荧光传感器体系的构建及其在几种重要生命物质检测中的应用

发布时间：2017-12-26 18:37

本文关键词：荧光传感器体系的构建及其在几种重要生命物质检测中的应用　出处：《华南理工大学》2016年博士论文　论文类型：学位论文

【摘要】：某些生命物质在生物体中起着重要作用,其含量的变化对生命活动具有重大的影响;因此,对这类物质的检测、监控和实时成像,在生理学和病理学研究中极具意义。在本文中,针对几种代表性的生命物质,选择合适的机理,设计并制备了四种荧光传感器,实现了对硫离子(S2-)、γ-谷酰胺转肽酶(GGT)及碱性磷酸酶(ALP)的便利、灵敏检测和细胞(或活体)成像。首先,针对硫离子,我们制备了利用配体分子改性的碳点(CD-S)作为探针前驱体,与铜离子(Cu2+)形成络合物并淬灭CD-S的荧光,作为检测S2-的荧光增强型探针。由于S2-与Cu2+之间有很高的结合能力,会夺取探针中的铜离子而生成稳定的CuS(Ksp=3.63×10-36);由此脱除了Cu2+的碳点的荧光得以恢复,该探针对硫离子的检测下限达0.78μM。该CD-S/Cu2+探针具有良好的水溶性、生物相容性、选择性和灵敏度,可以成功用于自来水中硫离子浓度的检测和活细胞中硫离子的检测、成像。其次,我们设计并合成了检测γ-谷酰胺转肽酶(GGT)的荧光探针Glu-TPE;作为四苯乙烯衍生物,探针Glu-TPE具有聚集诱导发光(AIE)效应;分子中的两个γ-谷酰胺基团使得探针Glu-TPE具有良好的水溶性与生物相容性;由于GGT催化探针Glu-TPE分子中γ-谷酰胺基团的水解,疏水性的酶解产物聚集诱发AIE效应而发出蓝色荧光,从而实现对GGT的增强型荧光检测。基于GGT酶促反应的专一性和高效性,探针Glu-TPE具有良好的选择性和灵敏性;能够用于对人血清样品中GGT含量的检测和A2780细胞中Qg源性GGT的检测、成像。针对GGT的检测,我们还制备了另一种基于分子内电荷转移(ICT)机理的荧光探针PEG-NA-Glu;当探针分子PEG-NA-Glu中γ-谷酰胺基团被GGT催化水解后,在这种ICT发光机理的荧光团中,电子供体的电子云密度发生变化,进而导致荧光发射波长的变化,由此实现了对GGT的比率型荧光检测。探针PEG-NA-Glu具有高灵敏度和生物相容性,其检测下限为0.76 U/L,可用于尿液和血清中检测GGT含量,且比ELISA Kit方法操作简便很多;并成功地实现了对活细胞内的GGT的双色荧光成像。再者,针对碱性磷酸酶(ALP),我们利用萘二甲酰亚胺衍生物的ICT荧光机理,设计并制备了探针AO-NA-P;ALP催化探针分子AO-NA-P的脱磷酸反应,使得探针中萘环4号位上的供电子基团发生变化,其结果增强了荧光分子的“推-拉”电子效应,荧光发射波长红移;基于此原理成功实现了对ALP的比率型荧光检测。由于ALP催化水解反应具有专一性和高效性,探针AO-NA-P具备高灵敏性和选择性,探针对ALP的检测下限达0.38 U/L;另外,探针分子AO-NA-P具有极好的水溶性和生物相溶性;可用于人血清样品、活细胞、活体中的ALP检测。我们首次实现了活体(斑马鱼)内ALP的比率型荧光成像,对斑马鱼内药物引致器官损伤而引发的ALP含量升高进行了成像和检测。相关结果可为研究器官损伤而引发的生命物质变化提供了有益的参考。
[Abstract]:Some life substances play an important role in organisms. The change of their contents has a great impact on life activities. Therefore, detection, monitoring and real-time imaging of these substances are of great significance in physiology and pathology research. In this paper, aiming at several representative life substances, we choose the appropriate mechanism to design and prepare four kinds of fluorescent sensors, realizing the convenient, sensitive detection and cell (or living) imaging of sulfur ion (S2-), gamma glutamyl trans peptidase (GGT) and alkaline phosphatase (ALP). First, for sulfur ion, we prepared the carbon dots (CD-S) modified by ligand molecules as the precursor of the probe, formed complexes with copper ions (Cu2+) and quenched the fluorescence of CD-S, which served as a fluorescence enhanced probe for detecting S2-. Because of the high binding ability between S2- and Cu2+, it will seize the copper ions in the probe and generate stable CuS (Ksp=3.63 * 10-36). Therefore, the fluorescence of the carbon dots of Cu2+ can be recovered, and the detection limit of the probe for sulfur ion is 0.78 M. The CD-S/Cu2+ probe has good water solubility, biocompatibility, selectivity and sensitivity. It can be successfully applied to the detection of sulfur ion in tap water and the detection and imaging of sulfur ions in living cells. Secondly, we design and detection of gamma glutamyl transpeptidase (GGT) synthesized Glu-TPE as fluorescence probe; four stilbene derivatives, Glu-TPE probe with aggregation induced emission (AIE) effect; two gamma molecules in the glutamine group makes Glu-TPE probe has good water solubility and biocompatiblity of; because of GGT catalyzed hydrolysis of Glu-TPE molecular probes in gamma glutamyl groups, hydrophobic enzymatic product aggregation induced AIE effect and emit blue fluorescence, so as to realize the enhanced fluorescence detection of GGT. Based on the specificity and efficiency of GGT enzymatic reaction, probe Glu-TPE has good selectivity and sensitivity. It can be used to detect GGT content in human serum samples and detect and imaging Qg derived GGT in A2780 cells. For the detection of GGT, we also made another based on intramolecular charge transfer (ICT) by fluorescence probe PEG-NA-Glu mechanism; when the probe molecule PEG-NA-Glu gamma glutamyl moiety catalyzed by GGT after hydrolysis, the fluorophore emitting mechanism in this ICT, the electron donor electron cloud density changes, leading to the change of fluorescence emission wavelength, thus achieving a ratiometric fluorescent detection of GGT. The probe PEG-NA-Glu has high sensitivity and biocompatibility. The detection limit is 0.76 U/L, which can be used for detecting GGT content in urine and serum. It is much simpler and more convenient than ELISA Kit method, and successfully realizes the two-color fluorescence imaging of GGT in living cells. Moreover, the alkaline phosphatase (ALP), we use the ICT fluorescence mechanism of naphthalene two phthalimide derivatives, design and probe AO-NA-P were prepared; phosphate removal catalyzed by ALP probe molecule AO-NA-P, makes the probe in naphthalene ring electron donating 4 position changes, the results of enhanced fluorescent molecules the "push-pull" electronic effect, fluorescence emission wavelength; based on this principle, the successful implementation of the ratiometric fluorescent ALP detection. Because the ALP catalytic hydrolysis with specificity and high efficiency, AO-NA-P probe with high sensitivity and selectivity, detection limit of ALP probe was 0.38 U/L; in addition, the probe molecule AO-NA-P has excellent water solubility and biocompatibility; can be used in human serum samples, ALP detection of viable cells, in vivo. We first realized the ratio type fluorescence imaging of ALP in vivo (zebrafish), and imaging and detecting the increase of ALP content induced by drug damage in zebrafish. The related results can provide a useful reference for the study of the changes of life substances caused by organ damage.
【学位授予单位】：华南理工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：O629.8;TP212

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