【作者】 虞伟；

【导师】 顾宁；

【作者基本信息】 东南大学 ， 生物医学工程， 2003， 博士

【摘要】 生物芯片是通过平面微细加工与生物分子的自组装技术相结合,在固体基片上组装成数目众多的不同生物分子微阵列,以实现对目标DNA、RNA或蛋白质分子信息的大规模检测。这种技术被认为是生命科学研究中的高效工具,因而成为目前国内外科学技术发展的热点。蛋白质免疫检测型芯片有别于一般的DNA芯片检测技术,其原理主要是基于抗原与抗体的特异结合反应来设计的,所测目的分子仅有结构上的专一性,而无序列特异性。本文在分离制备多种生物大分子的基础上,采用分子组装技术,探索出一种新型的适用于不同生物分子偶联的固相载体,即经氧化与化学修饰处理的琼脂糖凝胶基片,成功地在其表面制作了抗原与抗体分子微阵列,建立了3种不同免疫检测模式,包括直接模式、间接模式和双抗体夹心模式,可敏感特异检出抗原、抗体分子及酶与底物之间的相互作用;同时采用辣根过氧化物酶-二氨基联苯胺-双氧水（HRP-DAB-H₂O₂）、胶体金-硝酸银-对苯二酚和荧光素3种不同示踪剂作为蛋白质与抗原分子微阵列的信号显示系统,均取得了较好的效果。经初步对临床疾病标本的研究发现,本法制作的蛋白质与抗原分子微阵列用于自身抗体的检测表现出较好的方法学稳定性和重复性,与ELISA测定结果有较好的一致性,并具有操作简便、结果特异、实验条件易于控制、所需样本和试剂用量小等特点,对于实现高通量的生物分子检测具有重要的意义。 本论文的主要研究工作 1.利用生物化学、免疫学和分子生物学技术,具体的实施方法包括用饱和硫酸胺盐析、差速离心、凝胶色谱分析（分子筛、硅胶柱层析和亲和色谱）、薄层层析技术、聚丙烯酰胺凝胶电泳和琼脂糖凝胶电泳对待分离的组分进行分离纯化。制备了10余种不同的生物大分子抗原探针（包括蛋白质、DNA和磷脂分子等）,同时就有关自身抗体的检测方法、生物大分子抗原（主要为蛋白质）的一般理化特性、分离纯化原则和基本的实验技术进行了讨论。这些自身抗原分别是甲状腺球蛋白（TG）、甲状腺过氧化物酶（TPO）、肌红蛋白（Mb）、肝特异性细胞膜脂蛋白（LSP）、ENA抗原、精子膜抗原（SmAg）、热聚合兔IgG（AgRIgG）、子宫内膜抗原（EmAg）、人卵巢抗原（O-Ag）,心磷脂,dsDNA和ssDNA更多还原

【Abstract】 The slide-supported biochip is a microarray format of the assembly of various biological compounds. The field of microarray has attracted much interest with the developing techniques, such as microelectronic processing and molecular assembly. This novel and versatile array-technique allows high-throughput detection for DNA、 RNA and proteins within a single experiment. The preparation of chip for immunoassay of protein is more different than that of DNA. The principle of assay on protein chip is based on the interaction of antigen-antibody or partner-receptor. The target-molecules detected by protein array are only structure-specific but not sequence-specific. In the current study, the biomolecule of more than ten antigens were prepared, and a novel solid support, namely oxidized agarose-gel substrate with modification by glutaraldehyde (GA) was suitable for molecules immobilization. The antigen and antibody microarrays were subsequently fabricated on the solid support. Three kinds of experimental set-ups including direct, indirect and double-antibody sandwich immunoassays were also developed. Results showed that the techniques are able to detect responses of antigen-antibody and interactions of enzyme-substrate with high sensitivity and specificity. In the immunoassays, 3 different symbols, i.e. horseradish peroxidase (HRP), colloid gold (CG) and fluorescein (Cy3) were served as array tracer system. Satisfactory results of measurement were obtained. The detection for autoantibodies in serum by this microarray provided good stability, reproducibility, and good consistency with the result of ELISA. The main advantages of the microarray technology have been listed as follows: highly economical reagents and saving specimens are used, assay operation is easy to be performed, and experimental conditions are precisely controlled. It is very important that this approach can achieve large-scale assays for biomolecules.The major contributions are as followsSeveral biological methods, such as biochemical, immunological and molecule biological technologies, were utilized to prepare biological compounds. The concrete experiments performed in the course of purification contained the approaches of salt-out of saturated ammonium sulphate, differential speed centrifugation, gel chromatography (namely gel filtration, silica-gel chromatography and affinity chromatography), thin-layer chromatography, SDS-PAGE and agarose gel electrophoresis, etc. Furthermore, the assays for autoantibodis,the physical and chemical properties of the corresponding antigens (most of them were proteins), and the principles of purification approaches were discussed in detail. More than ten of auto antigens (include proteins, DNA and phospholipid etc.) were prepared respectively, such as thyroglobulin (TG), thyroid peroxidase (TPO), human myoglobin (Mb), liver specific cell membrane lipoprotein (LSP), extractable nuclear antigen (ENA), spermatozoa antigen (SmAg), heat aggregation rabbit IgG (HAgRIgG), endometrial antigen (EmAg), ovarian antigen (O-Ag), cardiolipin, double stranded DNA (dsDNA) and single stranded DNA (ssDNA) etc. Generally, these antigens could be served as immobilized probes and utilized for detection of corresponding autoantibodies. These results provided essential experimental materials for subsequent array preparation.To investigate the interactions of enzyme-substrate and antigen-antibody, the surface of 5 types of solid supports, including oxidized agarose-gel, APTES-GA, APTES, PLL-GA and PLL were successfully prepared. HRP and human IgG with different concentrations and status were subsequently immobilized and arrayed on these surfaces respectively, and the fix-effect of protein was further investigated. Results showed that immobilization of biomolecules were highly related to their characteristics, including molecular kinds, structures, molecular weight, and surface electric charge etc., and was directly associated with sample buffer with different pH. Among them, only the conditions i.e. oxidized agarose-gel film and carbonate buffer solution (0.05mol/L, pH 9.6) used for protein immobilization provide the optimal effect on measurement. AFM characterization demonstrated that the gel assembly film was a support of three-dimensional surface, and had a higher capacity for protein loading and linking than that of 2-dimensional surfaces (such as PLL/GA and APTES/GA modified slides). This gel surface will probably become a novel potential solid support adapted to other kinds of biomolecules immobilization.For the sake of evaluation and search after agarose-gel support, 3 kinds of gel surfaces, including agarose-gel assembly film prepared on surface of APTES-modified slides (surface-1), followed by oxidation with NaIC>4 (surface-2) and cross linking with glutaraldehyde (surface-3), were generated respectively. The performances of these gel assembly films were further investigated, such as the efficiency of HRP immobilization, the sensitivity of solid-phase molecules measurement and the stability of microarray in employment. Meanwhile, 3 kinds of techniques i.e., enzyme immunoassay (EIA), atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) were utilized to characterize and analyze the surface group modification, surface responsive characteristics as well as the process of protein immobilization. Results presented that reactive aldehyde groups were successfully induced onto the surface of gel-substrate, and surface-3 had an optimal performance of protein immobilization for measurements. There was no significant difference of influence on this gel-microarray in employment among different temperatures (i.e. 37°C, 25 "C and 4°C). The results of characterization for the surface provided direct evidences that proteins immobilized on gel surface could be clearly observed and Nls peak could be also detected after proteins attached. This investigation illustrated the mechanisms of which biomolecules could be firmly immobilized on gel-film. Therefore, it was important that surface derived active aldehyde groups and 3-dimensional structure were the main reasons forimprovement of biomolecules immobilization.Autoantigen or antibody microarrays were produced by attaching variety of proteins, peptides, DNA, cardiolipin or antibody to the surface of derived agarose-gel film (surface-3). Several patterns of enzyme immunoassays (EIA) based on this arrayed agarose-gel support were also successfully developed, such as direct immunoassay, indirect immunoassay and double antibody sandwich immunoassay, which were suitable for specific partner molecules detection, including the interactions of antigen-antibody and of enzyme-substrate. The arrays were incubated with serum samples, and resolvable HRP was utilized as a tagged molecule, which could perform qualitative and quantitative analysis for samples. In this section, autoantibodies were brought into arrayed system, it represented the first report of application of this technology in various human diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and mixed connective tissue disease (MCTD). The results indicated that higher prevalence of anti-ssDNA, anti-TG, anti-CaM and RF were found in these diseases, and the profile of autoantibodies in diversified diseases presented different combined characteristics. Also, this investigation had a good consistency with the result of ELISA technique, and laid a reliable foundation for large-scale and parallel autoantibodies analysis.Five types of colloidal gold (CG) nanoparticles with diameter ranging from 5nm to 50nm were synthesized by changing dosage of tannin and HAuCU. The proteins including GAH(goat anti-human)-IgG, BSA, SPA and rhCaM were successfully coated on the surface respectively. Meanwhile, agarose gel electrophoresis, isoelectrofocusing and capillary electrophoresis (CE) techniques were utilized to investigate electrophoretic behavior of particles. In brief, lOnm of CG capped with GAH-IgG was served as a probe and tracer, and the amplification of immunogold signal was achieved by AgNO3-hydroquinone. This silver enhancement technique (i.e., immunogold silver staining, IGSS) was firstly used for microarray detection based on reaction of antigen-antibody. In comparison with HRP and fluorescein (Cy3) labels used for microarray, IGSS had a similar or higher sensitivity. For example, the lower limitation of solid-phase IgG was 0.5 ng (direct immunoassay), and serum IgG detected by IGSS was at least 10 multiples higher than that of EIA. In 14 cases with HCV infection, the prevalence of positive autoantibodies was higher than that of EIA (38.6% vs 31.4%, respectively). The mean coefficient variation (cv) was of 7.14% by IGSS technique. Therefore, it is considered that a good reproducibility was achieved for autoantibodies based on this antigen microarray, and the results provided a new kind of experimental pattern for development of visible array technique.In conclusion, the significance of this investigation is as following: (1) The assembly gel film, i.e., oxidized agarose-gel surface followed by modification of GA was successfully prepared, and its surface group modification, surface responsive characteristics as well as the process of protein immobilization was detected and characterized systemial ly. (2) It is firstly reported that a novel gel assembly film was suitable for various kinds of molecules immobilization simultaneously, including proteins, small peptides, DNA, phospholipid and antibodies, etc. Therefore, it is an ideal solid support used for microarray preparation. (3) 3patterns of immunoassays based on arrayed agarose-gel substrate were successfully developed;which included direct;indirect and double-antibody sandwich immunoassays. (4) It is firstly reported that migration characteristic of CG nanoparticles and subsequently capped with proteins was investigated by using electrophoresis techniques under an applied electric field. (5) Autoantibodies were brought into arrayed detective system. As compared with other tracers;immunogold probe and silver staining amplification technique used for microarray measurement had a higher sensitivity. This application is also the first report of such technology for screening autoantibodies.更多还原