【作者】 肖方竹；

【导师】 邓健；

【作者基本信息】 南华大学 ， 卫生毒理学， 2008， 硕士

【摘要】 生物芯片（biochip）又称为生物微阵列（biologic microarray）技术,是20世纪90年代中期发展起来的一种具有划时代意义的微量分析技术。它是一门综合了微电子学、生物学、物理学、化学及计算机等学科的高新生物技术。生物芯片研制的第一步是要制备生物芯片载体,在此过程中,需要对载体表面进行化学处理,使之能特异、快速、高效地固定各种探针。载体的品质直接影响微阵列的性能,对芯片的制备至关重要。因此,对该领域的研究与开发,将有助于促进我国生物芯片的自主研发、规模生产和应用。纳米膜基底型生物芯片载体的研究就是在这种背景下提出的。实验选用表面平整的载玻片,先在玻片上组装具有超大比表面积和超强吸附性能的纳米薄膜,再对其进行功能化表面修饰,充分发挥物理吸附和化学偶联的双重优势,制备出表面带有功能基的生物芯片。通过试验优化了制备条件,从而建立了具有实用价值的纳米膜基底型生物芯片载体制备的新方法。1.将洁净的玻片,在以溶胶-凝胶法制备的TiO₂胶体溶液中浸渍-提拉成膜,经室温干燥、高温煅烧后,再进行硅烷化接枝处理,制得氨基化芯片载体。通过傅里叶变换红外光谱（FTIR）、扫描电镜（SEM）、荧光显微镜对样品进行表征。结果表明,带氨基的“手臂分子”可以均匀固定在纳米薄膜组装的玻片上。用模拟探针异硫氰酸荧光素（FITC）测试固载效果,其荧光信号强于仅用硅烷化处理的玻片。实验就加水量、滴加方式及煅烧条件等因素对TiO2膜性能的影响进行了研究。2.以溶胶-凝胶法制备SiO2胶体,浸渍-提拉法将其镀于经洁净处理的玻片表面,形成SiO2纳米膜,经高温煅烧后,对该膜进行硅烷化接枝处理,制成带氨基的生物芯片载体。用FTIR、SEM、原子力显微镜（AFM）等对氨基片进行表征,并分别以FITC及牛血清白蛋白（BSA）作为探针,测试其固载效果。结果表明,SiO2纳米膜基底型氨基片的固载效果较不镀膜仅硅烷化处理的氨基片有显著提高。实验就反应物配比、水解与缩聚反应pH值及反应温度等因素对SiO2纳米膜基底型生物芯片载体性能的影响进行了研究。3.在洁净玻片上组装纳米SiO2薄膜后,用γ-巯丙基三甲氧基硅烷对镀膜玻片表面进行巯基化处理,继而在以Frens法制备的胶体金溶液中浸镀,制得纳米金膜型生物芯片载体。该载体对蛋白质探针的固定效率达到参比载体的12.3倍。实验就试剂用量、反应时间、搅拌强度等因素对载体性能的影响进行了研究。结果表明:对于100 ml 0.01%氯金酸溶液,加入5 ml 1%的柠檬酸钠,在300r·min-1速率的搅拌下100℃反应10 min,可制得粒径为12-15nm的胶体金。在制备载体时,巯基化选用5%的γ-巯丙基三甲氧基硅烷的乙醇溶液为活化液,室温浸镀20min;浸提镀金膜时将巯基化玻片浸入金溶胶中5 min,提拉速度为6 cm/min,可以制得膜质均匀致密的纳米金膜型生物芯片载体。4.溶胶-凝胶法制得TiO2胶体,将洁净玻片以静电自组装（ESA）法在PSS溶液与TiO2胶体溶液中交替循环镀膜,制得ESA复合膜,继而在复合膜上进行硅烷化修饰,制备了接枝氨基的自组装膜基底型芯片载体。测试结果显示,该芯片载体对FITC的固定效率高,荧光信号强,分布的密度及均匀度等都要优于只用硅烷化试剂处理的玻片。TiO2薄膜制备选取酸性（pH=1）条件,可制得耐擦伤强度高和附着力良好的静电自组装多层复合膜;膜的硅烷化修饰选取10% 3-氨丙基三甲氧基硅烷的乙醇溶液中室温反应30 min,可获得最佳固载效果。更多还原

【Abstract】 Based on microelectronics, biology, physics, chemistry and computer sciences, biochip (or biologic microarray), which developed with Human Genome Project (HGP) and as first used in 1990s, marked a new era of micro-analysis technology.The first step of biochip manufacture is to prepare the carrier material. In this process, the glass slide need to be modified so that different probes can be immobilized on the slide surface with fastness, specificness and stability. Chemical modification on the glass slide surface is a key step in biochip fabrication, theoretical and applied research on the modification methods will therefore have great significance for the development of microarray technology.The study of biochip carrier based on nano-membranes has put forward under this background. The glass slides with flat and clean surface are used in these experiments. The surface modification includes nano-membrane assembling and then function-loading on the glass slides. By optimizing experimental conditions, new methods for preparation of biological chip carriers based on different nano-membranes have been established with the advantage of higher chemical stability and superior immobilizing efficency, and could find practical applications in the areas of life science reseach and related industries.1. Cleaned glass slides were dipped into the colloidal TiO2 solution prepared by sol-gel method. After coated with TiO2 nano-membrane, dried at room temperature and calcined at high temperature, the slides were silanized by treating with 3-aminopropyl trimethoxysilane reagent, thus the aminating biochip carriers were prepared and characterized by fourier transform infrared spectrometer (FTIR), scanning electron microscope (SEM) and fluorescence microscope. The results showed that“linkers”with amino groups can be immobilized uniformly on the modified surface. To test the immobilizing efficency, fluorescein isothiocyanate (FITC) was use as a simulating probe, the experimental results showed that the fluorescence intensity of the samples was superior to those slides treated only with silanization.2. Colloidal nano-SiO2 was prepared by the sol-gel method and coated on the surface of cleaned glass slides by a dip-coating technique to form SiO2 nano- membrane. After calcined at high temperature, the slides were silanized by treating with 3-aminopropyl trimethoxysilane in ethanol solution, and the biolochip carriers with amino groups were formed. The sample slides were characterized by FTIR, SEM and atomic force microscopy(AFM). The probe immobilization efficiency of these carriers were tested with FITC and bovine serum albumin (BSA). The results showed that the performance of improved carriers was superior to those carriers treated only with silanization. The influential factors on the performance of biochip carriers based on nano-SiO2 film, including component ratio of the reagents, acidity of hydrolysis and polycondensation, temperture of the reaction system were studied in detail.3. The slides coated with nanostructure SiO2 thin film were silanized byγ-mercaptopropyl trimethoxysilane and then dipped into the colloidal gold prepared by Frens method, thus the biochip carriers based on nano-gold film prepared. The immobilization efficiency of protein probes on these carriers was 11.3 times higher than those treated only with silanization. The influential factors on the performance of nano-gold film including dosage of reagents, reaction time and the stirring rate were studied, and the optimum conditions for preparation of nano-gold-film carriers were established.4. Colloidal TiO2 was prepared by the sol-gel method. Nano-TiO2 and poly(sodium 4-styrenesulfonate) salt (PSS) complex thin films were fabricated on clean slides by the electrostatic self-assembly (ESA) method. The slides with ESA films were then silanized by treating with 3-aminopropyl trimethoxysilane reagent. The results showed that these carriers had high efficiency of immobilizing FITC, and the fluorescence intensity, density and evenness were superior to those slides treated only with silanization. Effective connection in upper/lower layers between TiO2 films at acid conditions (pH=1) and PPS films provides the carrier high performaces in the scratch-resistance and cohesion tests. Silanization on the ESA film in 10% APS solution for 30 minutes makes the sample carriers better at the immoblization of probe.更多还原