摘要
金纳米粒子以优越的性能在生物分析中起到了重要作用，免疫金标记(IGS：Immuno-Gold Staining Method)是一种目前普遍应用的标记方法。IGS的不足和问题在于胶体金的制备对设备洁净度要求很高，颗粒大小和分散性不易控制，制备很小粒径的金纳米颗粒相对困难，关于蛋白质吸附于胶体金颗粒表面的机理目前有争议。因此，有必要研究一种新的能解决上述问题的金纳米粒子合成体系。本论文以蛋白质作用机理明确的树枝状大分子聚酰胺胺(NH_2-PAMAM)作为模板来合成粒度可控的金纳米粒子，利用在免疫学方面有独特性能的金黄色葡萄球菌蛋白A包覆制备SPA-PAMAM-Au。
论文主要分为三部分：1)2.0代NH_2-PAMAM(G2.0 NH_2-PAMAM)的合成和精制；2)SPA-PAMAM-Au纳米复合物的制备及表征；3)SPA-PAMAM-Au纳米复合物对典型抗原的特异性染色实验研究。
首先，使用发散法合成了PAMAM G2.0，应用色谱法对其进行分析和分离。合成的PAMAM鉴定结果与同行所报导的理论分析及研究结果一致，色谱法分析的结果证实了发散法合成得到的PAMAM具有乙二胺等的报道，分离后得到的PAMAM G2.0纯度较高。
其次，SPA-PAMAM-Au纳米复合物制备试验表明，在NH_2-PAMAM G2.0的水溶液中，运用硼氢化钠还原氯金酸，同时加入足量包覆的SPA，能够很好地制备粒径较小(～3nm)，粒度分布均匀，稳定的，具有良好抗体结合性的SPA-PAMAM-Au纳米复合物。对合成过程中化学行为的系统研究表明，pH值是制备过程的重要影响因素，最小蛋白质包覆用量较合理。复合物光学性质的研究表明，SPA-PAMAM-Au纳米复合物体系激发后均呈现了较强的蓝色荧光发射。这表明SPA-PAMAM-Au纳米复合物具有作为优良异光学标记的特殊性能。
最后，斑点金免疫渗滤法实验结果表明SPA-PAMAM-Au纳米复合物能够对癌胚抗原进行特异性染色，微量的抗原在膜上呈现可辨的红色斑点。荧光测试表明该纳米复合物与微量抗体作用或与抗体结合后再与微量癌胚抗原作用，荧光发生了较大的改变，其在荧光检测方面同样具有重要意义。SPA-PAMAM-Au纳米复合物粒子可望广泛用作生物分析和医学诊断高灵敏度光学探针。
Immuno-gold staining (IGS) which take advantage of the excellent properties of gold nanoparticles has been widely used in bioanalyses, in which some specific protein is used to warp colloidal gold for detecting the protein's target. The problems and shortages of IGS appear gradually along with enlargement of the marking area and requirement in improvement of detecting precision. First, the preparation of colloidal gold particles requires the equipment be very clean and the control of the size and distribution of the very small size of the gold particles is difficult. Second, the mechanism of protein adsorbing on the surface of colloidal gold is not completely elucidated at present. So it is necessary to study some new system by which the gold nanoparticles synthesized can be meet the requirements cited above. Such a system in this paper was chosen NH_2-terminated polyamidoamine (PAMAM), a water-soluble dendrimer which can adsorb on protein and the adsorbing mechanism is well elucidated to make the size of gold nanoparticles controllable. Staphylococcus aureus protein A (SPA) with excellent immunological properties was used to wrap the PAMAM-Au for preparing SPA-PAMAM-Au nanocomposites.
This paper mainly included three parts, i.e., 1) synthesis and purification, and characterization of NH_2-terminated PAMAM; 2) synthesis and characterization of SPA-PAMAM-Au nanocomposites; and 3) staining assay of a typical antigen by SPA -PAMAM-Au nanocomposites. The main results are as following.
Firstly, generation 2.0 PAMAM (G2.0 PAMAM) was synthesized in a relatively simple procedure. Structural characterization showed that the G2.0 PAMAM and its precursors self-synthesized have the same basic structural characters to the data reported. Chromatographic analyses showed some unnecessary components existing in the G2.0 PAMAM synthesized, which yet can basically be purified by further column chromatographic separation.
Secondly, the SPA-PAMAM-Au nanocomposites were synthesized by simply adding SPA into the PAMAM ( G2.0) aqueous solution where reducing HAuCl_4 into Au~0 with NaBH_4 at the same time. Characterization of the nanocomposites showed that the nanocomposites have a typical size of about 3nm and very narrow size-distribution, and were water-soluble and able to combine well with some specific antibody. The study on the solution chemistry of the formation process of the nanocomposites showed that pH is an important influencing factor, the least amount of protein to wrap PAMAM-Au is acceptable in terms of both the sensitivity and cost of the nanocomposites system. The study of optical properties of the nanocomposites showed that SPA-PAMAM-Au nanocomposites system emitted obvious bluish luminescence after excited. These specific optical properties make the nanocomposites an prosperous potential in being as an excellent optical marker.
Finally, the application of the SPA-PAMAM-Au nanocomposites in Dot Immuno Gold Filtration Assay (DIGFA) to stain Carcinoembryonic antigen showed that the nanocomposites could be detect supersensitively carcinoembryonic antigen. We find this probe is also significant on fluorescence detecting because there is a very apparent change in fluorescence spectra after SPA-PAMAM-Au nanocomposites bind a little of CEA antibody or CEA antibody plus CEA. This suggests that SPA-PAMAM-Au nanocomposites could be used as supersensitive optical probe for bioanalyses and medical diagnoses.
引文
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