本文關(guān)鍵詞： 核酸適配體 磁性微球 化學(xué)發(fā)光 凝血酶 96孔板 聚苯乙烯微球 放大　出處：《復(fù)旦大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 核酸適配體是近年來發(fā)展起來的一類SELEX篩選出的單鏈寡核苷酸序列,能高效、特異性地結(jié)合各種生物目標(biāo)分子,故它的出現(xiàn)為化學(xué)生物學(xué)界和生物醫(yī)學(xué)界提供了一種新的高效快速識(shí)別的研究平臺(tái)。目前生物分子檢測通常采用抗原抗體特異性識(shí)別模式,但由于受到抗體自身穩(wěn)定性、制備時(shí)間較長、對(duì)環(huán)境敏感等因素的影響,在一定程度上限制了抗體檢測技術(shù)的廣泛應(yīng)用。相比之下,核酸適配體自身穩(wěn)定性好、目標(biāo)分子范圍廣、特異性和親和性高、化學(xué)合成相對(duì)簡單、快速、容易獲得、易于修飾與標(biāo)記,且在生物傳感器設(shè)計(jì)中應(yīng)用靈活等優(yōu)點(diǎn),近幾年在生物分析檢測方面?zhèn)涫荜P(guān)注。目前已經(jīng)成為臨床診斷、環(huán)境監(jiān)測、生物醫(yī)學(xué)、藥學(xué)研究等許多領(lǐng)域中的研究熱點(diǎn)。 化學(xué)發(fā)光(CL)分析法具有不需光源,避免了雜散光的干擾,儀器設(shè)備簡單、操作簡便,具有極高的靈敏度,較寬的檢測范圍,可實(shí)現(xiàn)全自動(dòng)化等特點(diǎn),正逐漸成為分析檢測中極為有用的工具。隨著與其它學(xué)科的交叉研究和應(yīng)用領(lǐng)域的擴(kuò)展,目前已成功地應(yīng)用在藥學(xué)、生物學(xué)、分子生物學(xué)、臨床醫(yī)學(xué)和環(huán)境學(xué)等諸多領(lǐng)域。在本論文中,我們采用化學(xué)發(fā)光分析法,利用核酸適配體對(duì)目標(biāo)分子凝血酶的高分辨識(shí)別,發(fā)展了兩種具有創(chuàng)新意義的凝血酶化學(xué)發(fā)光適配體生物傳感器,由以下三部分組成： 第一章簡要概述了核酸適配體的制備、特點(diǎn)和優(yōu)勢,及其在相關(guān)檢測領(lǐng)域的應(yīng)用。第二節(jié)中著重介紹了基于核酸適配體的凝血酶生物傳感器的研究進(jìn)展,包括電化學(xué)、光學(xué)、色譜學(xué)和其它檢測方法,并列舉了多種檢測模式和檢測技術(shù)在近幾年的部分典型示例,最后簡單介紹了化學(xué)發(fā)光檢測技術(shù)基礎(chǔ)知識(shí)以及本課題的主要研究內(nèi)容。 第二章基于凝血酶的兩條核酸適配體與凝血酶的高親和力,構(gòu)建了三明治結(jié)構(gòu)、磁性分離的新型化學(xué)發(fā)光生物傳感器,可檢測凝血酶的線性范圍為0.5-100 nM (I=651.8C+1092.1,R2=0.9850),最低可檢測濃度為0.01 nM。該技術(shù)采用磁性微球作為生物富集分離載體,具有操作簡單、快速、靈敏度高的特點(diǎn)。 第三章以96孔板作為載體,基于核酸適配體對(duì)凝血酶的高分辨識(shí)別,構(gòu)建了三明治結(jié)構(gòu)的高通量化學(xué)發(fā)光生物傳感器,并進(jìn)而以490 nin的聚苯乙烯微球作為放大載體,構(gòu)建了一種超高靈敏度的凝血酶CL檢測平臺(tái)。不放大體系中凝血酶的線性范圍為0.9375-30 nM(I=6330.2C+1717.3,R2=0.99035),最低檢測濃度為0.46 nM；放大體系中凝血酶的線性范圍為7.8-250 pM(I=8711.5C-79898.1,R2=0.9937),最低檢測濃度為3.9 pM,檢測靈敏度提高了100倍以上。
[Abstract]:Nucleic acid aptamers are a class of single-stranded oligonucleotide sequences screened by SELEX in recent years, which can bind various biological target molecules efficiently and specifically. Therefore, it provides a new research platform for chemical biology and biomedical research. At present, antigen-specific recognition pattern is usually used in biomolecular detection. However, due to the autostability of antibody, long preparation time, sensitive to the environment and other factors, to a certain extent, the wide application of antibody detection technology is limited. In contrast, the aptamer of nucleic acid has good stability. It has many advantages such as wide range of target molecules, high specificity and affinity, relatively simple, rapid, easy to obtain, easy to modify and label, and flexible in biosensor design. In recent years, it has attracted much attention in many fields, such as clinical diagnosis, environmental monitoring, biomedicine, pharmaceutical research and so on. Chemiluminescence CLC analysis has the advantages of no light source, no stray light interference, simple instrument and equipment, simple operation, high sensitivity, wide detection range, full automation and so on. With the development of cross-research and application fields with other disciplines, it has been successfully applied in pharmacy, biology and molecular biology. In this paper, we use chemiluminescence analysis to identify the target molecule thrombin by nucleic acid aptamers. Two novel chemiluminescence biosensors for thrombin have been developed. The biosensors are composed of the following three parts: In the first chapter, the preparation, characteristics and advantages of nucleic acid aptamers and their applications in the field of detection are briefly reviewed. In the second section, the research progress of thrombin biosensors based on nucleic acid aptamers is introduced. It includes electrochemical, optical, chromatographic and other detection methods, and enumerates a number of typical examples of detection patterns and techniques in recent years. Finally, the basic knowledge of chemiluminescence detection technology and the main research contents of this subject are briefly introduced. Based on the high affinity between two nucleic acid aptamers of thrombin and thrombin, a new chemiluminescence biosensor with sandwich structure and magnetic separation was constructed in the second chapter. The linear range of detectable thrombin was 0.5-100nM Igna 651.8C1092.1R2O0.9850). The lowest detectable concentration is 0.01 nm. This technique uses magnetic microspheres as the carrier of bioconcentration separation, and has the advantages of simple operation, fast operation and high sensitivity. In chapter 3, a sandwich structure high throughput chemiluminescence biosensor was constructed based on the high resolution recognition of thrombin by nucleic acid aptamers. And then use 490 nin polystyrene microspheres as the amplification carrier. A high sensitivity detection platform for thrombin CL was constructed. The linear range of thrombin in unamplified system was 0.9375-30 nM(I=6330.2C 17.3. The lowest detection concentration was 0.46 nm. The linear range of thrombin in the amplified system was 7.8-250 pMU 8711.5C-79898.1C, R2O 0.99370.The lowest detection concentration was 3.9 pm. The sensitivity of detection was increased by more than 100 times.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R341

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