本文選題：配位競爭 + 無酶信號放大��； 參考：《山東師范大學》2017年碩士論文

【摘要】：活性氧(ROS)是維持組織和器官的結(jié)構(gòu)和功能穩(wěn)態(tài)的一類非常重要的生物活性分子。其中,過氧化氫(H2O2)不僅是細胞信號轉(zhuǎn)導的第二信使,也是氧化應(yīng)激的重要標記物。過量產(chǎn)生的H2O2會導致氧化損傷,從而引起衰老和心血管疾病、糖尿病、癌癥等。因此,監(jiān)控細胞及活體內(nèi)H2O2的濃度變化和時空分布變得尤為重要。熒光成像技術(shù)靈敏度高、選擇性好、對生物體無損壞,已成為H2O2檢測的最佳方法。但是由于H2O2具有壽命短、反應(yīng)活性較大、濃度低以及對環(huán)境敏感等特點,使得研究起來存在一定困難。此外,目前用于檢測H2O2的小分子熒光探針大多存在選擇性差、響應(yīng)速度慢,熒光量子產(chǎn)率低、易光漂白等不足,難以真實反映細胞及活體內(nèi)H2O2的水平變化。因此,迫切需要發(fā)展高選擇、高靈敏、快速響應(yīng)的H2O2熒光探針或傳感器,從而實現(xiàn)細胞及活體內(nèi)H2O2實時、動態(tài)的可視化示蹤。納米技術(shù)的發(fā)展恰為實現(xiàn)這一目標提供了契機。納米材料由于其納米級別的尺寸,使其具有超常的化學反應(yīng)活性、分散與團聚能力、催化性能以及吸附能力。因此,可與DNA分子通過π-π堆積、靜電力及金屬配位等多種分子間作用力組裝成納米熒光探針/傳感器,為生物分子的檢測提供了有力工具。雖然它們在蛋白質(zhì)、核酸等大分子檢測方面已有很多應(yīng)用,然而用于小分子H2O2檢測成像的研究還鮮有報道。鑒于此,本文綜述了熒光探針在H2O2檢測方面的研究現(xiàn)狀及發(fā)展趨勢,并基于配位競爭和無酶信號放大機理,利用氧化鈰納米線(CeO2 NWs)和氧化石墨烯(GO)兩種納米材料與DNA和H2O2之間的特殊吸附作用,構(gòu)建了以下兩種H2O2納米熒光傳感器實現(xiàn)了細胞及活體內(nèi)H2O2的快速、高選擇性和高靈敏度的檢測:1、基于配位競爭,發(fā)展了一種由CeO2 NWs與羧基熒光素(FAM)標記的單鏈(ss)DNA組裝而成的H2O2納米熒光傳感器CeO2 NWs-DNA(CNWD)。由于CeO2 NWs的高熒光猝滅效率,通過磷酸根與Ce4+配位而吸附的DNA的熒光被猝滅;當遇到H2O2時,由于H2O2與Ce4+的更高配位能力,吸附的DNA被競爭下來,熒光恢復。而且CeO2 NWs高長寬比的一維納米結(jié)構(gòu)使得DNA吸附密度提高,隨之靈敏度也得到提高�；谶@些優(yōu)點,所設(shè)計的CNWD對H2O2具有瞬時響應(yīng)和高選擇性的特點,且能夠?qū)罴毎麅?nèi)的H2O2進行示蹤并能對斑馬魚氧化損傷產(chǎn)生的H2O2實時成像。這種傳感器提供了一種全新的簡便的檢測H2O2的方法,對于發(fā)現(xiàn)和研究H2O2調(diào)控的信號通路尤為重要。更重要的是,通過利用合適的納米材料和功能化的DNA,這種配位競爭的機理能夠用于多種活性小分子的檢測。2、基于無酶信號放大,發(fā)展了一種組合納米熒光傳感器CeO2 NWs/GO(CG)來對細胞內(nèi)的H2O2進行高靈敏熒光成像。首先設(shè)計合成了3條花菁5(Cy5)標記的、可發(fā)生雜交鏈式反應(yīng)(HCR)的ssDNA,分別為Flare、發(fā)卡型H1和H2。由于CeO2 NWs和GO均具有良好的熒光猝滅能力,且都能吸附ssDNA,因此我們用CeO2 NWs和Flare、GO和H1/H2分別合成了CF以及GH,然后將二者2:1組合為CG納米熒光傳感器。當H2O2存在的時候,可以使CeO2 NWs表面吸附的Flare釋放下來,發(fā)生熒光的恢復;釋放下來的Flare會接著與GO上吸附的H1/H2發(fā)生HCR形成長的雙鏈(ds)DNA,由于GO對DNA雙鏈的吸附力遠遠弱于單鏈,因此雙鏈上的Cy5也隨之遠離GO,熒光信號進一步增強。與納米熒光傳感器CNWD相比,CG顯著提高了H2O2檢測的靈敏度,檢測限低至100 nM,可以更好的用于細胞內(nèi)源性H2O2的檢測及熒光成像。這種基于無酶信號放大的方法還為實現(xiàn)細胞內(nèi)活性小分子的高靈敏檢測提供了新的思路。
[Abstract]:Active oxygen (ROS) is a very important class of bioactive molecules to maintain the structure and function homeostasis of tissues and organs. Among them, hydrogen peroxide (H2O2) is not only the second messenger of cell signal transduction, but also an important marker for oxidative stress. Excessive production of H2O2 can lead to oxygen damage and cause aging and cardiovascular disease, diabetes, Therefore, it is very important to monitor the concentration changes and space-time distribution of H2O2 in cells and living bodies. Fluorescence imaging technology has high sensitivity, good selectivity and no damage to the organism. It has become the best method for H2O2 detection. But because H2O2 has the characteristics of short life, large reaction activity, low concentration and environmental sensitivity, it has made research. There are some difficulties. In addition, most of the small molecular fluorescent probes used to detect H2O2 are poor in selectivity, slow response, low fluorescence quantum yield, and easy to light bleaching. It is difficult to truly reflect the level changes of H2O2 in cells and living bodies. Therefore, it is urgent to develop a H2O2 fluorescent probe with high selectivity, high sensitivity and rapid response. The development of nanotechnology provides an opportunity to achieve this goal. Nanomaterials, due to their nanoscale size, make them have abnormal chemical reaction activity, dispersing and reunion capacity, catalytic properties and adsorption capacity. Therefore, it can be used with DNA molecules. Over pion pion accumulation, static electricity and metal coordination and other intermolecular forces are assembled into nanofluorescence probes / sensors, which provide a powerful tool for the detection of biomolecules. Although they have been widely used in the detection of large molecules such as proteins and nucleic acids, there are few reports on the detection of small molecule H2O2 imaging. The current research status and development trend of fluorescent probe in H2O2 detection are reviewed. Based on the mechanism of coordination competition and non enzyme signal amplification, the following two kinds of H2O2 nanofluorescence sensors are constructed by using the special adsorption between two nanomaterials, CeO2 NWs and GO, and DNA and H2O2. The rapid, high selective and highly sensitive detection of H2O2 in the living body: 1, based on coordination competition, a H2O2 nanofluorescence sensor CeO2 NWs-DNA (CNWD), composed of CeO2 NWs and carboxyl fluorescein (FAM) labeled single strand (SS) DNA, was developed. The fluorescence is quenched; when the H2O2 is encountered, the adsorbed DNA is competitive and the fluorescence is restored due to the higher coordination ability of H2O2 and Ce4+. And the CeO2 NWs high width ratio one-dimensional nanostructure makes the DNA adsorption density increase and the sensitivity increases. Based on these advantages, the designed CNWD has instantaneous response and high selectivity to H2O2. It is characterized by the ability to trace the H2O2 in living cells and to real-time imaging of H2O2 in zebrafish oxidative damage. This sensor provides a new and simple method for detecting H2O2, which is particularly important for the discovery and study of the signal pathways regulated by H2O2. More importantly, the use of suitable nanomaterials and functional DN is important. A, the mechanism of the coordination competition can be used to detect.2 for a variety of small active molecules. Based on the non enzyme signal amplification, a combined nano fluorescence sensor CeO2 NWs/GO (CG) is developed for high sensitive fluorescence imaging of H2O2 in the cell. First, 3 cyanine 5 (Cy5) markers are designed and synthesized, and the ssDNA of the hybrid chain reaction (HCR) can occur. Flare, H1 and H2. have good fluorescence quenching ability and can adsorb ssDNA because of CeO2 NWs and GO. Therefore, we use CeO2 NWs and Flare, GO and H1/H2, respectively. The release of the fluorescence is restored; the release of Flare will follow the HCR - shaped double chain (DS) DNA with the HCR - shaped growth of the H1/H2 adsorbed on the GO, because the GO absorbability of the double chain is far weaker than the single strand, so the Cy5 on the double chain is far away from GO and the fluorescence signal is further enhanced. Degree, the detection limit is low to 100 nM, can be better used for cell endogenous H2O2 detection and fluorescence imaging. This method based on no enzyme signal amplification also provides a new way of thinking for high sensitivity detection of small cell active molecules.

【學位授予單位】：山東師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：O657.3;TP212
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本文編號：1806910