摘要
自从1985年Tomalia报道聚酰胺-胺型(Polyamidoamine, PAMAM)树枝状大分子以来,由于PAMAM具有精确的分子结构、分子内存在大量的空腔、表面含有大量的官能团以及分子本身具有纳米尺寸等特点,引起了广泛的关注。但是在液态树枝状大分子的合成过程中,由于加入的原料大大过量,而且原料和产物都溶于反应中所使用的溶剂甲醇,以目前比较常用旋转蒸发的方法提纯得到产品纯度不是很高;同时液态也一定程度上限制了树枝状大分子的应用范围。将树枝状大分子固载到适当的载体上,不但获得的产品纯度高,而且分离非常简单,并且拓展了树枝状大分子的应用范围。通过硅胶负载树枝状大分子,得到一类新型的有机/无机杂化材料,它同时兼具了无机材料和树枝状大分子的结构特性,体现了有机和无机的结合。
常规合成PAMAM的方法往往需要花费大量的时间,而微波是近年来得到迅速发展与应用的一门新技术,直接微波加热可以显著地缩短反应时间,而且也能够减少副反应、提高产率、改善反应的重现性。所以本文尝试将微波辅助技术引入硅胶键合PAMAM的反应。在微波辅助下采用发散法,通过重复以下两步反应:(1)丙烯酸甲酯与表面氨基的Michael加成反应;(2)乙二胺同以酯基为端基的产物的酰胺化反应,得到了一系列PAMAM树枝状大分子修饰的硅胶。利用傅立叶红外光谱、酸碱滴定、荧光显微镜、元素分析等方法对产物进行了表征。结果表明,通过微波辅助的方法能够快速有效地合成PAMAM树枝状大分子修饰的硅胶。
因为PAMAM修饰的硅胶表面具有大量的氨基基团,可以和氨基酸、蛋白质产生不同的相互作用,同时,PAMAM具有极佳的生物相容性,因此本文对PAMAM树枝状大分子修饰的硅胶展开在生物固定化方面的研究。
以PAMAM树枝状大分子修饰的硅胶为载体,采用吸附和共价两种方式对牛血清白蛋白(BSA)的固定化。结果表明PAMAM修饰的硅胶对BSA的能力比未修饰的硅胶强,而且吸附量随着PAMAM代数的增加而增加;采用共价的方法PAMAM修饰的硅胶对牛血清白蛋白的固载量随着PAMAM代数的增加而增加。
以PAMAM树枝状大分子修饰的硅胶为载体,采用共价法对氨基酰化酶的进行了固定化。固定化酶是20世纪60年代发展起来的一项新技术,该技术克服了游离酶法稳定性的不足的缺点,提高了酶的储存稳定性,容易实现重复使用及连续自动化生产,降低了成本。结果表明,以PAMAM修饰的硅胶为载体将氨基酰化酶的固定化,酶的固载量和酶活均随着PAMAM代数的增加而增加。
本文将PAMAM修饰的硅胶用于表睾酮单克隆抗体的固定,制备得到的免疫亲和柱,对样品中的表睾酮和睾酮进行免疫亲和萃取。免疫亲和萃取是基于抗体与抗原的高度特异性结合的特点,将目标化合物从复杂的基体中特异性捕获和富集,具有高度灵敏和高特异性的优点。结果表明,制备的免疫亲和柱能够对睾酮和表睾酮特异性捕获,与毛细管电泳联用能够应用于实际尿样的分析检测,建立了快速、高效、高特异性地同时测定复杂体系中睾酮和表睾酮的方法。
Since Tomalia reported dendrimer polyamidoamine (PAMAM) in 1985, great attenation had been paid to PAMAM dendrimer due to its great potential appalication in many fields because of its well-defined structure, hollow cavities deep within the branching structure and high density of functional groups. However, it is difficult to obtain liquid dendrimers with high purity because both of the excessive raw materials and dendrimers are soluble in methanol and rotary evaporation method is not so effective. At the same time the liquid state limit the application of dendrimers. Immobilized dendrimers to the appropriate carrier can obtain dendrimers with high purity and the separation is very simple, meanwhile the application of PAMAM is expanded. Dendrimer-grafted silica is a type of organic-inorganic hybrid material which combins inorganic materials and the fascinating structural features of dendrimers, and it representes the combination of organic and inorganic.
However, the conventional synthesis of PAMAM grafted silica requires a long reaction time, and new technology is needed to accelerate the reaction. Microwave radiation is a new technology rapid development and application in recent years. This technology has the significant advantages of heating reactants and solvents directly with no temperature gradient, fast heating, minimal hot vessel surface, high efficiency, greatly accelerating reactions, and improving reaction yields compared with traditional heating. Microwave technology was adopted for grafting of PAMAM dendrimer onto the surface of silica. The PAMAM was grown from amino groups on the colloidal silica surface by repetition of two steps:(1) Michael addition of methyl acrylate (MA) to amino groups on the surface and (2) amidation of the resulting terminal ester groups with ethylenediamine (EDA). Fluorescence microscopy, elemental analysis (EA), Fourier transform infrared spectroscopy (FTIR) and titrimetry were used to characterize the structure and composition of the dendrimer-grafted silica surface. The results indicated that PAMAM were grafted successfully onto the silica surface with microwave assistance.
PAMAM provides large amount of NH2 which bring different interactions with amino acids and proteins. Besides, the biocompatibility of PAMAM is excellent, so in this paper, the PAMAM grafted silica was apply to the immobilization of biological technology.
Bovine serum albumin (BSA) was used as a model of biological molecule to react with the dendrimer-grafted silica by adsorption and chemical bonding through glutaraldehyde. The results showed that the immobilization efficiency for BSA increased with increasing PAMAM generation.
The PAMAM grafted silica was used to immobilize aminoacylase. Immobilized enzyme technology was developed from the 20th century 60s. The advantages of immobilized enzymes over free enzymes include resuse, enhanced stability, rapid separation of the catalyst from the reaction mixture and reduced operation cost. The results showed that the immobilization efficiency for aminoacylase increased with the generation of PAMAM increasing and the immobilized enzyme remained high activity.
The anti-epitestosterone monoclonal antibodies were immobilized onto the PAMAM grafted silica and prepared an off-line immunoextraction column. The column was employed to immunoaffinity extraction (IAE) of epitestosterone and testosterone from liquid sample. The immunoaffinity extraction is based on the specific interaction between antigen and antibody, therefore the target analytes are specific captured and enrich from complex sample matrices. The main advantages of IAE are the high sensitivity and exquisite selectivity. The results showed that the anti-epitestosterone monoclonal antibodies remained higher activity after immobilization and simultaneous determination of testosterone and epitestosterone in male urine was achieved by the immunoaffinity column coupling with capillary electrophoresis.
引文
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