Functionalized Nanofibers Embedded In A Microfluidic Chip For Cancer Cell Capture

Circulating tumor cells play an important role in cancer development,and are the hallmarks of cancer metastases.In recent years,CTCs isolation and enumeration have attracted much attention in clinical cancer studies,and the examination technologies have been improved continuously.Among them,microfluidic chips have been used increasingly in CTCs sorting due to their microscale dimension,easy integration,and accurate control of the fluid.In addition,nanofibers with large surface area and specific topological structures are suitable for cell adhesion,and thus have been used widely for cell capture applications.The integration of nanofibrous membrane with microfluidic technology would synergize the technological advantages of each method and result in higher efficiency in CTCs capture.In this work,a cell capture platform was developed by embedding a nanofibrous membrane in a microfluidic chip,and two representative ligands(folic acid(FA)and hyaluronic acid(HA))were used to specifically bind cancer cells.Two cancer cell lines(KB and He La)were chosen to evaluate the cell capture efficiency of the platform.Results showed that the platform had achieved satisfactory cell capture efficiency.And it is expected to become a new CTC capture technology and be applied to the early diagnosis of cancer.Previously,our research group has used dendrimers to link FA and HA to cellulose acetate nanofibers and captured tumor cells under static conditions.In this thesis,microfluidic chip and nanofiber membrane were integrated to capture tumor cells under dynamic conditions.Cell capture efficiency was evaluated by utilizing model cell lines,and optimized by using different flow velocities,chip lengths,and nanofibrous membrane thickness.Finally,diluted whole blood samples with spiked cancer cells were used to evaluate the integrated microfluidic chip platform.In the second chapter,cancer cells were captured with microfluidic chip embedded with folic acid-functionalized nanofibers.The structure of the chip was designed by Auto CAD software.The master mold of the microfluidic chamber was fabricated by photolithography technique using a negative photoresist SU-8 in a mask aligner.Finally,the PDMS cover slab was made by casting against the master mold using soft lithography.Meanwhile,poly(lactic-co-glycolic acid)(PLGA)nanofibrous membrane was prepared using electrostatic spinning technology.FA was modified on the surface of nanofibrous membrane through a series of EDC coupling reactions.Then the functionalized nanofibrous membrane was characterized by using scanning electron microscope(SEM),1H NMR,UV-Vis spectrophotometer,fluorescence microscope,water contact angle,and fourier transform infrared spectrometer.Results showed that the morphology of the nanofibers was uniform and FA was successfully modified to the surface of nanofibrous membrane.Next,the FA modified electrospun nanofibrous membrane was embedded into the microfluidic chip.KB cells,which is a FA receptor over-expressing cell line,were used to evaluate the capture efficiency of the device.L929 cells,which is a FA receptor low-expressing cell line,were used as control group.The capture efficiency and specificity were evaluated,and the effect of chip length,fiber membrane thickness,and flow rate on the cell capture efficiency were studied.Results showed that cell capture efficiency increased with reducing flow rate.The increasing membrane thickness increased capture efficiency.However,lower flow velocity and thicker membrane also increased nonspecific cell binding.After evaluated capture efficiency and purity,nanofibrous membrane spinned at 5 min,at 1 m L h-1 flow rate were determined to provide the best results.In the third chapter,we obtained morphologically uniform poly(lactic-co-glycolic acid)(PLGA)nanofibers with fiber diameter at about 900 nm through electrostatic spinning.Then PEI was used to modify HA onto the nanofiber surface through EDC coupling reaction to target CD44 receptor overexpressing He La cells.Then the HA functionalized nanofibrous membrane was embedded in the microfluidic chip.He La cells were captured under different flow velocities.Experimental results showed that the capture efficiency was lower with increasing flow velocity in culture medium,and cell capture rate reached about 90% under lowest flow velocity.Next,we investigated the capture efficiency using red blood cell-lysed blood.The capture efficiency of He La,KB,A549,and MCF-7 cells revealed that microfluidic chip embedded with HA-modified nanofibrous membrane would potentially be used to separate CTCs from various origins from the whole blood.In conclusion,I designed and fabricated a microfluidic chip embedded with ligand-functionalized nanofibrous membrane to capture cancer cells.This device provides a new method for the capture and enrichment of CTCs and is expected to be applied to the early diagnosis of cancer.