本文選題：表面等離子體共振成像　切入點：氧化石墨烯　出處：《西南大學》2015年碩士論文

【摘要】：表面等離子體共振成像(surface plasmon resonance imaging, SPRi)是近年來迅速發(fā)展起來的一種新型的光學檢測技術,在生物大分子相互作用和免疫檢測研究方面有著廣泛的應用。與其它檢測技術相比,SPRi具有無需標記、實時在線和高檢測通量等突出優(yōu)點。然而在生物分析中,與基于標記的技術如熒光標記法相比,SPRi的檢測靈敏度要低3個數(shù)量級以上。這是目前SPRi的一個主要的不足,限制了它在不同領域的實際應用。為了解決這一問題,許多研宄者致力于發(fā)展各種信號放大方法來提高SPRi的檢測靈敏度,如表面引發(fā)聚合法、酶催化沉積法以及生物偶合納米材料法等。雖然這些信號放大方法在一定程度上提高了SPRi免疫檢測的靈敏度,但是在實際應用中這些方法存在信號放大的定量關系不好、信號放大易產(chǎn)生假陽性信號等缺點,難以實現(xiàn)在寬濃度范圍內(nèi)靈敏檢測目標物并保持良好的檢測特異性。本碩士論文針對這一現(xiàn)狀,發(fā)展了一類連續(xù)信號放大方法,通過對信號放大物質的功能化和SPRi傳感界面的設計,保證了免疫檢測和隨后信號放大的特異性,避免了假陽性信號的產(chǎn)生。同時該信號放大方法還具有良好的定量關系,滿足了在火的濃度范圍內(nèi)準確定量目標物的要求。作為一種新型的單層碳材料,氧化石墨烯(graphene oxide, GO)具有很多優(yōu)良的性質。與其他的碳材料相比,GO易于制備,能穩(wěn)定分散于水中,比表面積大,而且攜帶大量的含氧官能團如羥基、羧基和環(huán)氧基等,易于進行各種生物偶聯(lián)或化學修飾。本論文將GO作為增強SPRi響應信號的載體和放大材料,通過催化沉積金屬銀或金,實現(xiàn)SPRi信號的連續(xù)放大,實現(xiàn)了靈敏、特異檢測人血清中的癌癥標記物。具體的研究內(nèi)容如下：(1)基于氧化石墨烯的催化銀沉積的特性,發(fā)展了SPRi串聯(lián)信號放大方法,并實現(xiàn)了在血清中靈敏的免疫檢測在本工作中,我們利用GO對金屬銀的沉積具有催化作用的特點,發(fā)展了一種連續(xù)的SPRi信號放大方法。通過親和作用,將GO與抗體偶聯(lián)得到GO-蛋白質復合物。該復合物與芯片上被捕獲的目標物(甲胎蛋白,AFP)相互作用形成三明治免疫結構,實現(xiàn)了SPRi第一次信號放大。隨后被結合到芯片表面的GO特異性的催化銀離子的還原沉積,產(chǎn)生銀納米粒子,形成第二次SPRi信號放大。通過這一設計,實現(xiàn)了在寬的濃度范圍內(nèi),高靈敏度和高特異性的檢測腫瘤標記物。只采用GO生物偶聯(lián)物做信號放大劑時,對人血清中的AFP的檢測限可達到5.0 ng mL-1。進一步的銀催化沉積產(chǎn)生的信號放大,可使檢測限達到100 pg mL-1。采用連續(xù)的信號放大法,可使動態(tài)檢測范圍在100 pg mL-1～μg mL-1的濃度范圍之內(nèi)。(2)基于聚多巴胺功能化的氧化石墨烯的SPRi雙重信號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)納米片,作為連續(xù)的信號放大材料,并將其應用在人血清中SPRi的免疫檢測。在含有GO的弱堿性溶液中,多巴胺在GO表面自發(fā)聚合,生成PDA-rGO納米片。通過蛋白質與聚多巴胺之間的自發(fā)反應,進一步制備了抗體與PDA-rGO的偶聯(lián)物。在免疫檢測中,抗體偶聯(lián)的PDA-rGO通過親和作用結合到SPRi芯片上,與被捕獲的目標物(癌胚抗原,CEA)形成夾心免疫復合物,形成第一次放大信號。再利用聚多巴胺具有的還原性和金屬離子結合能力,誘發(fā)氯金酸在PDA-rGO自發(fā)地還原沉積為金納米粒子,實現(xiàn)第二次SPRi信號增強。采用連續(xù)信號放大方法的SPRi芯片對10%人血清中CEA的檢出限可達到500pg mL-1,且動態(tài)檢測范圍可達到四個數(shù)量級。
[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.

【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R446.6;O613.71

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