用于细胞内阳离子检测的新型比率型荧光探针的研究

发布时间：2018-01-03 07:22

  本文关键词：用于细胞内阳离子检测的新型比率型荧光探针的研究　出处：《湖南大学》2016年硕士论文　论文类型：学位论文

  更多相关文章： 荧光分子探针 比率型 跨键能量转移 双光子 细胞成像 组织成像

【摘要】：研究发现,细胞内的微小化学变化都可能预示着生命体的重大改变,所以随着生命科学的快速发展,对单个细胞甚至细胞器内的小分子,离子,核酸或者蛋白质的精准,灵敏,快速检测等要求更加迫切。荧光传感技术因具有高选择性、高灵敏度、原位实时快速分析、以及对样品无损伤等优点,而成为目前生物分析的热点研究技术。事实证明不断涌现的新型荧光分子探针,实现了对细胞内的分子,离子等高灵敏的特异性检测,为生物分析技术的发展提供了进一步的技术支持。本文通过对荧光探针的研究,分别设计了一个基于氧杂葸衍生物的pH荧光探针FR-Lys,实现了定位溶酶体和动态检测其pH变化。基于跨键能量转移的双光子比率型的Cu2+离子荧光探针Np-Rh和Pd2+离子荧光探针Np-Rh-Pd,实现了细胞内金属Cu2+离子和Pd2+离子的特异性识别和组织深度成像。主要内容如下：1. 在第2章中,通过将氧杂葸,苯并噻唑,和吗啉有机结合起来,设计了一个定位溶酶体的pH荧光探针FR-Lys o在该探针中,吗啉起着定位溶酶体的作用,氧杂葸衍生物随pH变化而开关环,同时pH诱导邻羟基苯苯并噻唑发生或抑制激发态分子内质子转移(ESIPT)过程。实现了探针对pH 4.0-6.3范围内比率型响应。该响应过程迅速且可逆,并且此FR-Lys易合成,光稳定性好,低毒性等,这些优点都该探针适合于溶酶体pH动态成像,并取得好的效果。2.在第3章中,我们利用双光子荧光团萘衍生物与具有螺内酰胺环状结构的罗丹明B衍生物连接,构建了一个具有D-π-A结构的比率型双光子荧光探针平台。该荧光探针平台电子供体与受体通过跨键能量转移机理发生高效的能量传递90%,同时该探针有大的双发射峰位移为100 nna,大的双光子活性吸收截面。我们利用常见的Cu2+离子为检测对象构建了荧光探针Np-Rh,实验结果表明该探针对Cu2+离子特异性识别,并且检测下限达3.0×10-7 M,在生理条件下稳定性较好,同时实现了对活细胞的高分辨率双光子比率型成像和较深的组织深度(70-180μm)成像。3.在第4章中,基于第3章中构建的比率型双光子平台,我们设计了一个Pd2+离子的双光子比率型荧光探针Np-Rh-Pd。探针体系中存在Pd2+离子时,Pd2+离子与探针的受体罗丹明B的螺内酰胺上的N,O原子形成配合物,探针荧光发生波长红移,位移达到100 nm。对Pd2+离子的荧光检测信倍比高达31.2,检测限低至2.3×10μM,可以实现对活细胞和组织高分辨率的比率成像和深的组织深度(90-270μm)成像。
[Abstract]:Studies have found that small chemical changes in cells may indicate major changes in life, so with the rapid development of life science, the single cell and even the small molecules in the organelles, ions. Because of its high selectivity, high sensitivity, in situ real-time rapid analysis, and no damage to the sample, the fluorescent sensing technology is more urgent for the accuracy, sensitivity and rapid detection of nucleic acid or protein. It has become a hot research technology of biological analysis at present. It has been proved that the new fluorescent molecular probes, which have been emerging constantly, have achieved highly sensitive and specific detection of molecules and ions in cells. It provides further technical support for the development of biological analysis technology. In this paper, we designed a pH fluorescence probe FR-Lys based on oxa-anthracene derivatives through the study of fluorescent probe. Localization of lysosomes and dynamic detection of pH changes were achieved. Two-photon ratio Cu2 ion fluorescence probe Np-Rh and Pd2 ion fluorescence probe Np-Rh-Pd based on transbond energy transfer were achieved. The specific recognition and tissue depth imaging of intracellular metal Cu2 ions and Pd2 ions were realized. The main contents are as follows: 1. In Chapter 2, benzothiazole and benzothiazole were prepared by using oxazanthene and benzothiazole. Combined with morpholine organically, a pH fluorescence probe FR-Lys o for lysosomal localization was designed, in which morpholine acts as a lysosomal locator, and oxa-anthracene derivatives switch rings with the change of pH. At the same time, pH induced the occurrence or inhibition of excited intramolecular proton transfer (ESIPT) of o-hydroxybenzothiazole. The ratio response of the probe to pH 4.0-6.3 was achieved. The response process was rapid and reversible. And this FR-Lys is easy to synthesize, good photostability, low toxicity and so on. These advantages are suitable for lysosomal pH dynamic imaging, and achieved good results. 2. In Chapter 3. We use two-photon fluorescence group naphthalene derivatives to connect with rhodamine B derivatives with spirolactam ring structure. A ratio two-photon fluorescence probe platform with D- 蟺 -A structure was constructed. The electron donor and acceptor can transfer 90% energy efficiently through the transbond energy transfer mechanism. At the same time, the probe has a large double emission peak shift of 100nnaand a large two-photon active absorption cross-section. We have constructed a fluorescence probe Np-Rh using common Cu2 ions as the detection object. The results showed that the probe was specific for Cu2 ion recognition, and the detection limit was 3.0 脳 10 ~ (-7) M.The stability of the probe was good under physiological conditions. At the same time, the high resolution two-photon ratio imaging of living cells and the deep tissue depth of 70-180 渭 m) imaging. 3. In Chapter 4, based on the ratio of two-photon platform constructed in Chapter 3. We have designed a two-photon ratio fluorescence probe Np-Rh-Pd. for Pd2 ion, when there is Pd2 ion in the probe system. The Pd2 ion forms a complex with the N2O atom on the spirolactam B of the probe receptor Rhodamine B. the fluorescence of the probe is red-shifted. The shift reached 100nm.The fluorescence detection ratio of Pd2 ion was as high as 31.2, and the detection limit was as low as 2.3 脳 10 渭 M. High resolution ratio imaging and deep tissue depth 90-270 渭 m imaging of living cells and tissues can be achieved.
【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.3
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