基于核酸适配体的生物传感器的研究
摘要
蛋白质是生物体内含量最高,功能最重要的生物大分子。常规的蛋白质检测方法一般是基于传统的抗原-抗体之间的免疫反应进行的,由于抗原和抗体之间的特异性反应,免疫传感器具有很好的专一性和选择性。然而,近年来以适配体作为识别元件的蛋白质检测技术越来越受到人们的关注。适配体(aptamer)一词来源于拉丁语aptus,即to fit,适合的意思。核酸适配体是一类通过SELEX技术体外筛选而获得的,对蛋白质有很好的识别能力,核酸适配体对蛋白质的亲和力和特异性可与蛋白质的抗体相媲美,解离常数通常在纳摩尔到皮摩尔之间,且与抗体相比具有许多优越性.如核酸适配体是体外人工化学合成,合成简单,稳定性、重现性好,容易修饰,可以按需要对序列中的核苷酸定点标记各种官能团和报告基团,如荧光基团,各种酶,电活性物质等。便于核酸适配体的固定化和信号的检测,检测目标广泛等。本文基于核酸适配体的上述优点,在传统的免疫传感器的基础上,构建了一系列适配体生物传感器。具体内容如下:
(1)在第二章中报道了一种无标记电化学方法来检测凝血酶。在这个工作中,我们引进了一种新的夹心电化学方法,采用抗体作为捕获探针,适配体作为检测探针,亚甲基兰作为电活性物质嵌入适配体中来获取电信号,不需要对探针进行任何修饰,传感器对凝血酶的线性响应范围是1-60nM,检测下限是0.5nM。(2)在第二部分的夹心模式的基础上,在第三章中,报道了一种基于适配体的酶放大方法来检测免疫球蛋白E(IgE)。生物素化抗IgE适配体作为检测探针,亲和素标记的碱性磷酸酯酶与电极表面的适配体上的生物素相连,碱性磷酸酯酶催化抗坏血酸磷酸酯水解成强还原剂抗坏血酸,将溶液中的银离子还原成银单质,沉积到电极表面。沉积的银的量与电极表面固定的IgE成正比,用溶出伏安法来定量。(3)在第四章中报道了一种基于目标物诱导适配体置换技术的无标记电化学检测方法,用来检测腺苷。利用适配体与目标物的强亲和力,将电极表面与捕获探针杂交的互补链置换下来,使嵌入在双链中的电活性物质亚甲基兰量减少,从而使得电信号发生变化。(4)在第五章里,我们用一种简单的荧光方法来检测IgE,通过荧光基团标记的适配体与目标物先反应一段时间,再将淬灭基团标记的适配体的互补链(QDNA)与之反应,检测反应前后的荧光强度,通过强度的减弱程度来对IgE定量。此方法检测蛋白质的线性范围宽,下限低。
Protein is the most abundant and important macromolecule in our body. Conventional methods for detecting protein are based on traditional immunoassay. Because the reactions of antigen-antibody are highly specific, immunosensors possess great sensitivity and selectivity. However, recent decades have seen the increasing interest in developing the method of detecting protein based on aptamer. The word, aptamer, comes from Latin aptus, to fit. Aptamers are selected from SELEX (systematic evolution of ligands by exponential enrichment) and have good recognition ability to proteins. The affinity and specificity between aptamers and protein is as well as those between antibodies and antigens. The dissociation constant of these is 10-9~10-12. Comparing to antibodies, aptamers own great advantages, including easy synthesis, good stability, ease modification and so on. It is facile to label the nucleic acid with different kinds of functional and report groups, such as fluorescence and electroactive groups. Therefore, it facilitates the immobilization of nucleic aptamer. In this article, we created a series of biosensors based on aptamers. The details are as following:
(1) In the second chapter, we report a label-free electrochemical method to detect thrombin. In this work, we introduced a new kind of sandwich method to detect protein, utilizing antibody as the capturing probe, aptamer as the detecting probe and methylene blue as the electroactive substance intercalating in the nucleic acid, avoiding previous labeling. The sensor shows the linear response for thrombin in the range of 1-60nM with a detection limit of 0.5nM.
(2) Based on the principle of antibody-aptamer sandwich in the second part, we reported a new kind of electrochemical sensor, utilizing the enzyme linked aptamer assay to detect immunoglobulin E (IgE). In this method, the biotinlynated anti-IgE aptamer is used as the detection probe. The specific interaction of streptavidin-conjugated alkaline phosphatase to the surface-bound biotinlynated detection probe mediates a catalytic reaction of ascorbic acid 2-phosphate substrate to produce a reducing agent ascorbic acid. Then, silver ions in the solution can be reduced, leading to the deposition of metallic silver on the electrode surface. The amount of deposited silver, which is determined by the amount of IgE target bound on the electrode surface, can be quantified using the stripping voltammetry.
(3) A label-free electrochemical sensor based on target-induced displacement is reported with adenosine as the model analyte. Owing to the high affinity of aptamer binding to target, aptamer can displace the DNA which has bound to the capturing probe on the surface of the electrode, leading to the decreasing signal of the electroactive MB.
(4) We have designed a novel and simple fluorescence method to detect IgE. We labeled the aptamer with a fluorescence reporter group and its complementary DNA with a quencher (QDNA). After the aptamer reacting with IgE and QDNA, we measured the fluorescence intensity each step.
(1)在第二章中报道了一种无标记电化学方法来检测凝血酶。在这个工作中,我们引进了一种新的夹心电化学方法,采用抗体作为捕获探针,适配体作为检测探针,亚甲基兰作为电活性物质嵌入适配体中来获取电信号,不需要对探针进行任何修饰,传感器对凝血酶的线性响应范围是1-60nM,检测下限是0.5nM。(2)在第二部分的夹心模式的基础上,在第三章中,报道了一种基于适配体的酶放大方法来检测免疫球蛋白E(IgE)。生物素化抗IgE适配体作为检测探针,亲和素标记的碱性磷酸酯酶与电极表面的适配体上的生物素相连,碱性磷酸酯酶催化抗坏血酸磷酸酯水解成强还原剂抗坏血酸,将溶液中的银离子还原成银单质,沉积到电极表面。沉积的银的量与电极表面固定的IgE成正比,用溶出伏安法来定量。(3)在第四章中报道了一种基于目标物诱导适配体置换技术的无标记电化学检测方法,用来检测腺苷。利用适配体与目标物的强亲和力,将电极表面与捕获探针杂交的互补链置换下来,使嵌入在双链中的电活性物质亚甲基兰量减少,从而使得电信号发生变化。(4)在第五章里,我们用一种简单的荧光方法来检测IgE,通过荧光基团标记的适配体与目标物先反应一段时间,再将淬灭基团标记的适配体的互补链(QDNA)与之反应,检测反应前后的荧光强度,通过强度的减弱程度来对IgE定量。此方法检测蛋白质的线性范围宽,下限低。
Protein is the most abundant and important macromolecule in our body. Conventional methods for detecting protein are based on traditional immunoassay. Because the reactions of antigen-antibody are highly specific, immunosensors possess great sensitivity and selectivity. However, recent decades have seen the increasing interest in developing the method of detecting protein based on aptamer. The word, aptamer, comes from Latin aptus, to fit. Aptamers are selected from SELEX (systematic evolution of ligands by exponential enrichment) and have good recognition ability to proteins. The affinity and specificity between aptamers and protein is as well as those between antibodies and antigens. The dissociation constant of these is 10-9~10-12. Comparing to antibodies, aptamers own great advantages, including easy synthesis, good stability, ease modification and so on. It is facile to label the nucleic acid with different kinds of functional and report groups, such as fluorescence and electroactive groups. Therefore, it facilitates the immobilization of nucleic aptamer. In this article, we created a series of biosensors based on aptamers. The details are as following:
(1) In the second chapter, we report a label-free electrochemical method to detect thrombin. In this work, we introduced a new kind of sandwich method to detect protein, utilizing antibody as the capturing probe, aptamer as the detecting probe and methylene blue as the electroactive substance intercalating in the nucleic acid, avoiding previous labeling. The sensor shows the linear response for thrombin in the range of 1-60nM with a detection limit of 0.5nM.
(2) Based on the principle of antibody-aptamer sandwich in the second part, we reported a new kind of electrochemical sensor, utilizing the enzyme linked aptamer assay to detect immunoglobulin E (IgE). In this method, the biotinlynated anti-IgE aptamer is used as the detection probe. The specific interaction of streptavidin-conjugated alkaline phosphatase to the surface-bound biotinlynated detection probe mediates a catalytic reaction of ascorbic acid 2-phosphate substrate to produce a reducing agent ascorbic acid. Then, silver ions in the solution can be reduced, leading to the deposition of metallic silver on the electrode surface. The amount of deposited silver, which is determined by the amount of IgE target bound on the electrode surface, can be quantified using the stripping voltammetry.
(3) A label-free electrochemical sensor based on target-induced displacement is reported with adenosine as the model analyte. Owing to the high affinity of aptamer binding to target, aptamer can displace the DNA which has bound to the capturing probe on the surface of the electrode, leading to the decreasing signal of the electroactive MB.
(4) We have designed a novel and simple fluorescence method to detect IgE. We labeled the aptamer with a fluorescence reporter group and its complementary DNA with a quencher (QDNA). After the aptamer reacting with IgE and QDNA, we measured the fluorescence intensity each step.
引文
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