| In 2012, there were over 8 million people died of cancer and 8550 people became cancer patients every day in our country. Obviously, cancer has become the largest killer of human health. The major cause of cancer-related mortality was the so called mircometastasis, which could not be detected by conventional survey. In recent years, circulating tumor cells (CTCs) have attracted huge attentions from people and were regarded as biomarker for cancer research. CTCs are those cells falling down from the tumor tissue and slipping into the systemic circulation of blood. They are transformed from the ordinary cancer cells, and can grow into tumor tissue again by the retro-differentiation at any time, thus related to the proliferation and metastasis of cancer closely. As a reflection of the vane in cancer progression, it is most important to carry out detailed research of CTCs for the early diagnosis and treatment of cancer.In this dissertation, we employed circulating tumor cells in peripheral blood as a research subject and carried out research in three areas:First, the development of the CTC capture microchip based on nitrocellulose membrane. CTCs are rare, in only up to hundreds of cells mL-1, whereas comprising 109 common haematologic cells mL-1 in the peripheral blood of patients with metastatic cancer. Thus it requires us to develop highly selective and effective means to capture and detect in the blood. The second is the application of large-scale surface-enhanced Raman scattering (SERS) imaging technology in the sensitive enumeration of CTCs. We developed a novel and highly efficient SERS probe, ensuring the clarity of SERS imaging. The third is the development of a series of new technologies in proteomics research. With the rapid development of proteomics, conducting a detailed proteomics research of CTCs isolated from the blood was best for the deeper understanding of the molecular mechanisms in cancer progression. Consequently, we have developed the fow abundance proteins enrichment and effective proteolysis technologies. Based on the above ideas, this dissertation is divided into five chapters:The first chapter is a summary of the literature. It introduces the origin and biological significance of circulating tumor cells, the major role in current cancer diagnosis and treatment; the development process and latest research of the highly selective CTC capture technologies; the inherent advantages of SERS imaging technique and their bright future in cancer cell imaging application; the status of single-cell proteomics research and the urgent need to resolve technical problems. Finally leads to the purpose and significance of this dissertation:develop new technologies for the capture, detection and subsequent single-cell proteomics research of CTCs, to provide more theoretical basis for the diagnosis and treatment of cancer.The second chapter is about the nitrocellulose membrane microchip to capture circulating tumor cells in blood efficiently. The nitrocellulose membrane was low cost, bio compatible, and more importantly, it has a natural affinity to proteins, thus can immobilize antibody through self-assembled. We employed EpCAM CD326 antibody and non-small cell lung cancer NCI-H1650 to verify its target acquisition performance, more than 70 percent of cancer cells can be successfully captured. In the simulation experiment, we added 100 target cells into 1 mL human whole blood, still isolated and detected 34 cells.In order to solve the serious cell loss in the washing process, we constructed a microfluidic chip employing nitrocellulose membrane as the substrate. The microchip was 20 mm long and 4 mm wide, with many microspots in the middle. After the SDS-PAGE analysis of flowed antibody solution, we can calculate the amount of adsorbed antibodies. We have also examined the capture efficiency of cancer cells under different flow conditions. The optimized flow velocity was 0.3μL/min, when the capture efficiency was nearly 70 percent.45% of cancer cells were detected in the simulation experiment, indicating the bright prospect of CTC capture in human whole blood.The third chapter is the large-scale SERS imaging in the highly sensitive detection and enumeration of CTCs. Compared to the conventional fluorescence imaging, SERS imaging has a series of advantages, such as probe stability, low background noise, the narrow characteristic Raman peaks so as to multi-labelling detection and offering the intramolecular covalent bonding information. To this end, we have devefoped a highly sensitive SERS probes with a sandwich structure. We empployed 60nm gold nanoparticles as the core and modify the bi-functional molecule pMBA on the surface. On the one hand, it acted as a characteristic Raman signal molecules, on the other hand as an antibody linker molecule to allow carboxyl group react with the amino group of the antibody. This unique design endows the SERS probes with simplified structure and higher signal intensity. Furthermore, the prepared SERS probes were highly selective. Over 99 percent of target cells can be successfully labeled, while the the negative control cells displayed no Raman peaks. The cancer cells captured by CTC microchip were labeled and scanned in large-scale by Raman spectrometer. The SERS images were obtained through software simulation, which was very clear and can replace the traditional fluorescence imaging.The fourth chapter is the magnetic graphene composite materials for the enrichment of low-abundance proteins. In the proteomics research of small number of cells, the enrichment and concentration of low abundance proteins is critical for subsequent MS analysis. As an ideal adsorption substrate, graphene is mainly based on π electron interactions, hydrophobic interactions and electrostatic interactions, now widely used in organic contaminants, metal ions, peptides and DNA enrichment. However, due to the complexity of the spatial structure, the variety of hydrophobic and hydrophilic and charged status, there is an urgent need to explore novel sorbent materials. We have developed the core-shell structure magnetic graphene composites. The core of iron oxide, can achieve efficient separation under the applied magnetic field; the outside coated graphene shell can extend spatially, which has a large surface area and guarantees the enrichment efficiency and protein foading capability.According to different graphene coating approaches, we designed two types of magnetic graphene materials. The one is wrap silica on the surface of 200 nm Fe3O4 core, and then modify amino groups with APTES. Graphene oxide was linked with Fe3O4 through the reaction of carboxy and amino groups with the assistance of EDC and NHS. Finally, magnetic graphene oxide was reduced with hydrazine; another one is coat the magnetic nanosphere with a layer of chitosan, so let the nanosphere positively charged, then react with the negatively charged graphene oxide by electrostatic interactions. Both materials are coated with graphene shell and hold advantages of high capacity and high surface area. For the enrichment of Cyt-c, high efficiency was shown and the corresponding loading capabilities reached 0.117mg/g and 0.247 mg/g, respectively. Coupled with laser-assisted on-plate digestion techniques, the Cyt-c minimum detection limit reached 10 ng/mL. In human serum proteomics, we enriched the low abundance proteins with magnetic graphene materials instead of the commercial C18 protein-trap column. The number of identified proteins increased from 39 to 123 significantly, which proved its bright future in low abundance protein enrichment application.The fifth chapter is about laser-assisted immobilized enzyme reactor for fast and efficient protein digestion. Limited to the current mass spectrometry technology development, proteins were prior to be digested and then analyzed by mass spectrometry in proteome research. The classic in-solution digestion has some shortages, long hydrolysis time, low digestion efficiency and can’t work with HPLC and MS on line. We have developed a protein digestion monolith based on polyacrylamide substrate and in situ trypsin polymerization. It has good hydrophilicity and permeability. Moreover, it holds a high concentration of fixed trypsin. The standard proteins Myo, BSA and Cyt-c can be digested effectively in 5 min, and this technology has also been applied in human liver proteome research successful. To further improve the hydrolysis efficiency, we use 808 nm near infrared laser irradiation to aid the digestion of immobilized enzyme reactor. When the protein solution flowed through the whole column at a flow rate 0.5 μL/min, the proteolysis was achieved. This truly realized flowing and digesting, which meet the requirements coupling with chromatography and mass spectrometry online.In summary, focused on the CTCs in peripheral blood, we have developed a series of new technologies in our dissertation, which consisted of nitrocellulose membrane chips for the highly efficient capture of CTCs, large-scale SERS imaging for the highly sensitive detection of CTCs, core-shell magnetic graphene composite materials for the enrichment of low abundance proteins and laser-assisted immobilized enzyme reactor for fast digestion of proteins. These achievements can promote the CTCs research forward greatly. Moreover, we are looking forward to a more thorough understanding about the relationship between CTCs and cancer diagnosis and treatment for the health benefit of all mankind. |
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