基于金纳米材料介导电致化学发光的蛋白激酶检测研究

发布时间：2018-08-29 10:44

【摘要】：作为一门新兴的分析方法,近年来电致化学发光(ECL)受到了越来越多的关注,而新材料的开发在ECL发展过程中发挥了非常重要的作用。纳米材料具有独特的光电性能、比表面积大以及良好的生物相容性,已在ECL生物传感领域得到广泛应用。蛋白激酶调控的磷酸化作用与许多生命活动密切相关,而蛋白激酶的异常表达更会导致多种疾病及癌症的发生,因此精确分析激酶活性及筛选相关激酶抑制剂对于疾病诊断和临床治疗具有十分重要的意义。本论文制备了多种纳米材料,利用这些纳米材料的光电性质构建新型ECL生物传感器,并用于检测蛋白激酶活性以及筛选激酶抑制剂,主要分为以下三部分工作:1.首先,介绍了电致化学发光的定义、特点以及发光试剂的分类,电化学发光作为一种与电化学技术有关的光学分析方法,兼具电化学分析和光学分析的特点。随后,概述了金属纳米材料和碳氮化合物的结构和性质及其在电化学发光领域中的应用。最后,综述了蛋白质磷酸化反应和蛋白激酶的作用,蛋白激酶作为参与细胞活动的重要激酶,其活性的异常将导致多种疾病的发生,在激酶活性的众多检测方法中,基于纳米材料的ECL分析方法具有独特优势。2.利用Au NCs对g-C_3N_4的ECL信号的增强效应,构建了一种免标记、高灵敏且操作简单的新型生物传感器用于检测蛋白激酶A(PKA)的活性。首先将g-C_3N_4溶液滴涂于玻碳电极(GCE)表面并晾干,再滴涂一层壳聚糖(CS),在EDC和NHS作用下多肽与CS发生酰胺反应而偶联到CS/g-C_3N_4/GCE上;在PKA和巯基三磷酸腺苷(ATP-s)的作用下,多肽的丝氨酸位点发生磷酸化反应,从而将巯基磷酸根转移到多肽/CS/g-C_3N_4/GCE上;进而通过Au-S作用将Au NCs捕获于巯基磷酸化多肽修饰电极表面,使得g-C_3N_4的ECL信号大大增强。随着PKA浓度的增加,捕获到电极表面的Au NCs增多,对g-C_3N_4的ECL增强效应越显著,g-C_3N_4的ECL增强程度与PKA浓度呈正相关,据此可实现对PKA活性的高灵敏和选择性检测。此外,比较了多肽链长度以及可发生磷酸化的丝氨酸位置对g-C_3N_4的ECL强度的影响,结果表明,随着多肽链长度的增加以及磷酸化位点距离电极表面距离的增加,Au NCs对g-C_3N_4的ECL增强效应减弱,可见Au NCs对g-C_3N_4的ECL增强效应与二者之间的距离有关。本方法构建的ECL传感器还可用于对激酶抑制剂的筛选以及对MCF-7细胞溶解产物等复杂生物环境中PKA活性的灵敏检测。3.基于g-C_3N_4和Au NPs之间的共振能量转移效应(RET)构建新型ECL生物传感器并用于检测PKA活性。以S_2O_8~(2-)为共反应剂时,g-C_3N_4修饰电极能够产生强的阴极ECL信号;将多肽组装到g-C_3N_4修饰电极表面,在PKA和ATP-s的作用下,多肽发生巯基磷酸化,进而通过Au-S键将Au NPs捕获到多肽的巯基磷酸化位点上,拉近了Au NPs与g-C_3N_4之间的距离,且g-C_3N_4的ECL发射光谱和Au NPs的吸收光谱部分重叠,使得g-C_3N_4能够将能量转移给Au NPs,g-C_3N_4的ECL强度降低,g-C_3N_4的ECL信号降低程度与PKA的浓度呈正相关,据此可实现对PKA活性的定量检测,线性范围为0.1-80 U/mL,检测限为0.05U/mL。同时,还比较了多肽链长度以及Au NPs与g-C_3N_4之间的距离远近对ECL-RET的影响。此外,本方法还可用于血清样品中激酶活性的检测及其抑制剂的筛选,对生物化学基础研究和疾病诊断等领域都具有重要意义。
[Abstract]:As a new analytical method, electrochemiluminescence (ECL) has attracted more and more attention in recent years, and the development of new materials plays a very important role in the development of ECL. The phosphorylation regulated by protein kinase is closely related to many life activities, and the abnormal expression of protein kinase can lead to many diseases and cancers. Therefore, it is very important to accurately analyze the kinase activity and screen the kinase inhibitors for disease diagnosis and clinical treatment. Material, using the photoelectric properties of these nanomaterials to construct new ECL biosensors, and used to detect protein kinase activity and screen kinase inhibitors, the main work is divided into three parts: 1. First, the definition of electrochemiluminescence, characteristics and classification of luminescent reagents, electrochemiluminescence as a kind of electrochemical technology and electrochemical technology. The related optical analysis methods have the characteristics of both electrochemical analysis and optical analysis. Then, the structure and properties of metal nanomaterials and carbonitrides and their applications in the field of electrochemiluminescence are summarized. Finally, protein phosphorylation and the role of protein kinase are reviewed. Protein kinase is an important part of cell activity. The abnormal activity of kinases will lead to many diseases. Among the various methods of detecting kinase activity, the ECL analysis method based on nano-materials has unique advantages. 2. A novel label-free, highly sensitive and easy-to-operate biosensor was constructed to detect protein stimulation by using the enhancement effect of Au NCs on the ECL signal of g-C_3N_4. Enzyme A (PKA) activity. First, the g-C_3N_4 solution was dripped on the surface of glassy carbon electrode (GCE) and dried, then coated with a layer of chitosan (CS). Under the action of EDC and NHS, the peptides reacted with CS amide and were coupled to CS/g-C_3N_4/GCE. Under the action of PKA and ATP-s, the serine sites of the peptides were phosphorylated, which led to the phosphorylation of the peptides. The ECL signal of g-C_3N_4 was greatly enhanced by the transfer of mercaptophosphate to polypeptide/CS/g-C_3N_4/GCE and the capture of Au-NCs onto the surface of the electrode modified by mercaptophosphate through Au-S interaction. In addition, the effects of the length of polypeptide chain and the position of serine phosphorylated on the ECL strength of g-C_3N_4 were compared. The results showed that with the increase of the length of polypeptide chain and the distance between phosphorylated site and electrode surface, Au NCs could detect the activity of g-C_3N_4 with high sensitivity and selectivity. The enhanced effect of Au NCs on the ECL of g-C_3N_4 was weakened, which indicated that the enhanced effect of Au NCs on the ECL of g-C_3N_4 was related to the distance between the two. The ECL sensor constructed by this method can also be used to screen kinase inhibitors and detect the activity of PKA in complex biological environment such as MCF-7 cell lysates. The G-C_3N_4 modified electrode can produce strong cathodic ECL signal when S_2O_8~ (2-) is used as co-reactant, and the polypeptide is assembled on the surface of g-C_3N_4 modified electrode. Under the action of PKA and ATP-s, the polypeptide undergoes sulfhydryl phosphorylation, and then the polypeptide is captured by Au-S bond. The distance between Au NPs and g-C_3N_4 was shortened at the thiol phosphorylation site of Au-C_3N_4, and the ECL emission spectra of g-C_3N_4 and the absorption spectra of Au NPs were partly overlapped. As a result, the ECL intensity of g-C_3N_4 could be transferred to Au-NPs, the ECL intensity of g-C_3N_4 decreased, and the ECL signal intensity of g-C_3N_4 decreased positively correlated with the concentration of PKA. Quantitative detection showed that the linear range was 0.1-80 U/mL and the detection limit was 0.05 U/mL. The effects of the length of polypeptide chain and the distance between Au NPs and g-C_3N_4 on ECL-RET were also compared. In addition, this method can be used for the detection of kinase activity in serum samples and the screening of inhibitors, basic biochemical research and disease diagnosis. Both are of great significance.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.1;O629.8;TP212.3

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