| Biochip, which has advantages of miniature, automated, highly parallel and high throughput, has been broadly applied in many fields such as clinical diagnosis, drug screening, personalized medicine and biological engineering research, etc. Since the 1990s, although the preparation and detection technology of biochip have been grown fast, it still faces enormous challenges. For example, its manufacturing process is complicated and costly. Furthmore, the reproducibility of results is still poor, which greatly hinders its commercial apllication. Therefore, solving the existing disadvantages and developing promising biological molecule immobilization techniques is of importance to the advance of microarray technology.In this thesis, on the basis of surface grafting photopolymerization, two new strategies are developed to fabricate three-dimensional gene chip and protein microarray with hierarchical complex structures. DNA probe and protein molecules are fixed on these modified substrates to achieve a high-density immobilization. The application of three-dimensional gene chip for glioma diagnosis is also studied.The main results of this thesis are as follows:1. With commercial glass slide as substrate, double bond is introduced onto its surface by treatment with vinyl trichlorosilane. Under UV irradiation, copolymer of glycidyl methacrylat (GMA) and polyethylene glycol diacrylate (PEGDA) was grafted onto the surface containing double bond to form the three-dimensional crosslinked layer containing epoxy groups. Then, DNA probes labeled with fluorescent molecule Cy3 was immobilized to the substrate surface by the ring-opening reactions between the epoxy groups of PGMA and the amine groups of DNA probes. By changing the quantity of epoxy groups in the three dimensional cross-linked layers, thickness of cross linking layer and the concentration of sample spotting solution, the immobilization efficiency and density of DNA probe can be effectively adjusted. Compared to the existing two-dimensional gene chip, immobilization efficiency of the new three-dimensional chip can increase from about 30% to about 80%. Hybridization results of gene chip showed that DNA probes immobilized in the three dimensional structure can specifically match the target genes.2. Prepration of three-dimensional gene chip for glioma diagnosis. At first, the process for fluorescence labeling glioma sample was designed and time of amplification and labeling, dosage of the template cDNA, random primers and Klenow enzyme were optimized. Then the sequences of DNA probes for diagnosis of glioma were designed. Hybridization results showed that designed probes can successfully hybridize with the labeled sample, and showed good specificity and sensitivity.3. Protein microarray of hierarchical complex pattern on low density polyethylene film (LDPE) was prepared by visible light induced surface grafting polymerization. First, under UV irradiation, isopropyl thioxanthone (ITX) could be photo-reduced by surface H atoms of substrate and then in situ high efficiently coupled with polymer surface to form ITX-semipinacol (ITXSP) groups. The ITXSP could initiate surface living graft polymerization of various vinyl monomers under visible irradiation. By initiating PEGDA monomer polymerization, PEG 3D network structure was fabricated. ATR-FTIR spectra and microscopy images verified that first grafted layer still contains active dormant groups which may initiate regrafting polymerization. AFM images and the height measurements indicated that the growth rate of two parts of second pattern were almost same despite they grew from different substrates (LDPE-ITXSP or first pattern). Based on this method, with the help of photomask, a multiplex 3D protein microarray was fabricated by initiating different monomers. Fluorescence image verified that different protein molecules can be fixed in a specific area respectively. |
