The Capture And Detection Of Circulating Tumor Cells Based On Microfluidic And Magneto-optical Probe

Circulating tumor cells(CTCs)are tumor cells that are shed from tumor tissues and enter the circulation system.They play an important role in the process of tumor metastasis and are the main cause of death of malignant tumors.The enrichment and sorting of CTCs in patients’ blood are of great significance in the early detection and prognostic monitoring of malignant tumors.At present,the sorting of CTCs is mainly divided into the sorting based on physical properties and the sorting based on immune affinity.They have their advantages and disadvantages and can be combined with each other to become a comprehensive sorting method.However,among the existing liquid biopsy methods,there are still some problems to be solved in terms of CTCs capture purity,high-throughput detection,real-time monitoring/counting,etc.Based on this,this paper combined microfluidic chip and immunomagnetic beads,introduced upconversion fluorescence and intravascular stent capture technology,to achieve high efficiency and purity of CTCs sorting.The real-time fluorescence count and inactivation of CTCs were realized in microfluidic chip.In the design of tubular scaffolds,high speed enrichment,high purity capture and in situ inactivation of CTCs were achieved.Efficient sorting,fluorescence counting and multicolor imaging of CTCs were achieved in immunomagnetic beads.The specific results are as follows:(1)To solve the problem of capture purity,an inverted microfluidic chip combined with a multi-functional immunomagnetic bead was proposed.Through magnetic field selection,the contact between other blood cells and the capture surface is reduced,and the capture purity of CTCs is improved.In order to improve the capture efficiency of the microfluidic chip,a capture surface with three-dimensional macroporous biomimetic structure was designed and the antibody was modified.By combining the biomimetic characteristics with antibody specific recognition,the capture efficiency of up to 95% was obtained.In order to monitor the capture process of CTCs in real time,a unilateral transparent microcavity was designed in the microfluidic chip,and fluorescent molecules were integrated into the immunomagnetic beads bound with CTCs.To solve the problem of real-time quantity statistics,real-time fluorescence counting of CTCs in microfluidic chip was realized by measuring the ratio of rare earth upconversion fluorescence and immunomagnetic bead fluorescence on the capture surface of the chip.In order to solve the problem of low up-conversion luminescence efficiency,a double-layer structure of Inverse Opal photonic crystal(IOPC)was designed.The up-conversion luminescence of the captured surface was increased by 32 times by the bandedge effect of the photonic crystals.On this basis,the photosensitizing agent Ce6 molecules were integrated into the immunomagnetic beads.After binding to CTCs,the photodynamic effect was guided under the excitation of red light,and singlet oxygen was generated on the capture surface,and the CTCs captured on the capture surface was inactivated in situ.(2)Further research on CTCs detection in large flow samples was carried out.A seamless tubular fiber textile frame based on electrospinning technology is designed.Its size and internal thread structure can be adjusted,and it has a relatively large crosssectional area.The problem of small sample flux in CTCs enrichment strategy is solved.In capture efficiency,has flexible nano fiber structure was designed and the capture of surface modified antibody,through the bionic characteristics combined with antibody specificity increase the capture efficiency,in 3 mm diameter thread tube holder,single enrichment by more than 80% of the capture efficiency,and realize the circulation of the sample processing,to capture efficiency is as high as 98%;In order to improve the capture purity of CTCs,the inner wall of the scaffold with thread was designed to control the flow velocity distribution of the fluid in the tube,forming the pressure difference between the axial center and the tube wall,enhancing the axial property of the cells in the tube,reducing the contact probability of other cells with the capture surface,and achieving ultra-high capture purity of 98%.In the capture system of this tubular scaffold,the photodynamic effect is excited by red light,and the in situ inactivation of CTCs captured at large flux on the inner wall of the scaffold is realized.(3)Furthermore,an immunomagnetic bead was designed to recognize CTCs specifically,and the fixation and sorting of CTCs are realized under the external magnetic field,and the fluorescence counting and multicolor imaging of CTCs are explored.Combined with Perovskite nanocrystals(PNCs)as fluorescence probe,the immunomagnetic beads realized fluorescence counting of CTCs at the concentration range of 100-5000 CTCs· m L-1.Coating PNCs and Fe3O4 with polymeric PMMA does not produce obvious quenching of PNCs,and the immunomagnetic beads can inherit the fluorescence properties of PNCs well.Moreover,the coating isolated the contact between PNCs and the external solvent environment,inhibited the decomposition of PNCs in water,and ensured the stability of the fluorescence properties of immunomagnetic beads.The core-shell structure design can enhance the dispersion and stability of immune magnetic beads and isolate the direct contact between PNCs and CTCs,thus solving the problem of biotoxicity of PNCs.PMMA encapsulation also inhibits the exchange of different halogens between the lattices,thus inhibiting the luminescence fusion caused by PMMA encapsulation.Immuno-magnetic beads can produce stable multicolor emission in the same system,and realize multicolor imaging with multiple channels under fluorescence microscope.