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Study On Electrochemiluminescence Biosensor Based On Ru(bpy)32+

Posted on:2011-01-03Degree:MasterType:Thesis
Country:ChinaCandidate:P DongFull Text:PDF
GTID:2178360305998874Subject:Analytical Chemistry
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Aptamers are synthetic single-strand nucleic acids with high specificity and affinity to some given targets ranging from small molecules to large proteins and even cells. Apatmers have been demonstrated to have advantages over antibodies with regard to chemical stability, readily availability, simple modifiability and high flexibility in biosensor desigh for analyte detection. In the last decade, aptamers have been widely reported as highly promising recognition probes for disease diagnosis, new drug screening, protein analysis, biosensor and molecular switch development, etc. Onco protein platelet-derived growth factor, cocaine and IgE, for example, can be sensitively determined by fluorescence anisotropy, electrogenerated chemiluminescence and affinity pro capillary electrophoresis, respectively. When combined with all other analysis techniques, aptamer can be utilized to detect DNA sequence, correlative protein, enzyme and small molecules, which is of great significance on the research of functional gene, protein, cancer diagnose in the postgenome era.Electrochemiluminescence (ECL) is developed based on chemiluminescence (CL) and electrochemistry (EC) with a large number of advantages, such as high sensitivity and selectivity, rapid and convenient operation and relatively simple instrumentation system. Due to these inherent advantages, ECL method has attracted much attention from all analytical fields, especaily from biochemical analysis. As an important ECL substrate, Ru(bpy)32+ has been widely used in the traditiona solution-state ECL system. However, owning to the high price of Ru(bpy)32+, it becames significant to effectively immobilize Ru(bpy)32+ onto the electrode so as to reduce the consumption of expensive reagent.Chitosan is an amine-rich polysaccharide derived by deacetylation of chitin, which is abundant in nature. It has been wildly used in scientific and industial fields due to its excellent properties, such as biocompatibility, biodegradability, ability to form films and high reactivity. As an outstanding film material, chitosan has attracted more attention. The pKa of chitosan is about 6.3. Therefore, when the pH is lower than 6.3, most of the amino groups are protonated and positively charged so that chitosan is soluble in aqueous solution. As the pH is higher than 6.3, the amine groups become deprotonated and uncharged, thus making chitosan insoluble in water. According to its special characteristic, chitosan can function as a matrix to immobilize biomolecules by adjusting the pH value of the solution.The goal of the paper is to develop a novel solid-state ECL biosensor which combines the specific recognition of molecular recognition elements and electrochemiluminescence technique to detect thrombin with high sensitivity and selectivity. Moreover, the characteristics of chitosan is also used to selectively electrodeposite Ru(bpy)32+onto the array electrodes, which means a brand-new immobilization method of Ru(bpy)32+is structured.The dissertation is composed of four chapters as following:Chapter 1:Give a brief introduction on the principle and application of ECL, as well as several types of ECL reactions and ECL sensors in the field of analytical chemistry. DNA sensors including their principles, advantages and disadvantages are reviewed. Then, chitosan including its characteristics, advantages and application in biotechnology are detailed on the paper. At the end of chapter 1, the purpose of the dissertation is pointed out.Chapter 2:A solid-state electrochemiluminescence (ECL) biosensing switch system based on special ferrocenelabeled molecular beacon aptamer (Fc-MBA) has been developed successfully for thrombin detection. Such special switch system includes two main parts, an ECL substrate and an ECL intensity switch. The ECL substrate is made by modifying the complex of Au nanoparticle and Ruthenium (Ⅱ) tris-(bipyridine) (Ru(bpy)32+-AuNPs) onto Au electrode. A molecular beacon aptamer labeled by ferrocene acts as the ECL intensity switch. The loop bases of the ECL intensity switch are designed with special anti-thrombin aptamer sequence which can be combined with its target protein via the reaction between aptamer and thrombin. During the reaction, the molecular beacon aptamer opens its stem-loop, and the labeled Fc is, therefore, kept away from the ECL substrate. Such conformation adjustment results in an obvious ECL intensity increment due to the decreased quenching effect of Fc to the ECL substrate.Chapter 3:A solid-state electrochemiluminescence (ECL) biosensor based on the switch system of ferrocene-labeled double-stranded DNA (Fc-dsDNA) has been developed for thrombin detection. This special switch system contains an ECL substrate which is made by modifying the complex of Au nanoparticle and Ruthenium (II) tris-(bipyridine) (Ru(bpy)32+-AuNPs) onto Au electrode, and one dsDNA hybridized by a thrombin-aptamer and one ferrocene-labeled molecular beacon is used as the ECL signal switch. Before the reaction of thrombin and its aptamer, the molecular beacon opens its stem-loop and the labeled Fc, therefore, is kept away from the ECL substrate. After the specific recognition between aptamer and thrombin occurs, the Fc is pulled back onto the electrode so as to decline the ECL intensity due to the quenching effect of Fc to Ru(bpy)32+. The analysis results with sensitivity and specificity.Chapter 4:An effective method for the selective immobilization of tris(2,2'-bipyridyl)ruthenium(Ⅱ) (Ru(bpy)32+) onto one target electrode surface based on the electrodeposition of RuDS NPs Ru(bpy)32+ doped silica nanoparticles)/chitosan composite film is presented in this paper. Ru(bpy)32+ is selectively deposited onto four individual electrodes in an Au electrode array. The result demonstrates the possibility of selective immobilization of Ru(bpy)32+onto array electrodes and selective immobilization of different ECL composite. The method shows great potential of application in the field of bioanalysis, capillary electrophoresis and drug screening. RuDS NPs and the resulting composite film were characterized by UV-vis spectroscopy, transmission electron microscopy (TEM) and Field emission-scanning electron microscope (FE-SEM). The electrochemiluminescence (ECL) sensor based on the composite film modified electrode exhibites excellent reproducibility, stability and sensitivity for the detection of tri-n-propylamine (TPrA).
Keywords/Search Tags:Solid-state electrochemiluminescence biosensor, Aptamer, Thrombin, Molecular beacon, Quenching effect, Chitosan, Selective immobilization
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