以凝集素为分子识别物质的甘露聚糖电化学发光传感方法的研究

【作者】 杨娜；

【导师】 张成孝；

【作者基本信息】 陕西师范大学 ， 分析化学， 2011， 硕士

【摘要】 糖、蛋白质、核酸是涉及生命活动本质的三类重要生物分子。在蛋白组学和基因组学发展的同时,糖组学的研究也日益引起了人们的广泛关注。由单糖、寡糖或多糖与脂类或蛋白质连接形成的糖类物质广泛存在于生命体中,在生命活动过程中具有非常重要的意义。糖类对于生命体不仅仅是简单的能源物质,也是重要的信息分子。糖类参与许多生理和病理过程,与疾病的发生和治疗有着密切的关系。因此糖在生命科学、医学等研究领域中都具有重要的地位。结构多变、功能多样的聚糖几乎覆盖了生物有机体所有的细胞表面,不仅在正常的细胞粘附,细胞间信号转导等方面有重要的作用,而且在细胞病变、病原感染等方面亦具有十分重要的作用。糖与凝集素相互作用的研究及其检测是生化分析中的重要内容,在化学生物学、分子生物学、细胞生物学、药物分析学和环境科学等领域有着十分重要的意义。糖类的检测可为重大疾病的早期诊断、药物筛选以及糖组学的研究等生命科学中的重大研究提供新技术和新方法,对建立高灵敏和高选择性的糖或细胞的测定方法具有重要的作用。电化学发光法兼有电化学法和化学发光法的双重优点,具有极低的检测限、极宽的线性范围等优点,因此被广泛应用到免疫分析、DNA杂交检测等领域中。本论文旨在以凝集素伴刀豆球蛋白与甘露聚糖的特异性结合作用为模型,以电化学发光技术为检测手段,建立三种高灵敏检测甘露聚糖的传感方法。本论文构建了一种高灵敏、简单、快速均相电化学发光检测甘露聚糖的新方法,研制了一种通过树枝状高分子多负载电化学发光探针与羧基化单臂碳纳管双重信号增大的甘露聚糖电化学发光糖生物传感器,研制了以Nafion为成膜剂建立电化学发光平台分别实现对不同物质的区分。本论文由四章组成。第1章为引言部分,引言对糖和凝集素做了简要的概述,以伴刀豆球蛋白与甘露糖的结合为模型,简要介绍了糖-凝集素相互作用的机理；重点总结了糖-凝集素传感器的研究进展并介绍了树枝状聚酰胺-胺(PAMAM)在生物传感器中的应用,提出了本论文的立题依据、研究目的和研究内容。第2章均相电化学发光法测定甘露聚糖。该方法以钌联吡啶络合物为信号物质,以钌联吡啶络合物标记的伴刀豆球蛋白(Con A)为电化学发光探针,以甘露聚糖(Mannan)为目标分子。首先以钌联呲啶络合物标记Con A,制得探针Ru-Con A,当不存在甘露聚糖时,标记探针游离在电极周围,产生强的电化学发光信号；存在甘露聚糖时,与甘露聚糖结合,造成大的空间位阻以及分子量增大的复合物,标记物远离电极,电化学发光强度降低,基于Con A与Mannan结合前后电化学发光强度的变化实现对甘露聚糖的检测。实验中考察了电位、结合时间及探针浓度对发光信号的影响。在选定的实验条件下,电化学发光强度的变化值与目标物甘露聚糖浓度的负对数在1.0×10-10-5.0×10-g mol/L范围内呈良好的线性关系,线性方程为△I=-14751gC-9388(r=0.9979),检出限为6×10-11mol/L。本工作表明利用钌联吡啶络合物标记Con A作为电化学发光探针均相电化学发光检测甘露聚糖具有高灵敏度、简单、快速的优点。第3章介绍了我们建立的一种基于树枝状高分子探针电化学发光高灵敏检测甘露聚糖的新方法,首先以羧基化的单臂碳纳米管(SWCNTs)修饰石墨电极表面,将负载有钌联吡啶络合物的聚酰胺-胺树枝状分子(PAMAM-Ru)共价键合于SWCNTs修饰的石墨电极上,最后通过戊二醛交联法固定识别分子Con A。PAMAM在该传感器中起着双重的作用,既可作为信号物质的负载物,又可作为连接臂。该传感器利用羧基化单臂碳纳米管和负载有钌联吡啶衍生物的PAMAM的双重信号增大作用,实现了甘露聚糖的高灵敏检测。与之前检测糖的方法相比,我们构建的新型电化学发光糖生物传感器检测限较低,可以达到4 pmol/L。第4章以Nafion为成膜剂,将负载有钌联吡啶衍生物的聚酰胺-胺树枝状分子(PAMAM-Ru)修饰在热裂解石墨电极上,建立了信号非常稳定的电化学发光平台。该平台分别对甘露聚糖和伴刀豆球蛋白具有响应信号,主要以抑制型为主,实现了对不同物质的区分,具有宽的线性范围,高的灵敏度。并对该检测平台的信号响应的抑制机理进行了初步探讨。本论文研究了均相、纳米材料/多负载信号增大电化学发光生物传感方法,为分析检测糖类甚至细胞提供了性能优良的分析器件和分析新方法,为电化学发光糖生物传感器的研究提供了一些新思路和基础性研究资料。更多还原

【Abstract】 Saccharides, protein and nucleic acid are three kinds of molecules concerning life. Glycomics has increasingly attracted our attention with the development of genomics and proteomics. The saccharides connecting with the lipid or protein which exists extensively, play a significant role in biological processes.Saccharide is not only simple energy substance, but also important informational molecules.lt engages in various physiological and pathological processes. It is closely related with the occurrence and treatment of serious deseases. All the cells surfaces of living organisms are almost covered by the variable-structure, rich-function glycans.Those glycans of cell surface play key roles not only in cells recognition,cell adhesion and cell-cell signal transmission,but also in cell diseases and pathogen infections. The saccharides-lectin interaction and detection of them are the important parts of biochemical analysis, which play an impartant role in chemical biology, molecular, cell biology, drug analysis, environmental science and other fields of great importance. New technologies and methods for the detection of saccharides will improve early diagnosis of major diseases, drug screening and glycomics research.It is important to establish highly sensitive and selective methods for the determination of saccharides and cells.Electrogenerated chemiluminescence (abbreviated as ECL) method has many distinct advantages over fluorescence method, because it avoids the attendant problems of impurities luminescent and scattered light, and does not involve a light source. Compared to electrochemical method, it has low detection and is less affected by the electrodes pollution. ECL has been widely used in many fields related to biochemical and chemical applications such as immunoassay, food and water testing, DNA hybridization detection as well as explosive material or biowarfare agent detection.The aim of the thesis is to develope highly sensitive, convenient and rapid method for the detection of saccharides. The thesis mainly includes two parts. The first part, Chapter 1, is the general introduction while the second part consisting of three chapters, is a research report. In Chapter 1, general introduction to saccharides, lection and saccharides-lectin interaction including their research development, lastly the purpose of this research work was presented.In Chapter 2, a novel homogeneous ECL method for the determination of Mannan was developed by employing an ECL probe consisting of Concanavalin A as a recognition molecule and tris (2,2’-bipyridyl) ruthenium derivatives served as an ECL tag. It was found that a strong ECL emission was electrochemically generated at a glassy carbon electrode in a solution of the probe and markedly decreased when target analyte Mannan was added into the probe solution. The changes of ECL intensity has a linear relationship with logarithm of concentration of Mannan in the range from 1.0×10-10 to 5.0×10-8 mol/L and the linear regression equation wasΔI=-9388.3+1475.91gC (unit of C is mol/L, r=0.9979). The detection limit of Mannan was 6×10-11 mol/L.This work demonstrates that a homogeneous ECL model based on the specific interaction between Con A and Mannan is a promising approach to estabilsh simple and sensitive ECL methods for the determination of glucoprotein.It is expected that the developed method is potentially applied in the detection of glycosylation levels or patterns on the cell surfaces.In Chapter 3, a novel ECL biosensor for the determination of Mannan is designed based on graphite electrode (GE) modified by polyamidoamine dendrimers(PAMAM,G4.0-NH2) surface-functionalized with polypyridyl ruthenium complexes (PAMAM-Ru) and carboxyl single-wall carbon nanotubes (SWCNTs). A GE was firstly modified with SWCNTs by dropping, and then reacted with an amino-terminated PAMAM-Ru to obtain a thin film. Lastly, the plant lectin Concanavalin A (Con A) was immobilized onto the film to obtain a stable recognition layer by using the classic glutaraldehyde coupling reaction to detect target Mannan. It was found that a strong ECL emission was electrochemically generated at a modified electrode without the target Mannan.An weak ECL signal is generated upon recognition of the target Mannan, which is possiblely attributed to a change electron transport rate in close proximity to the sensor interface. Under the optimum condition, the ECL intensity versus the ligarithm of target Mannan concentration has a good linear relationship in the range from 1.0×10-11 to 5×10-9 mol L-1. The linear regression equation wasΔIECL=12898+1150.91gC(r=0.9963). The detection limit was 4×10-12 molL-1.In Chapter 4, an ECL platform was constructed by immobilizing PAMAM-Ru on the surfaces of pyrolytic graphite electrode by Nafion membrane, which has a very stable ECL response. This platform can response to different substances, in a sigal-off model. Using the platform, we successfully develope an ECL method for the discrimination different substances. The signal response mechanism of the platform was tentatively discussed. The method is simple, fast and cheap. It is expected that the developed method is potentially applied for the detection of cells.更多还原