本文關(guān)鍵詞：基于核酸染料和銅納米材料的熒光傳感新方法　出處：《湖南大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 核酸染料 G-四鏈體 銅納米顆粒 細胞色素c 胰蛋白酶 堿性磷酸酶

【摘要】：熒光技術(shù)伴隨功能性染料和標記物的使用,使得生命科學(xué)中許多基本過程的檢測得以實現(xiàn),并且伴有高效、高靈敏度、可靠性強和重現(xiàn)性好等特點,例如生命活動中分子或離子之間的相互作用等。靈敏地檢測生物樣品中微量的目標分析物通常需要將探針標記熒光基團或在其中嵌入功能性的熒光染料等,SYBR GreenⅠ就是其中的一個例子。利用SYBR Green Ⅰ這種新型功能性染料可與核酸結(jié)合發(fā)射熒光的性質(zhì),我們可將其與探針結(jié)合對微量的待測物進行檢測。另外,由于納米材料所帶有的獨特的量子尺寸效應(yīng)和表面結(jié)構(gòu)效應(yīng)因而具有許多普通材料難以比擬的優(yōu)越性質(zhì),本文以納米材料中的一個分支金屬納米材料作為熒光標志物來分析小分子物質(zhì)。金屬納米材料具有許多優(yōu)越的性能,例如易于合成,生物相容性好等特點,受到了科學(xué)界的廣泛關(guān)注,這其中就包含了銅納米顆粒。銅納米顆粒具有合成簡單、制備時間短,其熒光強度隨著顆粒尺寸的變化可以得到調(diào)節(jié)的特點,因此適用于熒光生物傳感器及傳感方法的改進,在生物傳感器應(yīng)用的研究中獲得了較多關(guān)注。綜上所述,為了對生物傳感器的穩(wěn)定性和靈敏性做出改進,本文構(gòu)建了幾組熒光生物傳感器來實現(xiàn)對腺苷三磷酸、胰蛋白酶和堿性磷酸酶的檢測。主要內(nèi)容如下：(1)第2章,我們構(gòu)建了基于G-四鏈體的,無標記的熒光傳感方法用于三磷酸腺苷(ATP)的檢測。首先由ATP適配體與其互補鏈形成的雙鏈DNA被用作與ATP結(jié)合的核酸鏈。另外,SYBR Green Ⅰ (SGI)核酸染料被用作本方法的熒光探針,與此同時核酸外切酶Ⅲ (Exo Ⅲ)的加入降低了本方法的背景信號。當(dāng)沒有ATP存在時,SG Ⅰ會嵌入到雙鏈DNA中,隨后被核酸外切酶ⅢⅡ水解,產(chǎn)生一個較低的背景信號。當(dāng)有ATP分子存在時,雙鏈DNA中的ATP適配體鏈在遇到ATP分子時即會折疊成一個內(nèi)部含有ATP分子的G-四鏈體結(jié)構(gòu),該G-四鏈體結(jié)構(gòu)的形成抵抗了Exo Ⅲ的水解作用。SG I隨即嵌入到G-四鏈體結(jié)構(gòu)當(dāng)中,顯示了相比于水溶液中顯著增強的熒光信號。由于背景噪音的降低,本方法可以得到一個較高的信噪比,該傳感器在50 μM-5 mM的范圍內(nèi)呈現(xiàn)了一個較好的線性關(guān)系,檢測限為5 μM。(2)第3章,我們以銅納米顆粒(CuNPs)作為熒光探針,以低濃度的Cyt c作為胰蛋白酶水解的基質(zhì)構(gòu)建了一個熒光傳感方法檢測胰蛋白酶。這里以低濃度的Cyt c作為胰蛋白酶的水解基質(zhì),避免了Cyt c到CuNPs之間的電子轉(zhuǎn)移過程引發(fā)的CuNPs熒光猝滅。當(dāng)加入胰蛋白酶時,Cyt c被水解成小的多肽片段,釋放自由的半胱氨酸殘基。CuNPs遇到半胱氨酸殘基時,其中的Cu原子和半胱氨酸中的S原子之間形成金屬配位鍵得到一個不發(fā)光的復(fù)合物引起CuNPs熒光強度的降低。這個定量檢測胰蛋白酶的傳感方法在0.25 μg ml-1-1000 μg ml-1之間有一個線性的檢測范圍,并且檢測限可以達到42 ng ml-1。(3)第4章,我們開發(fā)了一個無需標記的檢測堿性磷酸酶(ALP)的方法。該方法是基于焦磷酸鹽(PPi)對聚T-DNA為模板合成的熒光銅納米簇(CuNPs)的抑制作用開發(fā)的熒光生物傳感器,是一個熒光增強的傳感方法。本方法取決于Cu2+與PPi之間強烈的相互作用,這將有效地阻止發(fā)光CuNPs的形成,使得體系的熒光強度降低。ALP作為PPi的水解酶,將會破壞Cu2+與PPi之間的絡(luò)合作用,從而加速CuNPs的形成。因此,這個體系的熒光強度與ALP的濃度大小呈正比關(guān)系。此方法簡單有效并且不需要熒光素的標記或者復(fù)雜的操作過程,顯示了較高的靈敏度,對ALP的傳感顯示了較高的選擇性。另外,我們也研究了磷酸鹽對ALP酶水解的抑制作用。本方法以PPi為基質(zhì),在診斷與ALP酶相關(guān)的疾病時可能會有潛在的應(yīng)用價值。
[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.

【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：O657.3
，

本文編號：1371702