本文關(guān)鍵詞： 銀納米顆粒 熒光相關(guān)光譜 熒光增強(qiáng) 均相免疫分析 甲胎蛋白　出處：《上海交通大學(xué)》2010年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 免疫分析是利用抗原與抗體之間高特異性的反應(yīng)實(shí)現(xiàn)對(duì)抗體、抗原或相關(guān)物質(zhì)進(jìn)行檢測(cè)的分析方法。免疫分析是一種非常重要的生物分析方法,它被廣泛地應(yīng)用于臨床診斷,食品和環(huán)境分析,生物及醫(yī)學(xué)研究等領(lǐng)域。目前免疫分析主要以微孔板為實(shí)驗(yàn)平臺(tái)的異相分析模式。這種方法需要包埋、沖洗等多步操作。因此,這種方法操作繁瑣,分析時(shí)間長(zhǎng),不能滿(mǎn)足某些快速檢測(cè)和診斷的要求。近年來(lái),均相免疫分析以其快速便捷的操作和低廉的成本,受到越來(lái)越多的關(guān)注。然而,由于這種方法的靈敏度較低,不能滿(mǎn)足高背景噪音的臨床檢測(cè)要求。 本文旨在利用貴金屬納米顆粒合成生物探針,并結(jié)合熒光相關(guān)光譜,發(fā)展了一種高靈敏的均相免疫分析新方法。主要研究工作包括如下兩個(gè)方面: 1、銀納米顆粒(SNP)具有很強(qiáng)的共振散射光,并對(duì)熒光染料有信號(hào)增強(qiáng)的效果,是性能優(yōu)良的標(biāo)記分子。但是其在均勻性與穩(wěn)定性上的劣勢(shì)阻礙了它的應(yīng)用。本文采用巰基十一烷基酸(MUA)對(duì)銀納米顆粒進(jìn)行了表面修飾,有效地改善了它的均勻性與穩(wěn)定。并通過(guò)控制抗體與銀納米顆粒的物質(zhì)的量比例(2:1與10:1),實(shí)現(xiàn)了銀納米顆粒的生物連接,合成了低抗體-銀納米結(jié)合物與高抗體-銀納米結(jié)合物。同時(shí),本文還利用共振光散射相關(guān)光譜(RLSCS),對(duì)銀納米顆粒及其表面修飾和生物連接過(guò)程進(jìn)行了表征研究,并優(yōu)化了實(shí)驗(yàn)條件。通過(guò)與其他表征結(jié)果的比較,證明RLSCS是一種表征銀納米顆粒的有效的單分子檢測(cè)手段。 2、本文選擇甲胎蛋白抗原(AFP)與其抗體(AFP1A6)作為實(shí)驗(yàn)?zāi)Ｐ瓦M(jìn)行均相免疫研究,并利用Alexa Fluor 647標(biāo)記的AFP作為均相競(jìng)爭(zhēng)免疫的示蹤抗原。低抗體-銀納米結(jié)合物和高抗體-銀納米結(jié)合物分別與抗原進(jìn)行免疫反應(yīng)而成的免疫復(fù)合物,同自由的染料標(biāo)記抗原相比,擴(kuò)散時(shí)間分別增加了60倍(低抗體銀納米探針)與120倍(高抗體銀納米探針)。同時(shí),這些免疫產(chǎn)物的熒光強(qiáng)度分別增加了18倍與49倍,信噪比也增加了10倍與20倍�；阢y納米顆粒在熒光增強(qiáng)與擴(kuò)散時(shí)間增加上的作用,本文結(jié)合雙組分熒光相關(guān)光譜,發(fā)展了納米銀增強(qiáng)熒光相關(guān)光譜均相競(jìng)爭(zhēng)免疫分析方法。在優(yōu)化的條件下,這種方法具有良好的靈敏度,其檢測(cè)限為1.5 pM,線性范圍從6 pM至60 pM(R0.99)。 在人體血清AFP水平的測(cè)試中,檢測(cè)的相對(duì)標(biāo)準(zhǔn)偏差(RSD)約為5%,回收率大于90%。從而實(shí)現(xiàn)了均相免疫在實(shí)體生物樣品中應(yīng)用的目標(biāo)。綜上所述,利用納米銀增強(qiáng)熒光相關(guān)光譜方法進(jìn)行的免疫分析,不僅能夠提高抗原與免疫結(jié)合物的區(qū)分度,而且能夠顯著的增加免疫分析的靈敏度。這一技術(shù)必將在臨床診斷、食品與環(huán)境分析以及生物和生物醫(yī)藥等研究領(lǐng)域展現(xiàn)其巨大的發(fā)展?jié)摿Α?br/>[Abstract]:Immunoassay is a method to detect antibodies, antigens or related substances by using highly specific reactions between antigens and antibodies. Immunoassay is a very important biological analysis method, and it is widely used in clinical diagnosis. Food and environment analysis, biological and medical research and other fields. At present, immunoassay is mainly based on microporous plate as the experimental platform of heterogeneous analysis model. This method needs to be embedded, flushing and other multi-step operation. Therefore, the operation of this method is cumbersome. In recent years, homogeneous immunoassay has attracted more and more attention due to its rapid and convenient operation and low cost. However, the sensitivity of this method is low. Can not meet the high background noise clinical detection requirements. The aim of this paper is to synthesize biological probes using noble metal nanoparticles and develop a highly sensitive homogeneous immunoassay method combined with fluorescence correlation spectroscopy. The main research work includes the following two aspects:. 1. Silver nanoparticles SNPs have strong resonance scattering light and signal enhancement effect on fluorescent dyes. In this paper, silver nanoparticles were modified by mercaptodecyl acid MUAs. The homogeneity and stability of the silver nanoparticles are improved effectively, and the biological connection of silver nanoparticles is realized by controlling the ratio of antibody to substance of silver nanoparticles in the ratio of 2: 1 to 10: 1. The low antibody silver nanoconjugate and the high antibody silver nano bond were synthesized. At the same time, the silver nanoparticles, their surface modification and biological bonding processes were characterized by resonance light scattering correlation spectroscopy (RLS). The experimental conditions were optimized and compared with other characterization results, it was proved that RLSCS is an effective single molecular detection method for the characterization of silver nanoparticles. 2. AFP6 and AFP1A6) were selected as the experimental model to study the homogenous immunity of alpha-fetoprotein antigen (AFP) and its antibody (AFP1A6). AFP labeled by Alexa Fluor 647 was used as a tracer antigen for homogeneous competition immunity. The low antibody silver nanoconjugate and the high antibody silver nanoconjugate were used to react with the antigen respectively. Compared with free dye labeled antigens, the diffusion time was increased by 60 times (low antibody silver nanoprobes) and 120 times (high antibody silver nanoprobes), respectively. Meanwhile, the fluorescence intensity of these immune products increased by 18 and 49 times, respectively. The SNR is also increased by 10 and 20 times. Based on the effect of silver nanoparticles on fluorescence enhancement and diffusion time, two component fluorescence correlation spectra are combined in this paper. A homogeneous competitive immunoassay method for silver enhanced fluorescence correlation spectroscopy has been developed. Under the optimized conditions, this method has a good sensitivity with a detection limit of 1.5 pm and a linear range from 6 pm to 60 pm R0.99. In the test of human serum AFP level, the relative standard deviation (RSD) is about 5, and the recovery rate is more than 90. Thus, the goal of applying homogeneous immunization in solid biological samples is achieved. Immunoassay using silver nanoparticles enhanced fluorescence correlation spectroscopy can not only improve the differentiation of antigens from immune conjugates, but also significantly increase the sensitivity of immunoassay. This technique is bound to be used in clinical diagnosis. Research fields such as food and environment analysis and biology and biomedicine show great potential for development.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R392.1

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本文編號(hào)：1500034