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1、硕士学位论文 II 摘 要 由于压电免疫传感器的操作简便、灵敏度高、价廉、无污染等特点，使其在生物学和环境分析等领域逐步得到了应用。但是如何增加抗体或抗原的吸附量，提高传感器的灵敏度和重现性以及降低传感界面的非特异性吸附，使压电免疫传感器能够在临床检测方面得到应用，仍然是当前研究的热点。本文基于实验室先前的工作，结合纳米金混合自组装膜、电聚合膜和超支化化合物，提出了生物分子新的固定化方法，并以此构建了几种新型的压电免疫传感器，具体内容如下： (1)研究结果表明在纳米金表面自组装巯基混合膜，不仅能够提高生物分子的固定量，而且能很好地抵制非特异性吸附。但是，一般在纳米金表面组装的是长链巯基化合物，
2、当长链巯基化合物在纳米金表面组装的时候可能发生弯曲、缠绕的现象；而且整个过程是通过层层自组装完成的，这些都会影响生物活性组分的固定。据此，本文提出了先将纳米粒子表面组装一层短链巯基化合物的混合膜(半胱胺，巯基已醇)，再将被混合膜修饰了的纳米金粒子组装到被巯基丙酸修饰的晶振金电极上(用 N-乙基-N-(3-二甲氨基丙基)碳二亚胺 EDC、N-羟基琥珀酰亚胺NHS 活化羧基)的新方法，基于此方法研制了甲状腺素 T4 压电免疫传感器(第 2章)。 (2) 提出了一种结合酪胺电聚合膜和纳米金为界面的甲状腺激素压电免疫传感器(第 3 章)。先在晶振金电极表面电聚合一层酪胺聚合膜，然后通过聚酪胺一端的氨基
3、和纳米金间的作用力在聚合膜表面自组装一层纳米金，再在纳米金上固定 T4 抗体，研制的传感器实现了对 T4 的检测。考察了一些实验条件，如免疫反应时间，抗体浓度等对传感器响应性能的影响。经过测定该传感器对 T4 具有很好的线性响应，并将此传感器与采用戊二醛交联法和半胱胺吸附界面的传感器进行了比较。 (3) 通过超支化化合物(HB)和壳聚糖体系实现金标抗体在石英晶振上的固定，制得的压电型免疫传感器，用于人 IgG(hIgG) 的检测(第 4 章)。首先在晶振表面上自组装一层半胱胺，再将被活化了的超支化化合物(HB)通过化学键的键合作用连接到晶振表面， 由于它的三维立体结构，得到一带有大量负电荷晶振
4、表面。通过静电作用将壳聚糖分子连接到晶振上用于固定纳米金标记的抗体，实现对hIgG 的检测。结果表明，所制备的传感器操作简便，选择性好，频率响应大。 基于混合自组装和超支化化合物膜的压电免疫传感器 III(4) 将超支化合物(HB)和壳聚糖在 EDC 和 NHS 活化下形成混合液。将石英晶体金电极浸入 HB 和壳聚糖的混合液中进行自组装，使在晶振表面形成一带有氨基和羧基基团的传感界面，通过静电吸附作用吸附蛋白 A 分子从而实现抗体分子的定向固定化。所制备的压电免疫传感器具有操作简便，响应信号大，选择性好等特点(第 5 章)。 关键词：压电免疫传感器；甲状腺素 T4；超支化化合物 HB；混合自组
5、装膜；纳米金颗粒 硕士学位论文 IV Abstract Recently, the piezoelectric immunosensors have been applied in the biological and environment analytes areas with simple-operation, high-sensitivity, low-cost and non-pollution etc. But the research point still is how to make them to be applied in clinical detection by im
6、proving the immobilization and lowing the non-specific absorption. In this paper, new immobilized methods and immunosensors were developed based on the lab previous research. The detail contents are as follows: (1) Mixed self-assembled monolayer (SAM) modified on the surface of Au nanoparticles can
7、resist nonspecific adsorption and enhance the immobilization amount of the antigens. The thiols forming mixed self-assembled monolayer are long-chain and the strategy includes layer-by-layer scheme. But the long-chain thiols maybe curve round Au nanoparticles and therefore decrease the immobilizatio
8、n amount of the antigens and produce nonspecific adsorption. In the meantime, it requires multiple preparation steps during which possible corruption of the film may occur via the repetitive immersion and rinsing process. In this paper (Chaper 2), the short-chain thiols (Cysteamine and 6-mercapto-1-
9、haxanol) were self-assembled onto Au nanoparticles to form the mixed SAM-capped Au nanoparticles which were further covalently bound on the 3-Mercaptopropionic acid (MPA)-modified gold electrode. Based on the strategy, a novel piezoelectric immunosensor for the detection of thyroid hormone T4 was de
10、veloped. (2) A new quartz crystal microbalance (QCM) immunosensor based on self-assembling gold nanoparticles to the electropolymerized polytyramine (Pty) film was developed for the detection of thyroxine (T4) (in Chaper 3). A Pty film was firstly electropolymerized on the surface of the QCM; subseq
11、uently a self-assembled nano-gold layer was applied on the Pty film in order to immobilized anti-T4 antibodies; finally through the immunoreaction of anti-T4 antibodies with T4, the detection of T4 was realized. A series of experimental conditions were investigated including immunoreaction time, con
12、centration of anti-T4 antibody etc. Comparing to the glutaraldehyde binding and Cysteamine methods, the self-assembling gold nanoparticles showed larger frequency response and less non-specific adsorption. (3) A new QCM immunosensor based on cysteamine self-assembled monolayer and Hyperbranched poly
13、mer (HB)/chitosan adsorption procedure has been developed for the detection of hIgG (in Chaper 4). Cysteamine self-assembled monolayer firstly 基于混合自组装和超支化化合物膜的压电免疫传感器 Vwas applied to the surface of the QCM, and a strong negatively-charged HB monolayer, which had been actived with 1-Ethyl-3-(3-dimeth
14、ylamino-propyl) carbodiimide hydrochloricde (EDC), and N-hydroxysuccinimide (NHS) previously, was attached on the cysteamine by the strong force between -NH2 and -COOH. The positively-charged chitosan was then electrostatically posed on the crystal in order to immobilized Au-(goat anti-hIgG antibodi
15、es). Finally through the immunoreaction of goat anti-hIgG antibodies with hIgG, the detection of hIgG was realized. A series of experimental conditions were optimized in detail. The immunosensor fabricated with proposed method can offer large frequency response, good selectivity etc. (4) In this pap
16、er, a QCM immunosensor based on a novel biosensing interfacial by the absorption of self-assembled HB/Chitosan mixed-SAM monolayer and protein A has been developed for the determination of IgG (in Chaper 5). With the activation of EDC/NHS, the positively-charged chitosan and negatively-charged HB were linked each other by the strong force between -NH2 and -COOH and formed the mixture of HB and chitosan. HB/Chitosan mixed-SAM monolayer was first applied to the gold electrode surface by dippin
