基于核酸染料和铜纳米材料的荧光传感新方法

发布时间：2018-01-03 01:06

  本文关键词：基于核酸染料和铜纳米材料的荧光传感新方法　出处：《湖南大学》2016年硕士论文　论文类型：学位论文

  更多相关文章： 核酸染料 G-四链体 铜纳米颗粒 细胞色素c 胰蛋白酶 碱性磷酸酶

【摘要】：荧光技术伴随功能性染料和标记物的使用,使得生命科学中许多基本过程的检测得以实现,并且伴有高效、高灵敏度、可靠性强和重现性好等特点,例如生命活动中分子或离子之间的相互作用等。灵敏地检测生物样品中微量的目标分析物通常需要将探针标记荧光基团或在其中嵌入功能性的荧光染料等,SYBR GreenⅠ就是其中的一个例子。利用SYBR Green Ⅰ这种新型功能性染料可与核酸结合发射荧光的性质,我们可将其与探针结合对微量的待测物进行检测。另外,由于纳米材料所带有的独特的量子尺寸效应和表面结构效应因而具有许多普通材料难以比拟的优越性质,本文以纳米材料中的一个分支金属纳米材料作为荧光标志物来分析小分子物质。金属纳米材料具有许多优越的性能,例如易于合成,生物相容性好等特点,受到了科学界的广泛关注,这其中就包含了铜纳米颗粒。铜纳米颗粒具有合成简单、制备时间短,其荧光强度随着颗粒尺寸的变化可以得到调节的特点,因此适用于荧光生物传感器及传感方法的改进,在生物传感器应用的研究中获得了较多关注。综上所述,为了对生物传感器的稳定性和灵敏性做出改进,本文构建了几组荧光生物传感器来实现对腺苷三磷酸、胰蛋白酶和碱性磷酸酶的检测。主要内容如下：(1)第2章,我们构建了基于G-四链体的,无标记的荧光传感方法用于三磷酸腺苷(ATP)的检测。首先由ATP适配体与其互补链形成的双链DNA被用作与ATP结合的核酸链。另外,SYBR Green Ⅰ (SGI)核酸染料被用作本方法的荧光探针,与此同时核酸外切酶Ⅲ (Exo Ⅲ)的加入降低了本方法的背景信号。当没有ATP存在时,SG Ⅰ会嵌入到双链DNA中,随后被核酸外切酶ⅢⅡ水解,产生一个较低的背景信号。当有ATP分子存在时,双链DNA中的ATP适配体链在遇到ATP分子时即会折叠成一个内部含有ATP分子的G-四链体结构,该G-四链体结构的形成抵抗了Exo Ⅲ的水解作用。SG I随即嵌入到G-四链体结构当中,显示了相比于水溶液中显著增强的荧光信号。由于背景噪音的降低,本方法可以得到一个较高的信噪比,该传感器在50 μM-5 mM的范围内呈现了一个较好的线性关系,检测限为5 μM。(2)第3章,我们以铜纳米颗粒(CuNPs)作为荧光探针,以低浓度的Cyt c作为胰蛋白酶水解的基质构建了一个荧光传感方法检测胰蛋白酶。这里以低浓度的Cyt c作为胰蛋白酶的水解基质,避免了Cyt c到CuNPs之间的电子转移过程引发的CuNPs荧光猝灭。当加入胰蛋白酶时,Cyt c被水解成小的多肽片段,释放自由的半胱氨酸残基。CuNPs遇到半胱氨酸残基时,其中的Cu原子和半胱氨酸中的S原子之间形成金属配位键得到一个不发光的复合物引起CuNPs荧光强度的降低。这个定量检测胰蛋白酶的传感方法在0.25 μg ml-1-1000 μg ml-1之间有一个线性的检测范围,并且检测限可以达到42 ng ml-1。(3)第4章,我们开发了一个无需标记的检测碱性磷酸酶(ALP)的方法。该方法是基于焦磷酸盐(PPi)对聚T-DNA为模板合成的荧光铜纳米簇(CuNPs)的抑制作用开发的荧光生物传感器,是一个荧光增强的传感方法。本方法取决于Cu2+与PPi之间强烈的相互作用,这将有效地阻止发光CuNPs的形成,使得体系的荧光强度降低。ALP作为PPi的水解酶,将会破坏Cu2+与PPi之间的络合作用,从而加速CuNPs的形成。因此,这个体系的荧光强度与ALP的浓度大小呈正比关系。此方法简单有效并且不需要荧光素的标记或者复杂的操作过程,显示了较高的灵敏度,对ALP的传感显示了较高的选择性。另外,我们也研究了磷酸盐对ALP酶水解的抑制作用。本方法以PPi为基质,在诊断与ALP酶相关的疾病时可能会有潜在的应用价值。
[Abstract]:The fluorescence technology with the use of functional dyes and markers, which makes the detection of many basic processes in life science can be realized, and with high efficiency, high sensitivity, strong reliability and good repeatability, for example between molecules or ions in the life activity of interaction. The sensitive detection of trace analytes in biological samples usually needs to be labeled fluorophores or the embedding of functional fluorescent dyes, SYBR Green I is one example. Properties by using SYBR Green I of this new type of functional dyes can emit fluorescence with a nucleic acid binding, it can be combined with the probe of trace analytes were detected. In addition, due to the superior properties of nano materials with unique quantum size effect and surface effect and structure has many common materials incomparable, the nano materials in A branch of metal nano materials as fluorescent markers for the analysis of small molecules. The metal nano material has many superior properties, such as easy synthesis, good biocompatibility and other characteristics, has been widespread concern in the scientific community, which contains the copper nanoparticles. The copper nanoparticles with simple synthesis, preparation time in short, the fluorescence intensity changes with the particle size can be adjusted, so it is suitable for improved fluorescence biosensor and sensing method, received more attention in the research of biological sensor applications. In summary, in order to make improvements on the biological sensor stability and sensitivity, this paper constructs several groups of fluorescence biosensor three detection of adenosine phosphate, trypsin and alkaline phosphatase. The main contents are as follows: (1) the second chapter, we construct a G- chain based on the four body, fluorescent marker free Sensing method for adenosine triphosphate (ATP) detection. First by ATP double stranded DNA ligand complementary chain formation is used as a nucleic acid chain combined with ATP. In addition, SYBR Green 1 (SGI) nucleic acid dye was used as fluorescent probe of this method at the same time, exonuclease III (Exo III) to reduce the background signal this method. When there is no ATP, SG I will be embedded into the double stranded DNA, followed by exonuclease II hydrolysis, produces a lower background signal. With the existence of ATP molecules, the double stranded DNA ATP aptamer chain is folded into a chain structure of G- four the internal ATP containing molecules in ATP molecule, forming the G- four chain structure resistance to hydrolysis.SG Exo III I was embedded into G- four chain structure, shows the fluorescence signal compared to the aqueous solution significantly enhanced. Due to background noise reduction, the The method can get a higher signal-to-noise ratio, the sensor shows a good linear relationship in the range of 50 M-5 mM, the detection limit is 5 M. (2) in Chapter third, we use copper nanoparticles (CuNPs) as a fluorescence probe with low concentration of Cyt C as a matrix of trypsin the hydrolysis of construct a fluorescence sensing method for detection of trypsin. Here a low concentration of Cyt in the C matrix as the hydrolysis of trypsin, avoid the Cyt C to the electron transfer process between CuNPs CuNPs fluorescence quenching caused. When adding trypsin, Cyt C was hydrolyzed into small peptide fragments, the release of cysteine residues.CuNPs encountered free cysteine residues, metal ligand bond has a luminescent compound cause a decrease in the fluorescence intensity of CuNPs between S and Cu atom in the cysteine sensing. The quantitative detection of trypsin. Method has a linear detection range between 0.25 g ml-1-1000 g ml-1, and the detection limit can reach 42 ng ml-1. (3) in Chapter fourth, we developed a label free detection of alkaline phosphatase (ALP) method. The method is based on pyrophosphate (PPi) of poly T-DNA fluorescence the copper nanoparticles synthesized by the template (CuNPs) inhibition fluorescence biosensor development, is a method of sensing fluorescence enhancement. This method depends on the interaction between Cu2+ and PPi strongly, which will effectively prevent the formation of luminescence of CuNPs, the fluorescence intensity decreased the.ALP system as a PPi hydrolase complex the role of Cu2+ and PPi between will be destroyed, thus speeding up the formation of CuNPs. Therefore, the fluorescence intensity and the concentration of ALP in the system is proportional to the size. This method is simple and effective and does not require the fluorescein marker or the complex operation process,. It shows high sensitivity and high selectivity for ALP sensing. In addition, we also studied the inhibition effect of phosphates on ALP enzyme hydrolysis. This method takes PPi as substrate and has potential application value in diagnosing diseases related to ALP enzymes.

【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.3
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本文编号：1371702