Microfluidic Chip Research For Rare Cells Isolation From Peripheral Blood

Blood test is an effective method for clinical analysis of various diseases.In recent years,two kinds of important biomarkers,namely circulating tumor cells（CTCs）and fetal nucleated red blood cells（fNRBCs）,have emerged in the circulation of the blood system.Detection,enrichment and analysis of these two rare cells from peripheral blood can be respectively applied to non-invasive cancer diagnosis or non-invasive prenatal diagnosis（NIPD）.According to studies,CTCs are tumor cells that shed from primary or metastatic tumor and entered the circulation of peripheral blood,which have the potential to form new tumor metastasis.Clinically,counting CTCs in patients’blood can serve as an important prognostic indicator for a variety of cancers.Further,cell genome,transcriptome and proteome analysis of CTCs can promote the relevant research on the mechanism of tumor metastasis.In addition,in vitro culture of CTCs can be used for personalized drug response testing.Similar to CTCs,the number of fNRBCs in maternal peripheral blood is very rare.These cells are directly derived from the fetus and contain the entire fetal genome information,which can be used for genetic testing related to fetal congenital diseases.However,there are a large number of white blood cells（WBCs）and red blood cells（RBCs）in the peripheral blood,which cause great interference in the separation and detection of these two rare cells for biomedical research.With the development of microfluidic technology,CTCs or fNRBCs can be efficiently sorting from a large number of normal blood cells by microfluidic chip,which promotes the biomedical application of these two rare cells.The application of microfluidic chip in rare cell enrichment and sorting has unique advantages:（1）The limited volume of microchannel can effectively reduce the sample consumption and improve the reaction speed;（2）The geometric dimensions of microfluidic channels are very close to the size and shape of cells,enabling fixation and analysis of individual cells;（3）The microfluidic chip can flexibly manufacture many different microstructures as functional units to improve the sorting efficiency;（4）Being of miniaturization,integration and automation that has great potential in commercialization.In this paper,we first summarize the research progress in the field of CTC or f NRBC enrichment,and considering the difficulty of rare cell sorting,we carry out the studies of cell isolation based on the advantages of microfluidic technology mentioned above.The main work is summarized as follows:1.Enrichment of fNRBCs for non-invasive prenatal diagnosis based on a new density gradient centrifugation method.First,a kind of large,high-density silicon microsphere（30μm,2.0 g/m L）was introduced and coated with a biodegradable gelatin nanofilm,and then grafted with specific antibody（anti-CD147）.fNRBCs were captured onto the surface of microspheres by specific recognition of antigen and antibody,thus enlarging the density difference between target cells and the normal blood cells in peripheral blood.Then,the cell-attached microspheres were purified and enriched by an improved percoll solution（1.15 g/m L）.Finally,the captured fNRBCs were released from the microspheres utilizing the matrix metalloproteinase（MMP-9）to degrade the gelatin nanofilm.Based on this method,we counted fNRBCs in peripheral blood of pregnant women at different gestational weeks（10 to 30 weeks）,and further detected fetal polyploidy including trisomy 21 and trisomy 13 by fluorescence in situ hybridization（FISH）technique.2.Detecting fNRBCs from peripheral blood of pregnant women during early pregnancy using a new microfluidic chip.In this work,we propose a specially designed microfluidic chip,which combined the optimized herringbone structures with the curved channel to generate vortex effect,greatly enhancing the collision probability and interaction between cells in the fluid and the inner wall of the microchannel.Meanwhile,gelatin nanoparticles were utilized to form a rough nano-surface inside the chip,which provided more binding sites both for antibody molecules and cell adhesion,and facilitated the subsequent release of captured cells from the chip as well.We successfully detected fNRBCs in the peripheral blood of pregnant women in early pregnancy（7 to 13 weeks）,and successfully applied them to detect different types of fetal chromosomal abnormalities.This facilitates early non-invasive prenatal diagnosis based on fNRBCs in the fetus.3.An acoustic droplet-induced enzyme responsive platform for the capture and on-demand release of single CTCs.First,gelatin nanoparticles（GNPs）were coated onto the glass substrates to prepare the GNP-chip,and then grafted with specific antibody（anti-Ep CAM）to efficiently capture CTCs in peripheral blood of cancer patients.The acoustic droplet dispenser was employed to generate oxidized alginate microdroplets containing the MMP-9 enzyme（OA-MMP-9）with controllable size and precise positioning upon the cell-attached GNP-chip,allowing controlled cell-surface biodegradation under enzymatic reactions,followed by calcium chloride（Ca Cl₂）solution treatment to form single-cell encapsulated calcium alginate hydrogels.By optimizing the concentration and incubation time of nanoparticles,the optimal capture effect was achieved.By adjusting the pulse width,the size of OA-MMP-9 droplets on the GNP-chip could be controlled,and then hydrogels of different sizes were obtained for the release of single CTCs.Based on the optimal experimental parameters,we collected peripheral blood of one breast cancer patient for CTC capture and controlled release,and conducted subsequent genomic analysis to successfully detect the 3140A/G（H1047R）mutation in the PIK3CA gene of the patients.