Microfluidic Chip Combined With Nanofibers For Capture And Release Of Circulating Tumor Cells

Circulating tumor cells(CTCs)in peripheral blood can be regarded as a new "liquid biopsy" for tumors,which can ban the traditional biopsy of pathological tissues and reduce the suffering of patients.On the other hand,whether it is in the early detection of cancer,the guidance and adjustment of the protocol in the treatment process,or the prognosis guidance after the completion of treatment,these aspects have important significance.However,the extremely scarce number and heterogeneity of CTCs in peripheral blood lead to the problems of low capture efficiency,low capture purity,low cell activity,and low release efficiency of various methods for CTCs capture to a greater or lesser extent.Many methods are difficult to achieve a good balance or solve these problems at the same time.With the research and application of microfluidic chips in CTCs capture in recent years,breakthroughs are expected in these areas.The design of various micro-patterns and flow channels in microfluidic chips can sort CTCs according to their physical characteristics,such as volume and density.However,the research on nanomaterials has revealed that the topology of nanofibers can well simulate the extracellular matrix,and larger specific surface area can provide more contact sites.In addition,the coating of antibodies can also achieve good specific capture of CTCs.Besides,a disulfide bond can be connected during the loading of the antibody,and efficient release can be realized within a short time under the stimulation of glutathione(GSH)after the capture is completed.Therefore,inspired by the respective advantages of the two methods,a microfluidic chip with a new size is designed in this paper.The micro-patterns in the chip are cylindrical micro-pillars,which are arranged in a parallel and staggered manner in the chip.The number of micropillars in the working area is about 18,000.This design can change the Reynolds number of blood in the microchannel,change the original motion trajectory of CTCs,and improve the collision probability of CTCs with each capture site.At the same time,PLA nanofibers with good biocompatibility were loaded into the microfluidic chip for increasing the grafting site of the antibody.The combined chip can realize in-situ capture,detection and release of CTCs.Firstly,a cylindrical PDMS microfluidic chip with parallel staggered arrangement was designed and prepared.The morphology of the samples was characterized by confocal microscopy and scanning electron microscopy(SEM),which demonstrated the successful preparation of the chip micropattern.Then the polylactic acid(PLA)nanofibers were prepared by electrospinning technology.The morphology characterization by SEM proved that the nanofibers were successfully prepared,and the majority of the diameters were distributed in the range of 400–750 nm.The fibers were loaded into the micro-pillar array and characterization by fluorescence labeling and scanning demonstrated that a maximum fiber binding density of 14 was achieved at 30 s of reception.Subsequently,the chip after the binding is completed is subjected to poly-dopamine(PDA)modification,disulfide bond modification,biotin modification,streptavidin modification,antibody coating,and the like.The structure was characterized by Fourier transform infrared spectroscopy(Fourier transform infrared spectroscopy),scanning electron microscopy(SEM),energy dispersive spectroscopy(EDS),fluorescence microscopy,and water contact angle.The results demonstrated the successful modification at each step,the distribution of antibody grafting sites,and the change from the surface structure to hydrophilic structure.Finally,the co-incubation of human breast cancer cells(MCF-7),liver cancer cells(HepG2)and cervical cancer cells(He La)with the material proved the good compatibility of the material and the changes of adhesion to cells before and after modification.After the fibers were loaded onto the chip,the cell capture efficiency of Ep CAM(+)could reach more than 85%,while that of Ep CAM(-)was very low.Besides,it can achieve 85% release efficiency in GSH and the cell activity can also be maintained at about 90%.