基于氧化石墨烯的SPRi串联免疫信号放大新策略

发布时间：2018-04-03 23:49

本文选题：表面等离子体共振成像　切入点：氧化石墨烯　出处：《西南大学》2015年硕士论文

【摘要】：表面等离子体共振成像(surface plasmon resonance imaging, SPRi)是近年来迅速发展起来的一种新型的光学检测技术,在生物大分子相互作用和免疫检测研究方面有着广泛的应用。与其它检测技术相比,SPRi具有无需标记、实时在线和高检测通量等突出优点。然而在生物分析中,与基于标记的技术如荧光标记法相比,SPRi的检测灵敏度要低3个数量级以上。这是目前SPRi的一个主要的不足,限制了它在不同领域的实际应用。为了解决这一问题,许多研宄者致力于发展各种信号放大方法来提高SPRi的检测灵敏度,如表面引发聚合法、酶催化沉积法以及生物偶合纳米材料法等。虽然这些信号放大方法在一定程度上提高了SPRi免疫检测的灵敏度,但是在实际应用中这些方法存在信号放大的定量关系不好、信号放大易产生假阳性信号等缺点,难以实现在宽浓度范围内灵敏检测目标物并保持良好的检测特异性。本硕士论文针对这一现状,发展了一类连续信号放大方法,通过对信号放大物质的功能化和SPRi传感界面的设计,保证了免疫检测和随后信号放大的特异性,避免了假阳性信号的产生。同时该信号放大方法还具有良好的定量关系,满足了在火的浓度范围内准确定量目标物的要求。作为一种新型的单层碳材料,氧化石墨烯(graphene oxide, GO)具有很多优良的性质。与其他的碳材料相比,GO易于制备,能稳定分散于水中,比表面积大,而且携带大量的含氧官能团如羟基、羧基和环氧基等,易于进行各种生物偶联或化学修饰。本论文将GO作为增强SPRi响应信号的载体和放大材料,通过催化沉积金属银或金,实现SPRi信号的连续放大,实现了灵敏、特异检测人血清中的癌症标记物。具体的研究内容如下：(1)基于氧化石墨烯的催化银沉积的特性,发展了SPRi串联信号放大方法,并实现了在血清中灵敏的免疫检测在本工作中,我们利用GO对金属银的沉积具有催化作用的特点,发展了一种连续的SPRi信号放大方法。通过亲和作用,将GO与抗体偶联得到GO-蛋白质复合物。该复合物与芯片上被捕获的目标物(甲胎蛋白,AFP)相互作用形成三明治免疫结构,实现了SPRi第一次信号放大。随后被结合到芯片表面的GO特异性的催化银离子的还原沉积,产生银纳米粒子,形成第二次SPRi信号放大。通过这一设计,实现了在宽的浓度范围内,高灵敏度和高特异性的检测肿瘤标记物。只采用GO生物偶联物做信号放大剂时,对人血清中的AFP的检测限可达到5.0 ng mL-1。进一步的银催化沉积产生的信号放大,可使检测限达到100 pg mL-1。采用连续的信号放大法,可使动态检测范围在100 pg mL-1～μg mL-1的浓度范围之内。(2)基于聚多巴胺功能化的氧化石墨烯的SPRi双重信号放大在本部分工作中合成了聚多巴胺(polydopamine, PDA)功能化的氧化石墨烯(PDA-rGO)纳米片,作为连续的信号放大材料,并将其应用在人血清中SPRi的免疫检测。在含有GO的弱碱性溶液中,多巴胺在GO表面自发聚合,生成PDA-rGO纳米片。通过蛋白质与聚多巴胺之间的自发反应,进一步制备了抗体与PDA-rGO的偶联物。在免疫检测中,抗体偶联的PDA-rGO通过亲和作用结合到SPRi芯片上,与被捕获的目标物(癌胚抗原,CEA)形成夹心免疫复合物,形成第一次放大信号。再利用聚多巴胺具有的还原性和金属离子结合能力,诱发氯金酸在PDA-rGO自发地还原沉积为金纳米粒子,实现第二次SPRi信号增强。采用连续信号放大方法的SPRi芯片对10%人血清中CEA的检出限可达到500pg mL-1,且动态检测范围可达到四个数量级。
[Abstract]:Surface plasmon resonance imaging (surface plasmon resonance imaging, SPRi) is a new type of optical detection technology has been developed rapidly in recent years, it has wide application in the study of interaction between biological macromolecules and immune detection. Compared with other detection technology, SPRi has no label, real-time and high detection flux prominent in the analysis of biologic advantages. However, compared with the labeling techniques such as fluorescence labeling method based on the sensitivity of SPRi to more than 3 orders of magnitude lower. This is one of the main problems of the current SPRi, limits its practical application in different fields. In order to solve this problem, many researchers devoted to research the development of signal amplification method to improve the detection sensitivity of SPRi, such as surface initiated polymerization, enzyme catalyzed deposition method and biological coupling method. Although these nano materials in a signal amplification method Certain extent improve the sensitivity of SPRi assay, but in the practical application of these methods are the quantitative relationship between signal amplification and signal amplification is not good, easy to produce false positive signals and other shortcomings, it is difficult to achieve in a wide concentration range and sensitive detection of the target and maintain good detection specificity. In this thesis, aiming at the present situation and the development of a continuous signal amplification method, through the design of signal amplification material function and SPRi sensing interface, ensure the immune detection and subsequent signal amplification specificity, to avoid false positive signals. At the same time, the signal amplification method has good quantitative relationship, meet in the concentration range of fire in determining the amount of quasi target requirements. As a new type of single carbon material, graphene oxide (graphene oxide GO) has many excellent properties. Compared with other carbon materials, easy to GO Preparation and dispersion in water, large surface area, and carrying a large number of oxygen-containing functional groups such as hydroxyl, carboxyl and epoxy, easy to carry out a variety of biological coupling or chemical modification. This paper use GO as SPRi signal carrier and amplifying materials enhanced by catalytic deposition of metallic silver or gold, continuous amplification the realization of SPRi signal, the sensitive detection of human serum specific cancer markers. The specific contents are as follows: (1) the catalytic characteristics of silver deposition of graphene oxide based on the development of the SPRi series signal amplification method, and realizes the immune detection in serum and sensitive in this work, the characteristics of we use GO on deposition of metallic silver has the catalytic effect, the development of a continuous SPRi signal amplification method. Through affinity, coupling GO and GO- antibody protein complexes. The complexes were captured with on chip The target (AFP, AFP) interact to form a sandwich immune structure, to achieve the SPRi first. Then signal amplifier is coupled to the deposition of silver ion catalytic chip surface specific GO, produced silver nanoparticles, forming second SPRi signal amplification. Through this design, implemented in a wide concentration range in the detection of tumor markers with high sensitivity and specificity. Only by using GO bio conjugates do signal amplification agent, detection of human serum AFP in the limit can reach 5 ng mL-1. signal silver catalyzed deposition of further amplification, the detection limit of 100 pg mL-1. by continuous signal amplification method can make the dynamic detection range in the concentration range of 100 ~ PG mL-1 g mL-1. (2) SPRi dual signal graphene polydopamine functional amplification in this part in the synthesis of poly (dopamine based on polydopa Mine, PDA) functionalized graphene oxide (PDA-rGO) nano film, as a continuous signal amplification and detection of materials, the application of SPRi in immune serum. In alkaline solution containing GO, dopamine on GO surface spontaneous polymerization, generate PDA-rGO nanosheets. Through the spontaneous reaction between proteins with polydopamine, further preparation of conjugates with PDA-rGO antibodies. In immunoassay, PDA-rGO antibodies by affinity binding to the SPRi chip, and the object is captured (carcinoembryonic antigen, CEA) to form a sandwich immune complexes, the formation of the first amplified signal. Reduction and reuse polydopamine with metal ion binding capacity, chloroauric acid in PDA-rGO induced spontaneous deposition of gold nanoparticles, the realization of the second SPRi signal enhancement. SPRi chip with continuous signal amplification method for 10% human serum detection limit of CEA It can reach 500pg mL-1, and the dynamic detection range can reach four orders of magnitude.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.6;O613.71

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