高灵敏功能化荧光核酸探针在生化分析中的应用研究

发布时间：2018-06-17 21:30

本文选题：功能化荧光核酸探针 + 发夹型探针　；参考：《湖南大学》2011年博士论文

【摘要】：准确和灵敏地获取生物样品中小分子、核酸、蛋白质和细胞的相关信息对生物医学研究以及临床诊断和治疗都具有十分重要的意义。功能化荧光核酸探针,其功能超出了核酸传统的基因角色,受到广大研究者越来越多的关注,但是,如何获得更高的灵敏度,如何提高抗复杂环境干扰的能力,如何实时、动态、灵敏、准确地获取生命活动相关信息,仍然是分析化学工作者所面临的重大挑战。本论文瞄准上述挑战进行研究,基于分子识别和信号放大技术发展了一系列分别以小分子、核酸、蛋白质和癌细胞为检测对象的新型功能化荧光核酸探针,主要内容包括: (1)发展了一种基于荧光共振能量转移和链置换放大技术的核酸适体探针用于小分子可卡因的高灵敏检测。在此,含有核酸适体的发夹探针的3’端标记荧光受体Cy5,而引物的5’端标记荧光供体FAM,当核酸适体与可卡因结合会导致其构型发生变化从而打开发夹结构,引物便可以与打开的发夹探针杂交,在聚合酶作用下引物延伸成与发夹探针序列完全互补,产生新的双链使得供体和受体靠近发生荧光共振能量转移,同时可卡因被引物延伸的链竞争下来变成自由的可卡因分子,又可以与另外一条核酸适体结合,不断循环实现信号放大。该方法可以在16min内达到200nM的检测下限,选择性良好。 (2)发展了一种基于芘激发态二聚体和杂交链式放大技术的核酸探针用于DNA的高灵敏检测。首先对两个发夹探针两端分别进行芘分子标记,由于发夹探针粘性末端的长度使得两端的芘分子以单体形式存在,当目标DNA触发杂交链式反应发生后,会形成一条长的带缺口的双链,使大量的芘分子以二聚体的形式存在。通过稳态荧光分析和时间分辨荧光测量技术可以实现缓冲溶液和复杂生物样品中DNA的高灵敏检测。利用该方法在缓冲溶液中对DNA的检测下限可以达到250 fM。 (3)发展了一种基于芘激发态二聚体和竞争反应的核酸适体探针用于人血清中溶菌酶的高灵敏检测。该体系中含有一条两端标记芘分子的发夹探针和一条未标记的核酸适体:当没有目标分子时,两条链部分杂交可以使发夹探针打开,芘分子以单体形式存在;而当存在目标分子时,目标分子结合核酸适体并以竞争的方式将芘分子双标探针挤开,被挤开的探针呈发夹构型,使得芘分子以二聚体形式存在。我们结合稳态和时间分辨荧光测量技术可对缓冲溶液或血清中的溶菌酶进行检测。该方法在缓冲溶液中对溶菌酶的检测下限可以达到200 pM。利用该方法还实现了ATP的检测,说明这是一种通用的检测方法。 (4)发展了一种基于分子信标和缺刻酶信号放大技术的核酸适体探针用于目标细胞的高灵敏检测。一段能打开分子信标的单链DNA与核酸适体杂交形成核酸适体/单链DNA复合物。当没有靶细胞存在时,单链DNA不能打开分子信标;当有靶细胞存在时,核酸适体与其结合形成核酸适体/靶细胞复合物,导致原来的双链结构分散并将单链DNA释放出来。被释放的单链DNA序列含有缺刻酶的识别位点,当其与分子信标结合以后,缺刻酶会将分子信标切开,单链DNA随即又可以与新的分子信标杂交。以这种方式,每条单链DNA可以多次循环使多个分子信标被切开,从而实现信号放大。利用该方法检测Ramos细胞可以达到200 cells/mL的检测下限,且特异性良好。 (5)发展了一种基于人血管生成素介导进入细胞的核酸适体探针用于增强光动力学治疗的效果。合成并表征了光敏剂Ce6标记的血管生成素核酸适体,与血管生成素特异结合以后,在目标细胞膜表面受体蛋白的介导下进入目标细胞,经过特定波长光照后,Ce6激活周围的氧分子变成单态氧将目标细胞杀死。结果显示该方法具有较高的光动力学的治疗效果。
[Abstract]:Accurate and sensitive access to small molecules, nucleic acids, proteins and cells in biological samples is of great significance for biomedical research and clinical diagnosis and treatment. Functional fluorescent nucleic acid probes are beyond the traditional gene roles of nucleic acids. It is still a major challenge for analytical chemistry workers to obtain higher sensitivity, how to improve the ability to resist complex environmental interference, and how to obtain information about life activities in real time, dynamically, sensitively and accurately.
This thesis aims at studying the above challenges. Based on molecular recognition and signal amplification, a series of new functional fluorescent nucleic acid probes are developed with small molecules, nucleic acids, proteins and cancer cells. The main contents are as follows:
(1) a nucleic acid aptamer probe based on fluorescence resonance energy transfer and chain replacement amplification is developed for the high sensitivity detection of small molecular cocaine. Here, the 3 'terminal labeled fluorescent receptor Cy5 with a nucleic acid aptamer probe, while the 5' end of the primer is labeled with the fluorescent donor FAM, and the binding of the aptamer to cocaine will lead to its structure. The primers can hybridize with the open hairpin probe. The primers can be hybridized with the open hairpin probe. The primers are extended to complement the hairpin probe sequence under the polymerase chain reaction. The new double strands make the donor and the receptor close to the fluorescence resonance energy transfer, and cocaine is competing to become free by the chain that is extended by the primer. The cocaine molecule can also be combined with another aptamer to continuously circulate and amplify the signal. This method can reach the lower limit of 200nM in 16min, and the selectivity is good.
(2) a nucleic acid probe based on pyrene excited state two polymer and hybrid chain amplification technology is developed for high sensitivity detection of DNA. First, pyrene molecular markers are carried out on both ends of two hairpin probes. The pyrene molecules at both ends are in the form of monomers due to the length of the sticky ends of the hairpin probes, when the target DNA triggers the crosslinked reaction. After the occurrence, a long, notched double chain will be formed so that a large number of pyrene molecules exist in the form of two polymers. The high sensitivity detection of DNA in buffer solutions and complex biological samples can be achieved by steady fluorescence analysis and time resolved fluorescence measurement. The detection limit of DNA in buffer solution can reach 250 fM. by this method.
(3) a highly sensitive detection of lysozyme in human serum based on an aptamer probe based on pyrene excited state two polymer and competitive reaction is developed. The system contains a hairpin probe with a pyrene molecule at both ends and an unlabeled aptamer: when there is no target molecule, two strand part hybridization can open the hairpin probe and pyrene. The molecule exists in the form of a monomer, and when the target molecule exists, the target molecules combine with the aptamer to squeeze the pyrene molecular double probe in a competitive manner, and the extruded probe is in the hairpin configuration, which makes the pyrene molecule in the form of two polymer. The detection limit of the lysozyme in the buffer solution can be reached to 200 pM. in the buffer solution. This method is also used to detect the ATP, which shows that this method is a common detection method.
(4) a nucleic acid aptamer probe based on molecular beacon and short enzyme signal amplification technology is developed for high sensitivity detection of target cells. A single strand DNA that opens molecular beacons and nucleic acid aptamers can form an aptamer / single strand DNA complex. When there is no target cells, single strand DNA can not open molecular beacons; when there are target cells In existence, the nucleate aptamer combines with the complex of an aptamer / target cell that causes the original double stranded structure to disperse and release the single strand DNA. The released single strand DNA sequence contains the identification site of the absence of the enzyme, and when it is combined with the molecular beacon, the absence of the enzyme will cut the molecular beacon, and the single strand DNA can then be associated with the new molecule. Beacon hybridization. In this way, each single strand DNA can be repeatedly circulated to make multiple molecular beacons cut in order to achieve signal amplification. The method is used to detect Ramos cells to reach a lower limit of 200 cells/mL, and the specificity is good.
(5) a nucleo aptamer probe based on human angiopoietin into cells was developed to enhance the effect of photodynamic therapy. The angiopoietin aptamer was synthesized and characterized by a photosensitizer Ce6 labeled angiopoietin. After specific binding with angiopoietin, a target cell membrane surface receptor protein was introduced into the target cells. After a specific wavelength of light, Ce6 activates the surrounding oxygen molecules to become mono oxygen to kill the target cells. The results show that the method has a high photodynamic therapeutic effect.
【学位授予单位】：湖南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R341

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