本文選題：雙光子熒光探針 + 比率型　； 參考：《安徽大學(xué)》2017年碩士論文

【摘要】：近年來,熒光化學(xué)探針由于其具有簡(jiǎn)單方便、靈敏度高、選擇性好、實(shí)時(shí)檢測(cè)以及良好的生物相容性等優(yōu)點(diǎn),被認(rèn)為是痕量檢測(cè)生物體內(nèi)物種如陰陽離子、小分子信號(hào)分子以及蛋白質(zhì)等最有前途的方法之一。熒光探針是通過觀察和檢測(cè)目標(biāo)樣品溶液顏色或者熒光信號(hào)的變化來實(shí)現(xiàn)對(duì)特定樣品的定性或定量分析,愈來愈多地被應(yīng)用于環(huán)境檢測(cè)、生命醫(yī)學(xué)以及材料科學(xué)等領(lǐng)域。單光子熒光探針,由于激發(fā)波長(zhǎng)較短,穿透深度較淺,很難被應(yīng)用于實(shí)現(xiàn)在組織或者活體內(nèi)跟蹤檢測(cè)目標(biāo)離子或分子。最近,雙光子顯微成像(TPM)已經(jīng)成為一種領(lǐng)先而又成熟的科學(xué)技術(shù)。在生物研究中,它具有單光子顯微成像(OPM)所不具備的很多優(yōu)點(diǎn),如近紅外激發(fā),更優(yōu)異的空間定位效果,更深的穿透深度以及更低的光漂白和光致毒性等,成為在生物醫(yī)學(xué)研究中舉足輕重的成像技術(shù)。此外,比率型的熒光探針,通過測(cè)量?jī)煞N不同發(fā)射波長(zhǎng)的熒光強(qiáng)度的比值來消除環(huán)境因素如濃度、光漂白和背景干擾(pH值、粘度、極性、溫度等等)的影響以實(shí)現(xiàn)定量檢測(cè)。具有更加理想的光學(xué)信號(hào)的雙光子熒光探針的開發(fā)與應(yīng)用,已經(jīng)引起了科研工作者的關(guān)注,成為重要的研究領(lǐng)域和方向。焦磷酸根(PPi)是生物體中一種很重要的陰離子,它參與了許多細(xì)胞內(nèi)的生化反應(yīng)過程,如細(xì)胞內(nèi)ATP水解、DNA和RNA的聚合反應(yīng)以及其它許多生命代謝過程。此外,PPi的濃度水平還與許多疾病有很大的關(guān)系。當(dāng)PPi水平異常會(huì)引起血管鈣化,會(huì)導(dǎo)致嚴(yán)重的健康問題。而且,醫(yī)學(xué)研究表明患有脫水焦磷酸鈣沉積癥(CPPD)晶化和軟骨鈣化的患者體內(nèi)滑液中有較高濃度的PPi。因此,監(jiān)測(cè)生物體內(nèi)的PPi是具有重要的意義的。甲醛(FA)是一種常見的致癌和致畸物質(zhì),已經(jīng)被世界衛(wèi)生組織確定為潛在的變態(tài)反應(yīng)源。作為一種化學(xué)污染物,甲醛造成的室內(nèi)空氣、大氣環(huán)境和食品污染會(huì)導(dǎo)致甲醛中毒,造成頭暈、頭痛或惡心、嘔吐,嚴(yán)重的會(huì)引起記憶力減退,甚至死亡。在大多數(shù)生物有機(jī)體中,一些氨基酸和外源性物質(zhì),在脫甲基化酶或氧化酶的催化作用下,發(fā)生新陳代謝,也會(huì)產(chǎn)生內(nèi)源性的甲醛。在大腦組織內(nèi),正常濃度的甲醛通過DNA脫甲基過程,在長(zhǎng)期記憶的存儲(chǔ)、保存和檢索上有著至關(guān)重要的作用。然而,人體內(nèi)的甲醛含量超過一定劑量時(shí),會(huì)引起許多疾病,包括阿爾茨海默病、神經(jīng)源性炎癥、過敏性肺炎、哮喘癥狀,心血管疾病和癌癥等。因此,開發(fā)一種有效的監(jiān)測(cè)生物體系中甲醛的熒光探針也是一個(gè)非常有意義的課題。本文在查閱大量參考文獻(xiàn)和深入研究本課題組工作的基礎(chǔ)上,分別基于香豆素和喹啉熒光團(tuán)設(shè)計(jì)并合成了選擇性識(shí)別與檢測(cè)PPi和FA的雙光子熒光探針PC和MQAP,并通過核磁氫譜(1HNMR)、核磁碳譜(13CNMR)以及質(zhì)譜等方法對(duì)所得目標(biāo)化合物進(jìn)行了結(jié)構(gòu)表征。還進(jìn)一步研究了它們的光學(xué)性質(zhì),識(shí)別機(jī)理和在生物體系中活體成像應(yīng)用等。一、合成并深入探究了一種基于香豆素母體的用于檢測(cè)焦磷酸根的具有"ON-OFF-ON"熒光信號(hào)的雙光子熒光探針PC,通過向香豆素母體框架引入對(duì)甲氧基苯乙炔,增加了香豆素母體的共軛結(jié)構(gòu),從而增加了其有效雙光子共軛面積和吸收截面。探針PC的香豆素母體的羧基和二胺甲基吡啶結(jié)合后的識(shí)別基團(tuán),能實(shí)現(xiàn)對(duì)Cu2+的選擇性識(shí)別,并形成"ON-OFF"型的熒光淬滅的檢測(cè)信號(hào)。在PCCu配合物溶液中,加入焦磷酸根PPi后,體系的熒光隨著PPi濃度增大恢復(fù)到之前的熒光強(qiáng)度,形成"OFF-ON"型的熒光檢測(cè)信號(hào)。探針在PCR過程中也得到了成功地應(yīng)用。由于其細(xì)胞毒性小而且細(xì)胞通透性良好,可以應(yīng)用于細(xì)胞成像,也第一次應(yīng)用于PPi的組織成像和斑馬魚活體成像實(shí)驗(yàn)。二、設(shè)計(jì)并合成了一個(gè)以喹啉為母體的基于2-aza-cope重排的檢測(cè)甲醛分子的比率型雙光子熒光探針MQAP。我們同樣在喹啉母體框架六位上接一個(gè)對(duì)甲氧基苯乙炔,從而極大地拓展了喹啉的共軛結(jié)構(gòu),增大了熒光基團(tuán)的雙光子吸收截面。在喹啉母體的醛基位置直接引入烯丙氨基(homoallylamino)作為甲醛的選擇性反應(yīng)基團(tuán)。探針MQAP的烯丙氨基與甲醛反應(yīng)形成亞胺中間體,進(jìn)一步發(fā)生2-aza-cope重排和水解,最后生成強(qiáng)吸電子能力的醛基。在醛基和甲氧基的推電子的共同作用下,促進(jìn)了探針分子的分子內(nèi)電荷轉(zhuǎn)移(ICT)過程,從而探針的發(fā)射熒光光譜表現(xiàn)了紅移現(xiàn)象(85nm)。紫外吸收,熒光光譜,理論計(jì)算以及高效液相色譜和質(zhì)譜分析等方法驗(yàn)證了這一機(jī)理。探針MQAP的低毒性和較好的生物相容性,可成功應(yīng)用于在活細(xì)胞和斑馬魚活體內(nèi)的雙光子成像。
[Abstract]:In recent years, fluorescent chemical probes have been considered as one of the most promising methods for detecting species such as Yin and Yang, small molecule signal and protein, due to their advantages of simple convenience, high sensitivity, good selectivity, real-time detection and good biocompatibility. The change of the color of the sample solution or the change of the fluorescence signal to realize the qualitative or quantitative analysis of a specific sample, and more and more used in the fields of environmental detection, life medicine and material science. Single photon fluorescence probe is difficult to be applied to the tracking of tissue or in vivo due to the short excitation wavelength and the shallow penetration depth. Detection of target ions or molecules. Recently, two-photon microscopy (TPM) has become a leading and mature science. In biological research, it has many advantages that single photon microscopy (OPM) does not possess, such as near infrared excitation, better spatial positioning effect, deeper penetration depth, and lower photobleaching and photoluminescence. Toxicity, etc., has become an important imaging technique in biomedical research. In addition, the ratio type of fluorescence probe can eliminate the influence of environmental factors such as concentration, photobleaching and background interference (pH value, viscosity, polarity, temperature, etc.) by measuring the ratio of fluorescence intensity of two different emission wavelengths to achieve quantitative detection. The development and application of two photon fluorescence probes for optical signals have attracted the attention of researchers and become an important research field and direction. PPi is a very important anion in the organism. It participates in the biochemical reactions in many cells, such as intracellular ATP hydrolysis, the polymerization of DNA and RNA and its reaction. It has many life metabolic processes. In addition, the concentration level of PPi is also related to many diseases. When abnormal PPi levels cause vascular calcification, it can cause serious health problems. Moreover, medical studies have shown that there is a high concentration of PPi. in synovial fluid in patients with calcium dehydrated calcium phosphate deposition (CPPD) crystallization and cartilage calcification. Monitoring PPi in organisms is of great significance. Formaldehyde (FA) is a common carcinogenic and teratogenic substance that has been identified by the WHO as a potential source of allergy. As a chemical contaminant, formaldehyde caused by indoor air, atmospheric environment and food contamination can lead to formaldehyde poisoning, causing dizziness, headache, or nausea. Vomiting, seriously causing memory loss, and even death. In most biological organisms, some amino acids and exogenous substances metabolize and produce endogenous formaldehyde under the catalysis of demethylation or oxidase. In the brain tissue, the normal concentration of formaldehyde is demethmethylation through DNA, in long-term memory. It has a vital role in storage, storage and retrieval. However, when the content of formaldehyde in the human body exceeds a certain dose, it causes many diseases, including Alzheimer's disease, neurogenic inflammation, allergic pneumonia, asthma symptoms, cardiovascular diseases and cancer. On the basis of consulting a large number of references and studying the work of this group, this paper designs and synthesizes two photon fluorescence probes, PC and MQAP, which are selective identification and detection of PPi and FA based on coumarin and quinoline fluorophores respectively, and through nuclear magnetic hydrogen spectrum (1HNMR), nuclear magnetic carbon spectroscopy (13CNMR) and The structure of the target compounds was characterized by mass spectrometry. The optical properties, recognition mechanism and the application of living body imaging in the biological system were further studied. First, a two photon fluorescent probe PC based on the coumarin matrix for the detection of pyrophosphoric acid with "ON-OFF-ON" fluorescence signal was explored. By introducing Methoxybenzene into the coumarin matrix, the conjugated structure of the coumarin matrix is increased, and the effective two photon conjugation area and the absorption cross section are increased. The recognition group of the carboxyl group of the coumarin matrix of the probe PC and the two amine methyl pyridine can be identified, and the "ON-OFF" type can be formed. In the PCCu complex solution, after the addition of pyrophosphate PPi, the fluorescence of the system is restored to the previous fluorescence intensity as the concentration of PPi is increased to form a "OFF-ON" type of fluorescence detection signal. The probe has also been successfully applied in the PCR process. Cell imaging is also used for the first time in tissue imaging of PPi and in zebrafish living imaging experiments. Two, a ratio type two-photon fluorescence probe, MQAP., based on 2-aza-cope rearrangement based on quinoline, is designed and synthesized. We also connect a pair of methoxybenzenes on the six bit of the quinoline matrix. The earth expands the conjugated structure of quinoline and increases the two-photon absorption cross section of the fluorescent group. The alkenamine (homoallylamino) is introduced directly as the selective reaction group of formaldehyde in the aldehyde group of the quinoline matrix. The allyl amino group of the probe MQAP forms the intermediate body of the imide, and the 2-aza-cope rearrangement and hydrolysis will occur further. Finally The aldehyde group, which produces the strong electron absorption capacity, promotes the intramolecular charge transfer (ICT) process of the probe molecules under the interaction of aldehyde and methoxy electrons. The fluorescence spectra of the probe show the red shift (85nm), UV absorption, fluorescence spectra, theoretical calculation, and high performance liquid chromatography and mass spectrometry analysis. This mechanism, the low toxicity and good biocompatibility of the probe MQAP, can be successfully applied to two-photon imaging in living cells and zebrafish.
【學(xué)位授予單位】：安徽大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：O657.3

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本文編號(hào)：2103985