基于量子点的荧光传感器用于生物分析研究

发布时间：2018-06-12 20:36

本文选题：量子点 + 荧光生物传感器　；参考：《东南大学》2017年博士论文

【摘要】：量子点(QDs)是一种新型荧光纳米材料,具有众多独特的光物理特性,如高荧光量子产率、宽谱带吸收、窄谱带发射、发射峰连续可调,以及优良的抗光漂白性。基于QDs的荧光生物传感器具有高灵敏度和选择性,以及低成本、检测可视化等优势,对于开发高效的生物组分分析和疾病诊断方法具有重要的研究意义,目前成为近年来生物医学领域中研究的热点。本论文在合成了多种颜色、不同结构QDs的前提下,以QDs表面的生物偶联功能化为基础,构建了多种结构的荧光探针/传感器,并成功应用于DNA微阵列、编码微球阵列以及单细胞分析,分别实现了单核苷酸多态性(SNP)、microRNA(miRNA)以及细胞内Pb2+的检测。论文主要研究内容如下:1.合成了不同结构的多色量子点。利用金属有机法制备了 CdSe和InP单核量子点。使用连续离子层吸附反应(SILAR)法进行无机层包覆,制备了核/壳型CdSe/ZnS、CdSe/CdS/ZnS以及InP/ZnS量子点。此外,使用一锅法制备了三种合金型CdxSe1-xZnyS1-y、Cd1-xZnxSe和Cd1-xZnx/ZnS油溶性量子点。所制备的量子点具有较高荧光量子产率和较窄的半高全宽,并且发射波长连续可调(400nm-660nm),为量子点荧光传感器的构建提供了良好的材料基础。2.制备了链霉亲和素修饰的量子点(strAV-QD),并开发了该纳米荧光探针应用于分子信标(MB)微阵列分析的方法。所使用的MB在5'和3'末端分别修饰巯基和生物素,并通过巯基构建微阵列。当靶标DNA不存在时,MB处于"闭合"状态;当靶标DNA存在时,杂交反应"打开" MB。由于较大的尺寸,StrAV-QD只能标记"打开"状态而不标记"闭合"状态的MB,从而实现了对靶标DNA的高灵敏、高特异性检测,信噪比达到8.0。此方法避免了对靶标DNA的修饰并无需进行信号放大。通过应用StrAV-QD荧光探针标记MB微阵列进一步实现了对Ⅱ型糖尿病相关rs12255372 和 rs7903146 两种 SNP 的检测。3.制备了核酸适配体(apt)修饰的红色量子点(apt-rQD),并与绿色包硅量子点(gQD/SiO2)修饰的氧化石墨烯(GO)结合,构建了一种新型的双发射荧光传感器(apt-rQD@G0@gQD/SiO2),可实现对Pb2+的比率荧光检测。在该传感器中,InP/ZnS为荧光团,GO为淬灭剂,gQD/SiO2为参比荧光。当Pb2+不存在时,apt利用与GO之间的吸附作用拉近荧光团与猝灭剂之间的距离,通过纳米金属表面能量转移(NSET)作用导致量子点荧光猝灭。当apt与Pb2+特异性结合后,apt转化为G-四链体结构,使得apt-rQD脱离GO表面,NSET效率降低,rQD荧光增强,而gQD/SiO2的荧光保持不变,从而实现了比率荧光检测。由于apt-rQD、GO及gQD/SiO2良好的生物相容性,制备的传感器能够实现对细胞内Pb2+的检测。4.制备了二氧化硅包覆的量子点编码微球(Qbead@SiO2),并通过其表面的靶标结合以及链式杂交反应(HCR),结合小分子荧光染料染色技术,可用于对miRNA的多元、比色分析。在Qbead@SiO2表面,靶标DNA与捕获探针杂交后,可诱导两种发卡结构DNA发生约11个循环的HCR反应。HCR扩增导致小分子染料信号放大,其与Qbead@SiO2的编码荧光信号复合,进而实现了 Qbead@SiO2发光颜色变化的放大,从而允许进行有效的比色分析。这种Qbead@SiO2—HCR分析方法实现了对miRNA的高特异性、高灵敏分析,检测限降低至700 zmol,动态范围达到4个数量级。进一步基于四种编码微球可通过该比色分析方法实现对乳腺癌细胞中提取的四种miRNA的定量检测。
[Abstract]:Quantum dots (QDs) is a new type of fluorescent nanomaterial. It has many unique photophysical properties, such as Gao Yingguang quantum yield, wide band absorption, narrow band emission, continuous adjustable peak emission, and excellent photobleaching. QDs based fluorescent biosensor has the advantages of high sensitivity and selectivity, low cost, visual detection and so on. It has important research significance for the development of efficient biocomponent analysis and disease diagnosis methods. It has become a hot topic in the biomedical field in recent years. In this paper, based on the synthesis of various colors and different structures of QDs, based on the biological coupling function of QDs surface, a variety of fluorescent probes / sensing structures are constructed. It has been successfully applied to DNA microarray, coded microsphere array and single cell analysis to detect single nucleotide polymorphisms (SNP), microRNA (miRNA) and intracellular Pb2+. The main contents of this paper are as follows: 1. the polychromatic quantum dots with different structures were synthesized. The use of metal organic method was used to prepare the CdSe and InP mononuclear quantum dots. The nuclear / shell type CdSe/ZnS, CdSe/CdS/ZnS and InP/ZnS quantum dots were prepared by the continuous ion layer adsorption (SILAR) method, and three kinds of alloy CdxSe1-xZnyS1-y, Cd1-xZnxSe and Cd1-xZnx/ZnS oil soluble quantum dots were prepared by one pot method. The quantum yield and the narrower half height of the quantum yield were obtained. All wide and continuous tunable wavelengths (400nm-660nm) provide a good material basis for the construction of a quantum dot fluorescence sensor (.2.) to prepare a quantum dot (strAV-QD) modified by streptomycin, and develop a method for the application of the nanofluorescence probe to the molecular beacon (MB) microarray analysis. The MB is modified at the end of 5'and 3' respectively. When the target DNA does not exist, the MB is in a "closed" state. When the target DNA exists, the hybridization reaction "opens" MB. because of the larger size, StrAV-QD can only mark the "open" state and do not mark the "closed" state MB, thus realizing the high sensitivity, high specificity detection and signal-to-noise of the target DNA. To 8.0. this method avoids the need to amplify the target DNA modification. By using the StrAV-QD fluorescence probe labeled MB microarray, the red quantum dots (apt-rQD) modified with nucleic acid aptamer (APT) for the detection.3. of the two SNP of type II diabetes related rs12255372 and rs7903146 are further realized, and with the green package. A new type of double emission fluorescence sensor (apt-rQD@G0@gQD/SiO2) was constructed by the combination of silicon quantum dots (gQD/SiO2) modified graphene oxide (GO). The ratio fluorescence detection of Pb2+ was realized. In this sensor, InP/ZnS was a fluorophore, GO was a quenching agent, and gQD/SiO2 was a reference fluorescence. When Pb2+ did not exist, apt utilized the adsorption between GO. The distance between the fluorescence mass and the quenching agent is close to the fluorescence quenching of the quantum dots by the effect of energy transfer (NSET) on the surface of the nano metal. When apt and Pb2+ are specifically combined with Pb2+, apt is transformed into the structure of G- four chain body, which makes apt-rQD out of the GO surface, the efficiency of NSET is reduced, the rQD fluorescence is enhanced, and the fluorescence of gQD/SiO2 is kept unchanged, thus realizing the ratio fluorescein. Light detection. Due to the good biocompatibility of apt-rQD, GO and gQD/SiO2, the prepared sensor can realize the detection of intracellular Pb2+ by.4. to prepare the quantum dot encoded microsphere (Qbead@SiO2) coated with silica, and can be used in the combination of its surface target binding and chain hybridization reaction (HCR), combined with small molecular fluorescent dye dyeing technology. MiRNA's multivariate, colorimetric analysis. On the Qbead@SiO2 surface, after the target DNA is hybridized with the capture probe, it can induce two kinds of card structure DNA to produce about 11 cycles of HCR reaction,.HCR amplification leads to the amplification of the small molecule dye signal, which is combined with the coded fluorescence signal of Qbead@SiO2, thus realizing the amplification of the color change of the Qbead@SiO2 luminescence, thus allowing for the amplification of the color change of the Qbead@SiO2. Effective colorimetric analysis. This Qbead@SiO2 - HCR analysis method realizes high specificity of miRNA, high sensitivity analysis, the detection limit is reduced to 700 zmol, and the dynamic range reaches 4 orders of magnitude. Further quantitative detection of four kinds of miRNA extracted from breast cancer cells can be realized based on the four coded microspheres.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TP212

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