高灵敏功能化荧光核酸探针在生化分析中的应用研究
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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。
The essential information on small molecules, nucleic acids, proteins and cells obtained accurately and sensitively has great significance for biological medicine study, clinical diagnosis and therapy. Functional fluorescent nucleic acid probe,whose functions are beyond the conventional genetic roles of nucleic acids, had attracted researchers’increasing attention. However, how to get higher sensitivity, increase its ability to tolerate any interference from complex environment, get the dynamic data of these life processes sensitively and accurately in real time are still great challenges to analysts.
In this dissertation, a series of functional fluorescent nucleic acid probes based on molecule recognization and signal amplification technique have been developed for small molecule, nucleic acid, protein and cancer cell detection, respectively. The main researches of this dissertation are summarized as follows.
(1) An aptasensor for cocaine amplified detection based on a strand-displacement polymerization reaction and fluorescence resonance energy transfer (FRET) has been developed. Herein, the acceptor fluorophore (Cy5) is labelled at the 3’end of the hairpin probe, which contains the cocaine aptamer; and the donor fluorophore (FAM) is labelled at the 5’end of the primer. When the aptamer binds to cocaine and undergoes a conformational change, the hairpin structure opened. Then the primer anneals with the exposed stem of the hairpin probe. Following this, the primer will extend to form a complementary DNA of the hairpin probe in the presence of dNTPs/polymerase. The newly duplex will push the donor and acceptor in close proximity, resulting in FRET. Meanwhile, the target cocaine is displaced by the primer extension. To renew the cycle, the displaced free cocaine binds to another aptamer, making the signal amplification realized. The detection limit of this method is 200 nM in about 16 min and the specificity of this approach is excellent.
(2) A nucleic acid probe for highly sensitive DNA detection based on pyrene excimer and hybridization chain reaction (HCR) amplification has been developed. Two hairpin probes have been labeled two pyrene molecules at both ends, where the pyrene molecules are separated each other because of the sticky end. When the target DNA triggers HCR, there will be produce a long nicked duplex, allowing the formation of numerous pyrene excimers. Sensitive detection of target DNA in buffer and complex biological sample is achieved through steady-state fluorescence assay and time-resolved fluorescence measurement. The limit of detection for DNA is 250 fM in buffer.
(3) An aptasensor for sensitive lysozyme detection in human serum based on pyrene excimer and competition reaction has been developed. Herein, a dual-pyrene labeled hairpin probe and label-free lysozyme aptamer coexist in solution. In the absence of target, the aptamer hybridizes with part of the hairpin probe to form a duplex, resulting in two pyrene molecules spatially separated. However,in the presence of target, the dual-pyrene labeled probe is gradually displaced from the aptamer by the target, subsequently forming a hairpin structure, this brings the two pyrene moieties into close proximity and allows the formation of an excimer. Detection of target lysozyme in buffer and human serum is achieved through steady-state fluorescence assay and time-resolved fluorescence measurement. The detection limit of this method for lysozyme is 200 pM in buffer. Finally, the success of ATP detection suggests the generality of the strategy.
(4) A nucleic acid probe for highly sensitive target cell detection based on molecular beacon and nicking endonuclease signal amplification has been developed. A ssDNA , which can open the molecular beacon, is hybridized with aptamer to form a duplex. The formation of the aptamer/ssDNA complex inhibits the competitive hybridization of molecular beacon/ssDNA unless triggered by target cells. In the presence of the specific target cancer cells, the aptamer binds to the target cell and forms an aptamer/cell complex, resulting in the disassembly of the original duplex and release of the ssDNA. The released ssDNA sequence contains the nick endonuclease recognition site. One ssDNA hybridizes with one molecular beacon and then the nick endonuclease makes a nick in the beacon strand. After nicking, the molecular beacon is cleaved and the ssDNA can be reused for next cycle of cleavage. In this way, each ssDNA can go through many cycles, resulting in cleavage of many beacons, achieving a signal amplification. The detection limit of this method is 200 cells/mL and the specificity of this approach is excellent.
(5) An angiogenin-mediated cell-internalized aptamer probe for improve the efficiency of photodynamic therapy (PDT) has been developed. A Ce6-conjugated, angiogenin aptamer was sythesized and characterized, which can deliver the photosensitizer into target cells mediated by angiogenin. After irradiation by an appropriate light, the Ce6 active the surrounding oxygen molecules to form reactive oxygen species, that ultimately leads to tumor cell death. The results showed high efficiency of PDT.
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。
The essential information on small molecules, nucleic acids, proteins and cells obtained accurately and sensitively has great significance for biological medicine study, clinical diagnosis and therapy. Functional fluorescent nucleic acid probe,whose functions are beyond the conventional genetic roles of nucleic acids, had attracted researchers’increasing attention. However, how to get higher sensitivity, increase its ability to tolerate any interference from complex environment, get the dynamic data of these life processes sensitively and accurately in real time are still great challenges to analysts.
In this dissertation, a series of functional fluorescent nucleic acid probes based on molecule recognization and signal amplification technique have been developed for small molecule, nucleic acid, protein and cancer cell detection, respectively. The main researches of this dissertation are summarized as follows.
(1) An aptasensor for cocaine amplified detection based on a strand-displacement polymerization reaction and fluorescence resonance energy transfer (FRET) has been developed. Herein, the acceptor fluorophore (Cy5) is labelled at the 3’end of the hairpin probe, which contains the cocaine aptamer; and the donor fluorophore (FAM) is labelled at the 5’end of the primer. When the aptamer binds to cocaine and undergoes a conformational change, the hairpin structure opened. Then the primer anneals with the exposed stem of the hairpin probe. Following this, the primer will extend to form a complementary DNA of the hairpin probe in the presence of dNTPs/polymerase. The newly duplex will push the donor and acceptor in close proximity, resulting in FRET. Meanwhile, the target cocaine is displaced by the primer extension. To renew the cycle, the displaced free cocaine binds to another aptamer, making the signal amplification realized. The detection limit of this method is 200 nM in about 16 min and the specificity of this approach is excellent.
(2) A nucleic acid probe for highly sensitive DNA detection based on pyrene excimer and hybridization chain reaction (HCR) amplification has been developed. Two hairpin probes have been labeled two pyrene molecules at both ends, where the pyrene molecules are separated each other because of the sticky end. When the target DNA triggers HCR, there will be produce a long nicked duplex, allowing the formation of numerous pyrene excimers. Sensitive detection of target DNA in buffer and complex biological sample is achieved through steady-state fluorescence assay and time-resolved fluorescence measurement. The limit of detection for DNA is 250 fM in buffer.
(3) An aptasensor for sensitive lysozyme detection in human serum based on pyrene excimer and competition reaction has been developed. Herein, a dual-pyrene labeled hairpin probe and label-free lysozyme aptamer coexist in solution. In the absence of target, the aptamer hybridizes with part of the hairpin probe to form a duplex, resulting in two pyrene molecules spatially separated. However,in the presence of target, the dual-pyrene labeled probe is gradually displaced from the aptamer by the target, subsequently forming a hairpin structure, this brings the two pyrene moieties into close proximity and allows the formation of an excimer. Detection of target lysozyme in buffer and human serum is achieved through steady-state fluorescence assay and time-resolved fluorescence measurement. The detection limit of this method for lysozyme is 200 pM in buffer. Finally, the success of ATP detection suggests the generality of the strategy.
(4) A nucleic acid probe for highly sensitive target cell detection based on molecular beacon and nicking endonuclease signal amplification has been developed. A ssDNA , which can open the molecular beacon, is hybridized with aptamer to form a duplex. The formation of the aptamer/ssDNA complex inhibits the competitive hybridization of molecular beacon/ssDNA unless triggered by target cells. In the presence of the specific target cancer cells, the aptamer binds to the target cell and forms an aptamer/cell complex, resulting in the disassembly of the original duplex and release of the ssDNA. The released ssDNA sequence contains the nick endonuclease recognition site. One ssDNA hybridizes with one molecular beacon and then the nick endonuclease makes a nick in the beacon strand. After nicking, the molecular beacon is cleaved and the ssDNA can be reused for next cycle of cleavage. In this way, each ssDNA can go through many cycles, resulting in cleavage of many beacons, achieving a signal amplification. The detection limit of this method is 200 cells/mL and the specificity of this approach is excellent.
(5) An angiogenin-mediated cell-internalized aptamer probe for improve the efficiency of photodynamic therapy (PDT) has been developed. A Ce6-conjugated, angiogenin aptamer was sythesized and characterized, which can deliver the photosensitizer into target cells mediated by angiogenin. After irradiation by an appropriate light, the Ce6 active the surrounding oxygen molecules to form reactive oxygen species, that ultimately leads to tumor cell death. The results showed high efficiency of PDT.
引文
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