表面等离子共振传感膜的透明质酸修饰与应用

发布时间：2018-03-18 05:17

本文选题：抗污染　切入点：表面等离子体激元共振　出处：《天津大学》2015年硕士论文　论文类型：学位论文

【摘要】：透明质酸(hyaluronic acid,HA)是一种天然的两亲性材料,被广泛应用于生物传感器、航海、医学以及化妆品等领域。本文分别采用共价结合法和共混/聚合法将HA分子固定于表面等离子共振谱仪SPR的裸金(Au)传感芯片表面,在芯片表面形成一层抗污染“亲水层”。利用原子力显微镜、接触角测量仪等手段表征了抗污染芯片表面特性。采用SPR定量测定了抗污染表面对单一和实际复杂蛋白质体系的非特异性吸附量,并在实际体系中分析了抗原抗体相互作用动力学,考察了该抗污染表面的回收利用。(1)采用共价结合的方法将HA分子固定到Au芯片表面,获得具有良好亲水性能的HA-Au表面,该表面对单一蛋白以及复杂体系的蛋白质溶液可很好地抵抗非特异性的蛋白吸附。HA改性后,芯片表面从疏水性(102°)变为强亲水性(12°);表面粗糙度从8.36 nm降低到0.4 nm。HA-Au芯片对单一蛋白的吸附量为BSA 7.7 ng/cm2,溶菌酶4.6 ng/cm2;对复杂的蛋白质体系的非特异性吸附为豆浆0.67 ng/cm2,橙汁16.1 ng/cm2,牛奶9.8 ng/cm2;(2)在HA修饰的基础上,采用EDC/NHS法在HA表面连接抗体antiBSA,定量测定了抗体修饰后芯片(anti-BSA/HA-Au)对不同蛋白质体系的非特异性吸附量,结果表明anti-BSA/HA-Au芯片对单一蛋白(BSA、溶菌酶)和复杂蛋白体系(豆浆、10%血清)仍具有很好的抗蛋白吸附性能。同时,HA基质还具有较高的识别分子固定量,利用溶菌酶与豆浆作为参考蛋白来检测BSA分子,当浓度为15 nM时,对应的信噪比分别为94.6和25.8。在15 nM-700 nM浓度范围内,anti-BSA与BSA相互作用的响应信号与BSA浓度呈线性关系;且经过解离吸附循环后抗体仍然保持良好的生物活性;(3)利用聚多巴胺(ploydopamine,PDA)的“粘附”性能,通过共混/聚合法将HA固载到不同基质表面(树脂、聚苯乙烯、玻璃、Au和钢),即HA和多巴胺单体(DA)共混后,将基底浸入混合液中,聚合形成共混膜PDA/HA。当DA/HA比例为1,聚合时间为2 h时,形成的PDA/HA表面具有良好的抗非特异性吸附性能,对单一蛋白(BSA、溶菌酶、纤维蛋白原、β乳球蛋白)和复杂蛋白体系(豆浆、100%血清)均有较低的吸附量。进一步,利用NaOH使PDA发生解聚的特性,开发了一种芯片回收再利用的方法,提高芯片的利用率。
[Abstract]:Hyaluronic acid (HA) is a natural amphiphilic material that is widely used in biosensor, navigation, and navigation. In the field of medicine and cosmetics, HA molecules were immobilized on the surface of bare gold au (au) sensor chip of surface plasmon resonance spectrometer (SPR) by covalent binding method and blending / polymerization method, respectively. An anti-pollution "hydrophilic layer" is formed on the surface of the chip. The surface characteristics of antifouling microarray were characterized by contact angle measuring instrument. The nonspecific adsorption amount of antifouling surface on single and actual complex protein system was quantitatively determined by SPR. The antigen-antibody interaction kinetics was analyzed in the actual system. The recovery and utilization of the antifouling surface was investigated. The HA molecule was immobilized onto the au chip surface by covalent binding method, and the HA-Au surface with good hydrophilicity was obtained. The surface of single protein and protein solution of complex system can resist the modification of nonspecific protein adsorption. Ha. The surface of the chip was changed from hydrophobicity to hydrophobicity, the surface roughness was reduced from 8.36nm to 0.4 nm.HA-Au, the adsorption capacity of single protein was BSA 7.7 ng / cm ~ 2, lysozyme 4.6 ng / cm ~ 2, and the nonspecific adsorption of complex protein system was 0.67 ng / cm ~ (2). Orange juice 16.1 ng / cm 2, milk 9.8 ng / cm 2) based on HA modification, AntiBSAs were attached to HA surface by EDC/NHS method. The nonspecific adsorption of anti-BSA-HA-Au) on different protein systems was quantitatively determined by anti-BSA-HA-Au. The results showed that anti-BSA/HA-Au microarray still had good anti-protein adsorption properties for single protein BSA (lysozyme) and complex protein system (soybean milk 10% serum). Using lysozyme and soybean milk as reference proteins, the signal to noise ratio (SNR) of BSA was 94.6 and 25.8 respectively when the concentration was 15 nm. The response signal of anti-BSA interaction with BSA was linearly related to the concentration of BSA in the range of 15 nm. And after dissociation and adsorption cycle, the antibody still maintained good biological activity. By using the "adhesion" property of PDAs, HA was immobilized on different substrate surfaces (resin, polystyrene, polystyrene) by blending / polymerization. Glass-butadiene au and steel (HA and dopamine monomers) were mixed and the substrate was immersed in the mixture solution to form PDA-HA. when the ratio of DA/HA was 1 and the polymerization time was 2 h, the surface of PDA/HA formed had good anti-specific adsorption performance. The adsorption capacity of BSA, lysozyme, fibrinogen, 尾 lactoglobulin and complex protein system (100% serum of soybean milk) was lower. Further, a method of chip recovery and reuse was developed by using NaOH to depolymerize PDA. Improve the utilization rate of the chip.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TP212;TB306

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