| 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. |
PDF Full Text Request