Study Of Preparation Of High Sensitivity Polymer-Based Gene Chip Based On Photochemical Surface Modification

In recent years, polymer has very considerable prospects for the manufacture of biochip. It is important that these materials can be used in a variety of techniques and in some cases, the polymer can retain the original performance. Polymer has many advantages such as low cost, robust construction, easy fabrication, rapid prototyping. The technology for preparation of polymer biochip is a real alternative to ordinary glass substrate. At the same time, it can shorten time and reduce material consumption. DNA microarray system is usually formatted micro-DNA probes. DNA chips were favorable for the integration of diagnosis and treatment and the introduction of personalized medicine. The method of biomolecules bonding on surfaces can be divided into three categories: covalently binding, physical adsorption, or affinity binding interaction. Most binding mechanism often depend on N-hydroxysuccinimide (NHS), epoxy resin, an aldehyde, or a carbodiimide, which can be combined with amino groups on biomolecules. In this paper the different methods were used for covalently bonding DNA on polymer surface, which is important to improve the monitoring sensitivity and selectivity of the biochip for specific biological analysis.The following work was carried out based on the above background:1. Construction of DNA microarrays on cyclic olefin copolymer surfaces using confined photocatalytic oxidationDuring recent research on the fabrication of DNA microarrays, polymers have been intensively investigated as substrates for immobilizing oligonucleotides, due to their low cost, disposability and excellent processing flexibility. Among these, cyclic olefin copolymers (COCs) are of special interest because of their many favorable properties, including high glass transition temperature, low auto-fluorescence, optical clarity and resistance to organic solvents. In the present study a novel strategy has been developed by introducing epoxy groups on the COC surface, based on a confined photocatalytic oxidation (CPO) method. Firstly, a sulfate anion (-SO4-) was introduced on to the COC film by CPO, accompanied by brief (120 s) UV irradiation. The sulfate anions were then hydrolyzed to form hydroxyl groups (-OH), forming a glass-like surface, which readily reacts with a silane coupling agent. In the present study (3-glycidoxypropyl) trimethoxysilane was used as a model for introducing epoxy groups on to COCfilm, the result confirmed by X-ray photoelectron spectroscopy (XPS), water contact angle measurement and atomic force microscopy (AFM). DNA probes were subsequently spotted on the COC surface and immobilized by reaction between the epoxy groups and amino groups on strands of DNA. The immobilization efficiency of different concentrations of DNA probes on the COC surface ranged from 45% to 65%, comparable to a traditional epoxy-functionalized glass slide. Hybridization with complementary strands of this microarray was successfully achieved, and the fluorescence intensity after hybridization was readily tuned by adjusting the probe immobilization density, or the target DNA sequence concentration in a hybridization solution. This simple approach has considerable potential in the construction of low-cost polymer biochips.2. Preparation of three dimensional DNA microarrays on EVA surfaceEVA is a thermoplastic elastomer, due to the introduction of VA monomer in molecular chains, exhibits low crystallinity degree, improved flexibility, impact performance, compatibility with filler, has good resistance to environmental stress cracking resistance, high transparency (reduced background intensity) and good heat seal ability, and low temperature resistance and non-toxic characteristics. Isopropyl thioxanthone (ITX) is an initiator which can be covalently bound to the surface of polymer matrix under UV irradiation and isopropyl thioxanthone semi-pinacol (ITXSP) was formed. ITXSP can be used as reactive fragments to introduce vinyl monomers on polymer surface in visible light. Glycidyl methacrylate (GMA) was chosen as the functional monomer introduced on COC surface with epoxy groups. Firstly, PEGDA contamination layer was introduced on EVA surface under visible light and then a secondary functional layer was introduced on EVA surface under visible light. Preparation of DNA microarrays using 3D structure has a very important advantage:high immobilization capacity and more space for the fixed biomolecules. Characterization was carried out by water contact angle measurement, XPS, infrared spectroscopy and atomic force microscopy and biochip scanner. Surface hydrophilicity on EVA surface was improved after the successful introduction of epoxy groups. The surface morphology of EVA after secondary grafting of the mixture of PEGDA and GMA was characterized by atomic force microscopy showing regular reaction column with a certain height, then successful hybridization was carried out by the reaction between DNA probes with amino groups and epoxy groups on reaction column using a scanner. The method lays a solid foundation on biological applications of polymer microarrays.3. Hydrogel drop microchips with immobilized DNA on LDPE surface and hybirdizationCompared to two-dimensional microarray, three-dimensional hydrogel microchips has various advantages such as high capacity, homogeneous water surrounding, absence of immobilized molecules and hydrophobic substrate which increase the stability for probe immobilization. A novel copolymerization was developed for the fabrication of process for three-dimensional gel immobilization microchip. Aminated DNA and acrylamide were used as the unsaturated groups and monomer, respectively. Aminated DNA can bound to the growing polyacrylamide chains, followed by UV irradiation induced copolymerization of the droplets of the mixture on LDPE surface between DNA and acrylamide. The immobilization efficiency was more than 80%> showing that DNA probes was successfully introduced on LDPE surface and the mechanical and thermal properties of the gel immobilization microarray are very stable. This provides a potential approach for further clinical application.At present, the detection of glioma lesion is relatively complex and inaccurate. Hydrogel microchips aquired above was used to detect disease progression.25 specific grading of glioma associated gene were used as probes and the hybridized DNA was extracted from glioma tissues. Primary and level 4 lesion gliomas were used in this study. Firstly, RNA was extracted from glioma tissues, and then reverse transcribed into cDNA, and fluorescently labeled with Cy3 or Cy5 (target genes). Ultraviolet spectrophotometer was used to characterize the successful mark with fluorescent dye on cDNA. cDNA was used as target genes and hybridized with 25 specific DNA probes showing obvious hybridization signals. Finally, we can use different hybridization results and hybridization intensity to determine the unknown giloma lesion in early detection.