【摘要】：氧氣(O_2)對(duì)于所有的需氧生物來(lái)說(shuō)都是必需的,在酶或金屬離子等的作用下,O_2可以被轉(zhuǎn)化成一系列活性代謝產(chǎn)物,活性氧(ROS)就是其中最重要的一種產(chǎn)物。ROS包括過(guò)氧化氫(H202),超氧陰離子(O_2·-),羥基自由基(·OH),單線(xiàn)態(tài)氧(1C2),過(guò)氧自由基(ROO·),次氯酸及次氯酸鹽(HOC1/-OCl)等。許多研究表明,ROS在生命活動(dòng)中是重要的信號(hào)分子,但是,過(guò)量的ROS可以導(dǎo)致氧化損傷的累積,這些損傷可能會(huì)導(dǎo)致細(xì)胞凋亡、老化、癌變等疾病。H202是非常重要的一種ROS,相對(duì)于其它的ROS,H202比較穩(wěn)定。正常狀態(tài)下,細(xì)胞內(nèi)的H202處于相對(duì)低的水平。但是,如果細(xì)胞內(nèi)產(chǎn)生了過(guò)量的h2O_2,它將會(huì)與低價(jià)態(tài)的金屬離子(如:Fe2+、Cu2+)反應(yīng),產(chǎn)生高反應(yīng)活性的·OH,造成細(xì)胞功能紊亂。O_2·-是另一種十分關(guān)鍵的ROS,是其它ROS的重要來(lái)源。因此,探究檢測(cè)h2O_2/O_2·-的有效方法一直處于不斷的研究之中。其中,熒光分析因具有操作簡(jiǎn)單、靈敏度高、選擇性好等優(yōu)點(diǎn),成為近年來(lái)研究的熱點(diǎn)。比率型熒光探針是以?xún)蓚�(gè)波長(zhǎng)處熒光強(qiáng)度的比值作為響應(yīng)信號(hào),信號(hào)強(qiáng)度與探針濃度、光源強(qiáng)度、儀器的靈敏度等因素?zé)o關(guān),所以,能夠有效地防止背景干擾,提高檢測(cè)的靈敏度�；谝陨媳尘�,我們合成了基于硼酸酯的熒光分子探針,分別實(shí)現(xiàn)了對(duì)H202的比率型檢測(cè)和對(duì)O_2·-的雙功能靈敏性檢測(cè)。同時(shí)基于酶特異性催化氧化反應(yīng),比率型聚合物納米探針可用于能夠產(chǎn)生H202的生物基質(zhì)(如:葡萄糖、膽堿、膽固醇、乳酸)的檢測(cè)。進(jìn)而,利用比率型聚合物納米探針實(shí)現(xiàn)了對(duì)血清中葡萄糖的檢測(cè)。論文的具體內(nèi)容如下:第一章緒論首先本章介紹了 ROS及其相關(guān)研究,然后討論了 H202/02·-及其研究現(xiàn)狀。隨后介紹了聚合物膠束的相關(guān)知識(shí),最后對(duì)于本論文研究工作的目的和意義作了表述。第二章基于ARS-硼酸酯衍生物的熒光探針對(duì)H202的檢測(cè)本工作利用硼酸衍生物與ARS結(jié)合后可以產(chǎn)生熒光的特性及H202可以將苯硼酸酯轉(zhuǎn)化成苯酚從而使熒光淬滅的性質(zhì),實(shí)現(xiàn)了對(duì)H202的檢測(cè)。首先,選用帶有不同取代基的苯硼酸衍生物與ARS結(jié)合,形成ARS-硼酸酯衍生物熒光分子探針,檢測(cè)結(jié)合后的紫外吸收及熒光發(fā)射光譜。通過(guò)熒光光譜我們計(jì)算出了 ARS與苯硼酸衍生物及硼酸(BA)的結(jié)合常數(shù)。其次,將探針與H2O_2作用,測(cè)定反應(yīng)后的紫外吸收及熒光發(fā)射光譜。結(jié)果表明,苯硼酸衍生物取代基不同,與ARS結(jié)合后形成的探針對(duì)H202響應(yīng)的靈敏度也不同。我們選用有最大響應(yīng)的ARS-4-羧基-3-氟-苯硼酸(ARS-FPBA)實(shí)現(xiàn)了對(duì)H202的靈敏性和選擇性的檢測(cè)。第三章自組裝比率型聚合物納米探針對(duì)H2O_2的高選擇性檢測(cè)本工作我們構(gòu)建了一種基于膠束的比率型熒光納米探針。首先,將FPBA及7-羥基香豆素(HC)通過(guò)縮合反應(yīng)修飾在聚合物兩親性分子二硬脂酰磷脂酰乙醇胺-氨基聚乙二醇(DSPE-PEG-NH2)的親水端,形成DSPE-PEG-FPBA和DSPE-PEG-HC。PEG-FPBA及PEG-HC是親水端,DSPE是疏水端。當(dāng)聚合物加入到水相中時(shí),可形成球狀水溶性膠束。然后,將膠束與ARS混合后,ARS與FPBA形成ARS-FPBA而結(jié)合在膠束的表面。在405 nm波長(zhǎng)光的激發(fā)下,HC和ARS-FPBA的熒光發(fā)射波長(zhǎng)分別在450 nm和600 nm。ARS-FPBA的吸收峰在455 nm,與HC的發(fā)射峰(450 nm)有較大重疊,所以,HC與ARS-FPBA之間可以發(fā)生熒光共振能量轉(zhuǎn)移(FRET)。將膠束與H202共同孵育后,ARS從膠束上脫離,FRET效率降低,在450 nm處HC的熒光增強(qiáng),在600 nm處ARS-FPBA的熒光減弱,實(shí)現(xiàn)了對(duì)H202的比率型熒光檢測(cè)。此外,我們還實(shí)現(xiàn)了對(duì)血清中葡萄糖的檢測(cè)。第四章熒光/比色法雙功能探針的合成及其對(duì)于O_2·-的靈敏性檢測(cè)本工作中,通過(guò)在二氰亞甲基-4H-苯并吡喃(DCM)分子中引入苯硼酸基團(tuán),合成了一種既可以用比色法又可以通過(guò)熒光法檢測(cè)O_2·-的雙功能探針。我們通過(guò)甲基與醛基之間的縮合反應(yīng)合成了探針DCM-PBA,并檢測(cè)了其對(duì)O_2·-的響應(yīng)。與O_2·-作用后,隨著O_2·-濃度的增大,探針位于560 nm處的吸光度增加,溶液顏色由黃色變?yōu)樗{(lán)色。同時(shí),在波長(zhǎng)為560 nm光的激發(fā)下,探針位于700 nm處的熒光逐漸增強(qiáng)。因此,可以通過(guò)比色和熒光的方法來(lái)同時(shí)實(shí)現(xiàn)對(duì)O_2·-的檢測(cè)。
[Abstract]:Oxygen (O_2) is necessary for all aerobic organisms. Under the action of enzymes or metal ions, O_2 can be converted into a series of active metabolites. Reactive oxygen species (ROS), the most important product of which,.ROS includes hydrogen peroxide (H202), superoxide anion (O_2), hydroxyl radical (OH), single state oxygen (1C2), and superoxide self. ROO, hypochloric acid and hypochlorite (HOC1/-OCl). Many studies have shown that ROS is an important signal molecule in life activities. However, excessive ROS can lead to the accumulation of oxidative damage. These damage may lead to cell apoptosis, aging, cancer and other diseases,.H202 is a very important ROS, relative to the other ROS, H202 comparison. Stable. Under normal conditions, the H202 in the cell is relatively low. However, if the cell produces an excessive amount of h2O_2, it will react with the low valence metal ions (such as Fe2+, Cu2+) and produce a highly reactive. OH, causing cell dysfunction.O_2. It is another crucial ROS, an important source of other ROS. The effective methods for detecting h2O_2/O_2 - - have been in constant research. Among them, fluorescence analysis has become a hot topic in recent years because of its advantages of simple operation, high sensitivity and good selectivity. The ratio type fluorescence probe is a response signal with the ratio of fluorescence intensity at two wavelengths, signal intensity and probe concentration, and the intensity of light source. The sensitivity of the instrument is independent, so it can effectively prevent background interference and improve the sensitivity of detection. Based on the above background, we synthesized a fluorescence molecular probe based on borate ester, and realized the ratio detection of H202 and the dual functional sensitivity detection of O_2. The rate type polymer nanoprobe can be used for the detection of biological matrix (such as glucose, choline, cholesterol and lactic acid) that can produce H202. Then, the detection of glucose in serum is realized by using the ratio polymer nanoprobe. The contents of the thesis are as follows: first chapter of the chapter introduced the ROS and its related research, and then discussed it. H202/02 - and its current research status. Subsequently, the related knowledge of polymer micelles was introduced. Finally, the purpose and significance of the research work in this paper were expressed. The second chapter based on the detection of H202 based on the fluorescence probe of borate derivative ARS- in this work, the properties of fluorescence can be produced by the combination of boric acid derivatives with ARS and H202 can be used. H202 was detected by the conversion of benzyl borate to phenol and fluorescence quenching. First, the derivatives of benzboric acid with different substituents were combined with ARS to form a ARS- borate derivative fluorescent molecular probe, which was used to detect the UV absorption and fluorescence emission spectra after the combination. The ARS and benzene were calculated by fluorescence spectra. The binding constants of boric acid derivatives and boric acid (BA) are determined. Secondly, the UV absorption and fluorescence emission spectra of the reaction are determined by the action of the probe and H2O_2. The results show that the substituent of the benzyl boric acid derivatives is different, and the sensitivity of the probe formed after the combination with ARS is different. We choose the ARS-4- carboxyl -3- fluoro benzyl boric acid (A) with the maximum response (A). RS-FPBA) the sensitivity and selectivity of H202 were detected. In the third chapter, the high selectivity detection of H2O_2 by self assembled polymer nano probe, we constructed a ratio based fluorescent nano probe based on micelles. First, the FPBA and 7- hydroxyl coumarin (HC) were modified in the polymer two amphiphilic molecules by condensation reaction. Two the hydrophilic end of the two stearyl phosphatidyl ethanolamine aminopolyethylene glycol (DSPE-PEG-NH2), forming DSPE-PEG-FPBA and DSPE-PEG-HC.PEG-FPBA and PEG-HC are hydrophilic ends and DSPE is the hydrophobic end. When the polymer is added to the water phase, the spherical water-soluble micelles can be formed. Then, the micelle is mixed with ARS and ARS and FPBA form ARS-FPBA to combine in micelle. Surface. Under the excitation of 405 nm wave long light, the fluorescence emission wavelengths of HC and ARS-FPBA are at 450 nm and 600 nm.ARS-FPBA respectively, and the absorption peaks are 455 nm, and the emission peaks of HC (450 nm) are overlapped greatly. Therefore, the fluorescence resonance energy transfer (FRET) can occur between HC and ARS-FPBA. The rate was reduced, the fluorescence of HC was enhanced at 450 nm and the fluorescence of ARS-FPBA was weakened at 600 nm. The ratio of fluorescence to H202 was detected. In addition, we also realized the detection of glucose in the serum. The fourth chapter of the fluorescence / colorimetric dual function probe and the detection of the sensitivity of the O_2. A bifunctional group is introduced into the benzo - DCM molecule, and a bifunctional probe, which can be used to detect O_2 - - by the colorimetric method and the fluorescence method, is synthesized. The probe DCM-PBA is synthesized by condensation reaction between methyl and aldehyde groups. The response of the probe to O_2 - - is detected. After the action of O_2. The probe is increased with the concentration of O_2. The absorbance at 560 nm is increased and the color of the solution changes from yellow to blue. At the same time, the fluorescence of the probe at 700 nm is gradually enhanced under the excitation of the wavelength of 560 nm light. Therefore, the detection of O_2 by colorimetric and fluorescence can be achieved simultaneously.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：O657.3

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