separation instruments,our instrument has good capture efficiency and purity of cancer cells,and it also has advantages of low-cost,lightweight,and high-throughput,which demonstrates the good commercial prospect of the instrument. |
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