發(fā)布時(shí)間：2018-01-03 07:22

  本文關(guān)鍵詞：用于細(xì)胞內(nèi)陽(yáng)離子檢測(cè)的新型比率型熒光探針的研究　出處：《湖南大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 熒光分子探針 比率型 跨鍵能量轉(zhuǎn)移 雙光子 細(xì)胞成像 組織成像

【摘要】：研究發(fā)現(xiàn),細(xì)胞內(nèi)的微小化學(xué)變化都可能預(yù)示著生命體的重大改變,所以隨著生命科學(xué)的快速發(fā)展,對(duì)單個(gè)細(xì)胞甚至細(xì)胞器內(nèi)的小分子,離子,核酸或者蛋白質(zhì)的精準(zhǔn),靈敏,快速檢測(cè)等要求更加迫切。熒光傳感技術(shù)因具有高選擇性、高靈敏度、原位實(shí)時(shí)快速分析、以及對(duì)樣品無(wú)損傷等優(yōu)點(diǎn),而成為目前生物分析的熱點(diǎn)研究技術(shù)。事實(shí)證明不斷涌現(xiàn)的新型熒光分子探針,實(shí)現(xiàn)了對(duì)細(xì)胞內(nèi)的分子,離子等高靈敏的特異性檢測(cè),為生物分析技術(shù)的發(fā)展提供了進(jìn)一步的技術(shù)支持。本文通過(guò)對(duì)熒光探針的研究,分別設(shè)計(jì)了一個(gè)基于氧雜葸衍生物的pH熒光探針FR-Lys,實(shí)現(xiàn)了定位溶酶體和動(dòng)態(tài)檢測(cè)其pH變化�；诳珂I能量轉(zhuǎn)移的雙光子比率型的Cu2+離子熒光探針Np-Rh和Pd2+離子熒光探針Np-Rh-Pd,實(shí)現(xiàn)了細(xì)胞內(nèi)金屬Cu2+離子和Pd2+離子的特異性識(shí)別和組織深度成像。主要內(nèi)容如下：1. 在第2章中,通過(guò)將氧雜葸,苯并噻唑,和嗎啉有機(jī)結(jié)合起來(lái),設(shè)計(jì)了一個(gè)定位溶酶體的pH熒光探針FR-Lys o在該探針中,嗎啉起著定位溶酶體的作用,氧雜葸衍生物隨pH變化而開關(guān)環(huán),同時(shí)pH誘導(dǎo)鄰羥基苯苯并噻唑發(fā)生或抑制激發(fā)態(tài)分子內(nèi)質(zhì)子轉(zhuǎn)移(ESIPT)過(guò)程。實(shí)現(xiàn)了探針對(duì)pH 4.0-6.3范圍內(nèi)比率型響應(yīng)。該響應(yīng)過(guò)程迅速且可逆,并且此FR-Lys易合成,光穩(wěn)定性好,低毒性等,這些優(yōu)點(diǎn)都該探針適合于溶酶體pH動(dòng)態(tài)成像,并取得好的效果。2.在第3章中,我們利用雙光子熒光團(tuán)萘衍生物與具有螺內(nèi)酰胺環(huán)狀結(jié)構(gòu)的羅丹明B衍生物連接,構(gòu)建了一個(gè)具有D-π-A結(jié)構(gòu)的比率型雙光子熒光探針平臺(tái)。該熒光探針平臺(tái)電子供體與受體通過(guò)跨鍵能量轉(zhuǎn)移機(jī)理發(fā)生高效的能量傳遞90%,同時(shí)該探針有大的雙發(fā)射峰位移為100 nna,大的雙光子活性吸收截面。我們利用常見的Cu2+離子為檢測(cè)對(duì)象構(gòu)建了熒光探針Np-Rh,實(shí)驗(yàn)結(jié)果表明該探針對(duì)Cu2+離子特異性識(shí)別,并且檢測(cè)下限達(dá)3.0×10-7 M,在生理?xiàng)l件下穩(wěn)定性較好,同時(shí)實(shí)現(xiàn)了對(duì)活細(xì)胞的高分辨率雙光子比率型成像和較深的組織深度(70-180μm)成像。3.在第4章中,基于第3章中構(gòu)建的比率型雙光子平臺(tái),我們?cè)O(shè)計(jì)了一個(gè)Pd2+離子的雙光子比率型熒光探針Np-Rh-Pd。探針體系中存在Pd2+離子時(shí),Pd2+離子與探針的受體羅丹明B的螺內(nèi)酰胺上的N,O原子形成配合物,探針熒光發(fā)生波長(zhǎng)紅移,位移達(dá)到100 nm。對(duì)Pd2+離子的熒光檢測(cè)信倍比高達(dá)31.2,檢測(cè)限低至2.3×10μM,可以實(shí)現(xiàn)對(duì)活細(xì)胞和組織高分辨率的比率成像和深的組織深度(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.
【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：O657.3
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本文編號(hào)：1372950