Isolation And Enrichment Of Rare Cells By Epoxy Resin Microfluidic Chips

Specific screening, sorting and enrichment of single or small groups of cells from blood and tissues are required for many biological and medical researches, such as low-abundance stem cell sorting, detection and isolation of rare circulating tumor cells, and screening of cell-based libraries. Conventional cell separation techniques such as flow cytometry, immunomagnetic cell sorting and density gradient centrifugation are limited to application widely, which generally require large samples, expensive equipments and complicated operation. Compared to the conventional techniques, microfluidic used to isolate and enrich the rare cell offer several advantages. Microfluidic are generally in micro and nano-scale experiments, and can elucidate some new biological phenomena and results that traditional techniques do not give.Microfluidic have the advantage of small sample volumes, simple equipment, easy to handle, and meet the needs of common laboratory and non-technical personnel. Microfluidics for biological and medical applications has consistently faced a commercialization challenge: how to produce these one-off chips in large-scale while meeting the material demands of biological and medical use. A variety of polymeric materials can meet the requirements of the above two points is not much. In recent years, many research emerged on the surface modification of polymeric materials, which had the multi-function and compatibility on surface by a variety of physical and chemical methods.In this paper, a new polymeric materials-epoxy resin was used for microfluidics construction. The properties of low cure temperature, short cure time, good transparency, and hardness were suitable for the construction of microfluidics. The surface of microfluidics was treated by the technology of air-plasma treatment which has the advantage of treating various polymeric materials with complex shapes, and acquired the oxygen-containing groups on surface; and further modified by near-UV induced graft copolymerization with hydrophilic monomers, to obtain the desired carboxyl groups. Surface functionalisation was achieved through cross-linking reagent that coupled the specific antibodies, and interacted with the surface antigen of target cells. Aimed to isolation and enrichment the rare cells from blood or tissues, the channels of microfluidics were constructed regular arrangement of microposts that were made chemically functional with specific antibodies which interacted with the antigens of target cells, to achieve the rare target cells from blood or tissues to sorting and enrichment in one-step procedure.The methods of fabrication microfluidic are simple to fabricate which need simple equipments and low cost, and can achieve the production of microfluidic in large scale. The methods to modify the microfluidic surface are universal, and suited to most polymers materials. The modified surface with special functional groups could covalently bind to proteins, peptides, nucleic acid and other functional bimolecular. As to capture the rare cells by immunochemistry, the microposts were constructed to increase the effective functional areas to connect to specific biomolecular, and increased the probability of contact of cells with regular array. The channels with microposts could effectively capture target cells, and reduce the non-specific cell adhesion, to achieve to isolate and enrich the rare cells from complicated samples in one-step procedure.The thesis is composed of four parts:In chapter 1, the materials and fabrication methods of microfluidic, surface modification methods of polymeric materials, and the application of microfluidic were reviewed. The first part focused on the fabrication methods of polymeric microfluidic, included hot embossing, injection molding, casting, lithography electroforming micro molding, laser photoablation and soft lithography. As well as, a polymeric materials SU-8 photoresist used to fabricate microfluidic was introduced. The second part described several methods for surface modification of polymeric materials, included wet chemical, silanization, plasma treatment and grafting. The third part focused on the application of microfluidic, included the application in point-of-care testing and separation and enrichment of cells.In chapter 2, a new materials-low viscosity epoxy resin was used to fabricate microfluidic which was molded with regular arrangement of microposts. The microstructure of microfluidic was fabrication with SU-8 2075 photoresist, which was as positive mask to fabricate the PDMS-negative replicas. Epoxy resin was poured over the PDMS mold, cured and peeled off, and then an epoxy resin microfluidic with arrangement microposts was obtained. The epoxy resin microfluidic possessed fine microstructure and a simple production process. The PDMS-negative replicas which were used to fabricate microfluidic were used repeatedly that saved the cost and times.In chapter 3, the method of surface modification of epoxy resin was introduced. The hydrophobic property of epoxy resin had not availed to forming the special surface. The channels of microfluidic were treated with the low temperature plasma to induce the reactive groups on the surface, and then grafted acrylic acid monomers by UV light induced, and covalently linked with specific biomoleculas used to immunological detection. The modified epoxy resin were characterized by static water contact angle measurement, toluidine blue O staining test, X-ray photoelectron spectroscopy (XPS), bicinchoninic acid (BCA) test, and cell attachment. Our studies showed that the carboxyl groups were grafted onto the surface of epoxy resin, and covalently crosslinked with proteins. The results of cell attachment revealed that the modified epoxy resin could be applied to immunological detection.In chapter 4, the microfluidic of epoxy resin were used to test the capacity to capture specific cells from complex samples. Covalent immobilization of antibodies on the AAc-grafted epoxy resin surface was facilitated by the coupling reagents EDC/NHS,then the antibodies were used to capture A549 cells which expressed the antigens of CEA in membrane. After staining, the captured cells were directly observed by fluorescence microscopy and counting. The captured cells were detected by RT-PCR, which determined the expression of surface antigen CEA.The fabrication process for epoxy resin microfluidic chips is easy and low cost, that can achieve the large-scale production. The method of surface modification is universal, and suit to most of the polymer materials. The microfluidic device has simple structure and dose not require pretreatment of samples, which can achieve the capture and detection of specific cells. The microfluidic chips are hopeful to capture the circulating tumor cells and separation of immune cells from blood and tissue, which have great potential in the biomedical application...