本文選題：抗污染　切入點：表面等離子體激元共振　出處：《天津大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：透明質(zhì)酸(hyaluronic acid,HA)是一種天然的兩親性材料,被廣泛應(yīng)用于生物傳感器、航海、醫(yī)學(xué)以及化妝品等領(lǐng)域。本文分別采用共價結(jié)合法和共混/聚合法將HA分子固定于表面等離子共振譜儀SPR的裸金(Au)傳感芯片表面,在芯片表面形成一層抗污染“親水層”。利用原子力顯微鏡、接觸角測量儀等手段表征了抗污染芯片表面特性。采用SPR定量測定了抗污染表面對單一和實際復(fù)雜蛋白質(zhì)體系的非特異性吸附量,并在實際體系中分析了抗原抗體相互作用動力學(xué),考察了該抗污染表面的回收利用。(1)采用共價結(jié)合的方法將HA分子固定到Au芯片表面,獲得具有良好親水性能的HA-Au表面,該表面對單一蛋白以及復(fù)雜體系的蛋白質(zhì)溶液可很好地抵抗非特異性的蛋白吸附。HA改性后,芯片表面從疏水性(102°)變?yōu)閺娪H水性(12°);表面粗糙度從8.36 nm降低到0.4 nm。HA-Au芯片對單一蛋白的吸附量為BSA 7.7 ng/cm2,溶菌酶4.6 ng/cm2;對復(fù)雜的蛋白質(zhì)體系的非特異性吸附為豆?jié){0.67 ng/cm2,橙汁16.1 ng/cm2,牛奶9.8 ng/cm2;(2)在HA修飾的基礎(chǔ)上,采用EDC/NHS法在HA表面連接抗體antiBSA,定量測定了抗體修飾后芯片(anti-BSA/HA-Au)對不同蛋白質(zhì)體系的非特異性吸附量,結(jié)果表明anti-BSA/HA-Au芯片對單一蛋白(BSA、溶菌酶)和復(fù)雜蛋白體系(豆?jié){、10%血清)仍具有很好的抗蛋白吸附性能。同時,HA基質(zhì)還具有較高的識別分子固定量,利用溶菌酶與豆?jié){作為參考蛋白來檢測BSA分子,當(dāng)濃度為15 nM時,對應(yīng)的信噪比分別為94.6和25.8。在15 nM-700 nM濃度范圍內(nèi),anti-BSA與BSA相互作用的響應(yīng)信號與BSA濃度呈線性關(guān)系;且經(jīng)過解離吸附循環(huán)后抗體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,通過共混/聚合法將HA固載到不同基質(zhì)表面(樹脂、聚苯乙烯、玻璃、Au和鋼),即HA和多巴胺單體(DA)共混后,將基底浸入混合液中,聚合形成共混膜PDA/HA。當(dāng)DA/HA比例為1,聚合時間為2 h時,形成的PDA/HA表面具有良好的抗非特異性吸附性能,對單一蛋白(BSA、溶菌酶、纖維蛋白原、β乳球蛋白)和復(fù)雜蛋白體系(豆?jié){、100%血清)均有較低的吸附量。進一步,利用NaOH使PDA發(fā)生解聚的特性,開發(fā)了一種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.
【學(xué)位授予單位】：天津大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：TP212;TB306

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