本文選題：比率熒光探針　切入點：FRET　出處：《山東大學(xué)》2017年博士論文

【摘要】：在生物體內(nèi),二氧化硫(SO_2)可產(chǎn)生自含硫氨基酸的分解、硫化氫(H_2S)的氧化等酶促過程。研究發(fā)現(xiàn)低濃度的SO_2在血壓調(diào)節(jié)、炎癥反應(yīng)等生理與病理過程中發(fā)揮重要的作用。與一氧化氮(NO)分子類似,SO_2也被認(rèn)為是一種生物氣體信號分子。由于SO_2易溶于水形成亞硫酸鹽與亞硫酸氫鹽,因此其生理作用是通過這類SO_2衍生物實現(xiàn)的。另一方面,SO_2衍生物廣泛應(yīng)用于食品抗氧化劑、抗菌劑,例如SO_2作為葡萄酒的抗菌劑使用等。但是由于現(xiàn)有檢測方法的限制,SO_2及其衍生物具體的生理作用及轉(zhuǎn)化過程尚不十分明確。因此,亟待開發(fā)高靈敏的、高分辨率的、實時原位的檢測方法。熒光分析法具有良好的生物兼容性,在生物及醫(yī)學(xué)領(lǐng)域得到了廣泛關(guān)注。有機小分子熒光探針具有開發(fā)周期短,光穩(wěn)定性好,細(xì)胞毒性低、操作簡單及信號直觀等優(yōu)點,近年來得以飛速發(fā)展,包括許多基于有機熒光小分子的SO_2衍生物探針也有報道。目前報道的SO_2衍生物熒光探針多是基于單波長熒光強度的變化(增強或減弱)實現(xiàn)檢測的。單一信號容易受到測試體系、探針濃度、儀器參數(shù)等外界因素的干擾而影響分析結(jié)果的準(zhǔn)確度。比率熒光探針具有兩個熒光發(fā)射峰,在SO_2衍生物的作用下,至少一個熒光峰的強度發(fā)生變化。通過探針分子內(nèi)部的相互參照,能有效地消除來自外界因素的干擾,從而提高分析結(jié)果的準(zhǔn)確度。比率熒光探針一般含有一個熒光基團或者兩個不同的熒光基團。單熒光基團的比率熒光探針的信號調(diào)控機理主要有分子內(nèi)電荷轉(zhuǎn)移(ICT)和激發(fā)態(tài)分子內(nèi)質(zhì)子轉(zhuǎn)移(ESIPT)等,雙熒光團的比率熒光探針信號的主要調(diào)控機理有熒光共振能量轉(zhuǎn)移(FRET)和跨鍵能量轉(zhuǎn)移(TBET)等。黃酮醇結(jié)構(gòu)是ESIPT型熒光染料的典型代表。黃酮醇由于具有大的Stokes位移,好的生物膜透性和低的細(xì)胞毒性而受到廣泛關(guān)注�；邳S酮醇及其衍生物的熒光探針大多是通過控制其ESIPT過程的"開-關(guān)"實現(xiàn)的。FRET過程的實現(xiàn)必須要有兩個熒光基團的共同參與,其中一個作為能量供體,另一個作為能量受體。半花菁熒光團具有摩爾消光系數(shù)大、熒光量子產(chǎn)率高、可見光有效激發(fā)、長波光區(qū)域發(fā)射、結(jié)構(gòu)容易修飾等特點,有作為FRET體系中能量受體使用的潛質(zhì)。本論文中,我們致力于研究比率熒光探針的設(shè)計、合成與應(yīng)用。以FRET和ESIPT為主要比率信號變化機理,以半花菁和黃酮醇結(jié)構(gòu)為主要的熒光報告基團,設(shè)計合成了四個SO_2衍生物探針以及一個H_2S探針,并研究了它們的熒光性質(zhì)和在生物檢測方面的應(yīng)用。第一章,簡要介紹了熒光探針的基本概念,熒光信號的變化類型及其調(diào)控原理;總結(jié)了 SO_2衍生物熒光探針的最新進展;綜述了基于半花菁和黃酮醇結(jié)構(gòu)的熒光探針的最新進展。第二章,設(shè)計并合成了以半花菁為"能量-檢測對象"雙受體的FRET新模板并應(yīng)用與SO_2衍生物的比率檢測。該FRET體系具有高的能量傳遞效率與顯著的比率特性。同時探針HCy-D可靶向細(xì)胞內(nèi)線粒體,并通過對該細(xì)胞內(nèi)生亞硫酸氫鹽的檢測,成功區(qū)分正常肝細(xì)胞(L-02)與癌變的肝細(xì)胞(HepG2)。第三章,合成了一例基于FRET新模板的比率熒光及比色探針HCy-NBD,以檢測SO_2衍生物。該探針以半花菁基團為受體,以NBD基團為供體,可實現(xiàn)靈敏,快速的檢測及裸眼識別。第四章,以FRET模板為基礎(chǔ),引入具有ESIPT過程的黃酮醇基團作為能量供體,從而將ESIPT過程與FRET過程聯(lián)系起來。由于ESIPT效應(yīng),供體黃酮醇分子的Stokes位移高達185 nm;而由于ESIPT與FRET效應(yīng)的共同作用,受體會產(chǎn)生很大的偽Stokes位移值,故基于該模板的探針在與檢測對象作用前后都將體現(xiàn)大的(偽)Stokes位移。探針L-HF1選取對SO_2衍生物有高度反應(yīng)專一性的半花菁作為能量受體,具有好的選擇性與靈敏度。將黃酮醇結(jié)構(gòu)中的羥基轉(zhuǎn)化為酯基以抑制ESIPT過程,所得化合物L(fēng)-HF2分子中沒有檢測到FRET過程,這進一步印證了 EISPT對FRET過程的重要輔助作用。同樣,該探針可應(yīng)用于細(xì)胞內(nèi)亞硫酸氫鹽的成像,體現(xiàn)了良好的比率特性與低的細(xì)胞毒性。第五章,依托ESIPT輔助的FRET模板,為有效減少供體發(fā)射峰與受體發(fā)射峰的重疊,增加兩個熒光發(fā)射峰的分辨率,設(shè)計合成了探針L-HF3。由于受體熒光團TCF的長波或近紅外發(fā)射特性,該探針具有更低的熒光檢測背景與更小的光譜重疊程度,因此有更大程度的比率熒光變化及更可靠的檢測結(jié)果。此外,探針L-HF3可有效地檢測SO_2有機供體原位生成的SO_2及其衍生物。第六章,基于對ESIPT過程"開-關(guān)"狀態(tài)的調(diào)控,以黃酮醇為熒光團,以二硫鍵與酯基為反應(yīng)活性位點,設(shè)計合成了一例H_2S熒光探針HF-PBA。該探針本身由于ESIPT過程被阻斷,只在400 nm波長處有微弱的熒光。H_2S進攻二硫鍵引發(fā)的二步連串反應(yīng)將具有ESIPT效應(yīng)的黃酮醇分子游離出來,從而產(chǎn)生酮式(強)與醇式(弱)兩個結(jié)構(gòu)的熒光發(fā)射峰。該探針具有高的反應(yīng)活性、高的靈敏度與低的細(xì)胞毒性,可應(yīng)用于細(xì)胞內(nèi)H_2S的成像研究。本論文主要創(chuàng)新點:1.以半花菁、黃酮醇等結(jié)構(gòu)為基礎(chǔ),以FRET、ESIPT與ICT等原理為信號調(diào)控方式,以化學(xué)反應(yīng)為識別方式,設(shè)計合成了一系列在生理條件下檢測SO_2衍生物比率熒光探針及一個H_2S比率熒光探針。并成功地將這些探針應(yīng)用于細(xì)胞內(nèi)成像研究。2.首次將半花菁熒光基團引入FRET體系中,半花菁作為供體能量及檢測對象的受體,構(gòu)建出新的FRET模板。該FRET模板是通過對受體結(jié)構(gòu)內(nèi)ICT過程的阻斷來實現(xiàn)比率調(diào)控的,具有高的能量傳遞效率以及較大的Stokes位移。通過對細(xì)胞內(nèi)源亞硫酸氫鹽的檢測,首次揭示了肝癌細(xì)胞與正常肝細(xì)胞的內(nèi)源亞硫酸氫鹽濃度具有較大差異,為在細(xì)胞水平上診斷肝癌提供了新的思路。3.首次將ESIPT過程與FRET過程聯(lián)系起來,構(gòu)建了 ESIPT輔助的FRET模板,具有很大的(偽)Stokes位移值與高的能量轉(zhuǎn)移效率,并且豐富了 FRET型比率探針供體的結(jié)構(gòu)多樣性。
[Abstract]:In vivo, sulfur dioxide (SO_2) can be produced from decomposition of sulfur-containing amino acids, hydrogen sulfide (H_2S) oxidation enzymatic process. The study found that low concentration of SO_2 in blood pressure regulation, play an important role in inflammation and other physiological and pathological processes. And nitric oxide (NO) molecules are similar to SO_2, is also considered a biological gas signal molecule. Because the SO_2 soluble form of sulfite and bisulfite in water, so its physiological function is realized by this kind of SO_2 derivatives. On the other hand, SO_2 derivatives are widely used in food antioxidant, antibacterial agents, such as SO_2 Wine as antibacterial agent and so on. But because of the existing detection methods limit, physiological function and transformation process of SO_2 and its derivatives concrete is not very clear. Therefore, the development needs of high sensitivity, high resolution, real time detection method for in situ analysis has good fluorescent light. Good biological compatibility, has received wide attention in the field of biology and medicine. Organic small molecule fluorescent probe has a short development cycle, good light stability, low toxicity, simple operation and visual signal and other advantages, in recent years the rapid development of SO_2, including many organic small molecule fluorescent probe derivatives based on SO_2 have also been reported. The reported derivatives fluorescent probe is based on single wavelength fluorescence intensity change (increase or decrease) detection. A single signal is susceptible to test system, probe concentration, instrument parameters of external factors such as interference and affect the accuracy of the analysis results. The ratio of fluorescence probe with two fluorescence emission peaks, in SO_2 derivatives under the effect of changes in at least one fluorescence peak intensity. By cross referencing the probe molecule inside, can effectively eliminate the interference from external factors, so as to improve the analysis The accuracy ratio fluorescence probe contains a fluorophore or two different fluorophores. The signal regulation mechanism of ratiometric fluorescent probes of single fluorophores of intramolecular charge transfer (ICT) and excited state intramolecular proton transfer (ESIPT), the main control signal ratio fluorescent probe double fluorescence mechanism the group has the fluorescence resonance energy transfer (FRET) and cross bond energy transfer (TBET). Flaonols structure is a typical ESIPT type fluorescent dyes. Because of its large displacement Stokes flavonols, biofilm good permeability and low toxicity and attracted widespread attention. Based on the fluorescence probe of flavonols and its derivatives the most common is controlled by the ESIPT involved in the process of "on-off" implementation of the.FRET process must have two fluorescent groups, one as the energy donor and another half flowers as energy acceptor. Cyanine fluorophore with high extinction coefficient, high fluorescence quantum yield, visible light excitation and emission characteristics of long wavelength region, easy structure modification, as the use of energy in the system of FRET receptor potential. In this thesis, we are committed to the study of ratio fluorescent probe design, synthesis and application. The main mechanism of signal ratio changes in FRET and ESIPT, with hemicyanine and flavonols as the main structure of the fluorescent probe, four SO_2 derivatives and a H_2S probe was designed and synthesized, and studied their fluorescence properties and the application in biological detection. The first chapter briefly introduces the basic concept of the fluorescence probe changes the type and regulation principle of fluorescence signal; summarizes the latest progress of SO_2 derivatives as fluorescent probe; reviewed the latest progress of fluorescent probe hemicyanine and flavonols based on the structure. In the second chapter, design and synthesis The ratio of detection into a hemicyanine as "energy detection object" double receptor FRET new templates and application and SO_2 derivatives. The FRET system has the characteristics of high efficiency ratio and significant energy transfer. At the same time probe HCy-D targets mitochondria, and through the detection of the cell endogenous hydrogen sub salt sulfuric acid, distinguish between normal liver cells (L-02) and cancerous liver cells (HepG2). In the third chapter, the synthesis of a new template FRET cases ratiometric fluorescent and colorimetric probe based on HCy-NBD to detect SO_2 derivatives. The probe with hemicyanine groups as receptors, with NBD group as the donor, can be realized sensitive, rapid detection and identification of naked eye. In the fourth chapter, the FRET template based introduction of flavonol group has ESIPT as the energy donor, and thus linked to ESIPT process and FRET process. Due to the ESIPT effect, the displacement of Stokes donor flavonol molecules up to 185 Nm; and due to the combined effect of ESIPT and FRET effect, by the experience of pseudo Stokes displacement of large value, it is based on the template effect before and after the probe and detection of objects will be reflected in (pseudo) Stokes L-HF1 probe displacement. The selection of SO_2 derivatives with high selectivity of the reaction of hemicyanine as energy acceptor. Has good selectivity and sensitivity. The structure of the flavonols into hydroxyl ester to inhibit ESIPT process, not the compound L-HF2 molecule was detected in the FRET process, which further confirms the important role of EISPT assisted FRET process. Also, the imaging probe can be applied to the intracellular hydrogen sulfate, reflects the the ratio of good characteristics and low toxicity. In the fifth chapter, based on the ESIPT auxiliary FRET template, in order to effectively reduce the donor emission peaks overlap with the receptor of emission peaks, two increase in fluorescence emission peak resolution, design A probe of the L-HF3. due to the long wave acceptor fluorophore TCF or near infrared emission characteristics, spectral overlap and background fluorescence detection of the probe with smaller lower, so the ratio of fluorescence changes more and more reliable detection results. In addition, the probe L-HF3 can effectively detect SO_2 SO_2 in situ organic donor and its derivatives the sixth chapter, based on the regulation of ESIPT during the "on-off" state, with flavonols as fluorophore, with two disulfide bond and ester reactive sites, designed and synthesized a H_2S fluorescent probe HF-PBA. was blocked by the probe itself in the ESIPT process, the flavonol molecule two step consecutive reaction only at 400 nm wavelength with fluorescent.H_2S weak offensive of the two disulfide bonds with ESIPT effect free, resulting in the keto alcohol (strong) and type two (weak) structure of the emission peak. The probe with high response Activity, high sensitivity and low cytotoxicity, imaging studies can be applied to the H_2S cells. The main innovations of this dissertation: 1. hemicyanine, flavonol structure based on FRET, ESIPT and ICT principle of signal regulation, the chemical reaction for the identification, design and synthesis. In a series of physiological conditions to detect SO_2 derivative ratio fluorescence probe and a H_2S ratio of fluorescence probe. And successfully applied to these probes to study the intracellular.2. imaging for the first time hemicyanine fluorophore introduced in FRET system, hemicyanine as donor and energy detection object receptor, construct new FRET templates the FRET template by blocking the receptor structure of ICT in the process to achieve the ratio of regulation, has the advantages of high energy transfer efficiency and large displacement of Stokes. Through the detection of endogenous bisulfite, revealed for the first time in hepatocellular carcinoma cells With large differences in endogenous bisulfite concentration and normal liver cells, for the diagnosis of liver cancer at the cellular level provides a new idea of.3. for the first time will be linked to the ESIPT process and FRET process, construct the ESIPT assisted FRET template has great (pseudo) Stokes displacement efficiency and high energy transfer, and the rich the FRET type of structural diversity. The ratio of donor probe

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：O657.3

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