基于石墨烯氧化物-DNA传感器对凝血酶高灵敏检测的研究

发布时间：2019-05-22 13:53

【摘要】：凝血酶是一种重要的多功能蛋白酶,催化纤维蛋白原转化成纤维蛋白,促进凝血,浓度变化异常会导致一些疾病的发生如血栓、弥散性小管内凝血(DIC)等。传统的凝血酶检测方法技术复杂,时间长,不能满足临床的检测要求,因此快速、有效地检测凝血酶对临床诊断非常重要。近年来作为分子生物学研究工具的生物传感器对凝血酶的检测已有报道,但一些检测方法的灵敏性有待进一步提高,本研究基于石墨烯氧化物(GO)-DNA传感器发展了几种高灵敏的检测方法,所用的DNA序列是与凝血酶特异性结合的核酸适配体。1.采用聚乙二醇(PEG)修饰的GO-DNA传感器对凝血酶高灵敏检测。GO为荧光淬灭剂,FAM修饰的核酸适配体为识别元件,用PEG对裸露的GO进行表面修饰,去除对凝血酶的非特异性吸附,通过优化PEG的浓度提高检测灵敏性,检测限为4.8 pM,与裸露的GO表面凝血酶检测限51 pM相比提高了10倍。2.结合金纳米(GNPs)的GO-DNA传感器对凝血酶高灵敏检测。吐温80(tween 80)加快GNPs与巯基修饰核酸适配体的连接速度,还阻止GO表面对蛋白质的非特异性吸附,提高检测灵敏性,基于该特点发展了一种基于GNPs紫外吸收值与颜色变化为参考的GO传感器,通过比较加入蛋白前后溶液中组分、吸收值、溶液颜色的变化,计算凝血酶的含量,该方法无需荧光标记、简单、快速,检测限为0.68 pM。3.基于共价连接的GO-DNA传感器对凝血酶高灵敏检测。将FAM修饰的氨基化的核酸适配体共价连接到活化的GO表面,并去除物理吸附的核酸适配体,避免假阳性信号;另一方面PEG阻止GO表面的非特异性吸附,从而提高凝血酶检测的准确性和灵敏性,检测极限为0.034 pM。4.基于酶切循环放大的GO-DNA传感器对凝血酶高灵敏检测。荧光修饰的凝血酶核酸适配体(TBA)与互补核酸序列capture-DNA杂交,固定在GO表面,在核酸外切酶的催化下,凝血酶循环使用,TBA核酸适配体-凝血酶复合物不断形成,荧光信号逐渐放大,实现凝血酶的高灵敏检测,该方法对凝血酶的检测限为0.024 pM。
[Abstract]:Thrombin is an important multifunctional protease, which catalyzes the conversion of fibrin to fibrin and promotes coagulation. Abnormal concentration changes will lead to the occurrence of some diseases, such as thrombus, disseminated tubule coagulation (DIC) and so on. The traditional detection method of thrombin is complex, long time and can not meet the requirements of clinical detection, so rapid and effective detection of thrombin is very important for clinical diagnosis. In recent years, biosensors, as a tool for molecular biology research, have been reported for the detection of thrombin, but the sensitivity of some detection methods needs to be further improved. In this study, several highly sensitive detection methods were developed based on graphene oxide (GO)-DNA sensor. The DNA sequence used is a nucleic acid aptamer that specifically binds to thrombin. Polyethylene glycol (PEG) modified GO-DNA sensor was used for highly sensitive detection of thrombin. Go was used as fluorescent quenching agent, FAM modified nucleic acid aptamer was used as recognition element, and bare GO was modified by PEG. By optimizing the concentration of PEG to improve the detection sensitivity, the detection limit of 4.8 pM, was 10 times higher than that of 51 pM on the surface of exposed GO. The GO-DNA sensor of alloyed nano-(GNPs) is highly sensitive to the detection of thrombin. Tween 80 (tween 80) accelerates the bonding speed between GNPs and sulfhydryl modified nucleic acid aptamers, prevents the nonspecific adsorption of proteins on the surface of GO, and improves the sensitivity of detection. Based on this characteristic, a GO sensor based on GNPs UV absorption value and color change is developed. By comparing the changes of composition, absorption value and solution color before and after adding protein, the content of thrombin is calculated. The method is simple and rapid without fluorescence labeling, and the detection limit is 0.68 pM.3.. GO-DNA sensor based on covalent connection is highly sensitive to thrombin detection. The amino acid aptamers modified by FAM were covalently connected to the activated GO surface and the physically adsorbed aptamers were removed to avoid false positive signals. On the other hand, PEG prevents the nonspecific adsorption of GO surface, thus improving the accuracy and sensitivity of thrombin detection, and the detection limit is 0.034 pM.4.. The GO-DNA sensor based on enzyme digestion cycle amplification is highly sensitive to the detection of thrombin. The fluorescent modified thrombin nucleic acid aptamer (TBA) was crossbred with complementary nucleic acid sequence capture-DNA and fixed on the surface of GO. Under the catalysis of exonuclease, thrombin was recycled and the TBA nucleic acid aptamer-thrombin complex was continuously formed. The fluorescence signal was gradually amplified to realize the highly sensitive detection of thrombin. The detection limit of this method was 0.024 pM..
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O613.71;R446.1

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