凝血酶的新型适配体—悬浮芯片检测技术及作用机制研究

发布时间：2018-01-07 11:02

本文关键词：凝血酶的新型适配体—悬浮芯片检测技术及作用机制研究　出处：《天津大学》2015年硕士论文　论文类型：学位论文

【摘要】：核酸适配体由于其具有分子量小、制备简单、高亲和力、高特异性等多种优点,逐渐替代抗体作为一种新型的生物识别元件,有望广泛应用于生命科学研究、检测分析和临床诊断等诸多领域。悬浮芯片技术是一种高通量、低背景的生物芯片技术,以荧光编码微球作为检测探针载体进行靶标物的快速灵敏检测。本研究将适配体与悬浮芯片技术相结合,以经典模式靶标物凝血酶作为检测目标,建立高亲和力和高特异性的适配体-悬浮芯片检测新技术。将氨基化的15mer的适配体(Thrombin binding aptamer 1,TBA1)修饰在磁性羧基化微球表面制备成探针微球,用于捕获溶液和血液样品中的凝血酶,生物素化的29mer的适配体(Thrombin binding aptamer 2,TBA2)作为报告探针与凝血酶结合,形成在微球上稳定的三明治夹心复合物,经过磁分离,将此复合物与溶液中未偶联靶标物分离。通过链霉亲和素-生物素的反应系统将荧光报告蛋白链霉亲和素-藻红蛋白(Strepavidin-phycoerythrin,SA-PE)连接到报告探针TBA2上,由悬浮芯片系统检测得到中位荧光强度值(Median Fluorescence Intensity,MFI)。通过优化,获得了最佳检测孵育条件和最佳偶联方案。在最佳检测条件和偶联方案下,建立了凝血酶检测标准曲线,检测范围18.37-554.31 nM(IC10-IC90)最低检出限为5.4 nM,说明检测方法具有较高的检测灵敏度和精确度。经过特异性测定实验证明了适配体-悬浮芯片检测技术检测凝血酶具有良好的特异性。对人血清中进行凝血酶加标回收率的测定,加标回收率在82.6-114.2%之间,相对标准偏差在10%以内,获得了满意的回收率效果,该检测技术有望成功应用于临床诊断的高通量样品检测。在建立适配体-悬浮芯片检测新技术的同时,对TBAs与凝血酶之间的分子间相互作用机制进行了初步探讨。利用微量热泳动技术(Microscale thermophoresis,MST)证实TBAs与凝血酶间具有很高的亲和力。MST信号反映了TBA1与凝血酶的偶联模式,表明TBA1可同时结合纤维蛋白原和肝素位点。本研究将新型识别元件——适配体与灵敏的悬浮芯片技术相结合,建立了适配体-悬浮芯片检测新技术,初步实现了对人血清样本中凝血酶的分析,方法灵敏、特异和快速,展现了良好的应用前景。利用MST技术分析适配体与凝血酶间的相互作用,为深入探究适配体与靶标物的识别偶联机制奠定了基础,也可为药物靶向性和药物作用机制等的研究提供一定的数据支持。
[Abstract]:Aptamer due to its small molecular weight, simple preparation, high affinity, high specificity and other advantages, gradually replace the antibody as a new biological recognition element, is expected to be widely used in life science research, detection and analysis of clinical diagnosis and many other fields. Suspension array technology is a high-throughput biological. Chip technology, low background, rapid and sensitive detection of targets using fluorescent microspheres as detection probe vector encoding. This study will aptamer and suspension chip technology combined with classic model of target thrombin as detection target, new technology of aptamer suspension chip detection to establish high affinity and high specificity of the amino. The 15mer aptamer (Thrombin binding aptamer 1, TBA1) in the modified magnetic carboxylated microspheres were prepared by surface probe microspheres, used to capture the blood coagulation enzyme solution and blood samples, biotin Aptamers of 29mer (Thrombin binding aptamer 2, TBA2) report as a combination of probe and thrombin, form stable microspheres sandwich complexes, after magnetic separation, the complex with the solution without coupling target separation. By streptavidin biotin streptavidin fluorescent reporter protein reaction system streptavidin - phycoerythrin (Strepavidin-phycoerythrin, SA-PE) connected to the reporter probe TBA2, median fluorescence intensity values obtained by suspension chip detection system (Median Fluorescence Intensity, MFI). After optimization, the optimal detection of incubation conditions and the best coupling scheme is obtained. In the optimum conditions and the coupling scheme, a thrombin standard detection curve, detection range of 18.37-554.31 nM (IC10-IC90) the lowest detection limit was 5.4 nM, that method has high detection sensitivity and accuracy. After a specific test The experiment proved that the aptamer fixed suspension chip detection of thrombin has good specificity of thrombin. Spiked recovery tests performed in human serum, the recovery rate was 82.6-114.2%, the relative standard deviation is less than 10%, the recovery rate obtained satisfactory effect, high-throughput sample detection the detection technology is expected to be successful applied to the clinical diagnosis. In the establishment of the aptamer and new detection technology of suspension array, between TBAs and thrombin on the molecular interaction mechanism was discussed. The use of trace thermophoresis Technology (Microscale thermophoresis MST) has confirmed the high affinity.MST signal reflects the coupling model of TBA1 and thrombin TBAs with thrombin showed that TBA1 can at the same time, fibrinogen and heparin binding sites. This research will be the new recognition element, aptamer and suspension chip technology sensitive combination Together, established a new technique for the detection of aptamer suspension chip, realized the analysis of thrombin in human serum samples method is sensitive, specific and rapid, show a good application prospect. The analysis suitable ligand interaction between thrombin and the use of MST technology, laid the foundation for further exploration of the coupling mechanism and aptamer recognition target, also can provide the data support for targeted drug research and drug action mechanism.

【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.1

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