Study On The Novel DNA Electrochemical Biosensors Based On Nanomateirals And Cyclodextrin

With improved understanding of structure and function of human gene, and the development of the Human Genome Project, DNA separation and analysis has taken a more and more important role in the areas of clinical diagnosis, medicine, epidemic prevention, environmental protection and bioengineering. Wide-scale genetic testing requires the development easy-to-use, fast, inexpensive, miniaturized devices. Many new biological technologies emerged and found their applications in this field. Among them, DNA biosensors are rapidly developed and have received considerable attentions. Electrochemical DNA detection is a novel and developing technique that combining biochemical, electrochemical, medical and electronic techniques with the advantages of being simple, reliable, cheap, sensitive and selective for genetic detection, and has been a hot topic in the field of biochemistry and medicine.The emergence of nanotechnology is opening new horizons for the application of nanoparticles in analytical chemistry. In particular, nanoparticles are of considerable interest in the world of nanoscience owing to their unique physical and chemical properties. With these unique properties, they are widely used in the fields of catalysis, optical absorption, medicine, magnetic medium, new materials synthesis. Such properties offer excellent prospects for chemical and biological sensing. The power and scope of such nanoparticles can be greatly enhanced by coupling them with biological recognition reactions and electrical processes (i.e. nanobioelectronics). Nanoparticle-biopolymer conjugates offer great potential for DNA diagnostics.Supramolecular chemistry has been defined as the "chemistry of molecular assemblies and of the intermolecular bond". Combing with the material science, biological science, information technology and nanometer technology, it has been the supramolecular science and has been employed as a vital method to design, and prepare new materials and obtain novel properties. Therefore, supramolecular chemistry is believed to be the base of new concept and technology in the 21st century. Cyclodextrin (CD), as the most important host, have received considerable attention because of its particular characterization, and the studies on the host-guest interaction based on CD had been transferred from the processes and mechanism of inclusion complex between a pair of host and guest to the application in the fields such as analysis, medicine, environment protection and sensors.The goal of the present study is to design and optimize new DNA hybridization techniques with high sensitivity and selectivity. This dissertation focuses on fabricating novel electrochemical DNA biosensors based on eletrochemical analysis technique, combining nano-materials, layer-by-layer technology and supramolecular inclusion interaction, thus developing a sensitive, sequence-specific and quantifiable gene detection method, and establishing the bases, especially one base mutation, then for application of electrochemical DNA biosensor to clinic diagnose.Chapter 1: PrefaceFirstly, we introduce the progress of DNA biosensor, including its principle (probe identification principle and immobilization method of ssDNA on solid support) and its classification (electrochemical DNA biosensor, optical DNA biosensor and piezoelectric DNA biosensor). Among these, we emphatically review the principle, progress, the application and development trends of electrochemical DNA biosensors. Second, the application of nano-materials on biosensors was introduced. At last, we pointed out the purpose and significance of the dissertation.Chapter 2: The preparation of Ru(bpy)₃²⁺-doped nanoparticle and its application in electrogenerated chemiluminescence detection DNA hybridization analysisA sensitive electrogenerated chemiluminescence (ECL) detection of DNA hybridization, based on tris(2,2'-bipyridyl)ruthenium(â…¡) -doped silica nanoparticles (Ru(bpy)₃²⁺-doped SNPs) as DNA tags, is described. In this protocol, Ru(bpy)₃²⁺-doped SNPs was used for DNA labeling with trimethoxysilylpropy -diethylenetriamine (DETA) and glutaraldehyde as linking agents. The Ru(bpy)₃²⁺-doped SNPs labeled DNA probe was hybridized with target DNA immobilized on the surface of PPy modified Pt electrode. The hybridization events were evaluated by ECL measurements and only the complementary sequence could form a double-stranded DNA (dsDNA) with DNA probe and give strong ECL signals. A three-base mismatch sequence and a non-complementary sequence had almost negligible responses. Due to the large number of Ru(bpy)₃²⁺ molecules inside SNPs, the assay allows detection at levels as low as 1.0×10^-13 mol 1^-1 of the target DNA. The intensity of ECL was linearly related to the concentration of the complementary sequence in the range of 2.0×10^-13-2.0×10^-9 mol 1^-1.Chapter 3: Electrochemical DNA biosensors based on palladium nanoparticles combined with carbon nanotubesPalladium nanoparticles, in combination with multi-walled carbon nanotubes (MWCNTs), were used to fabricate a sensitivity-enhanced electrochemical DNA biosensor. MWCNTs and palladium nanoparticles were dispersed in Nafion, which were used to modify a glassy carbon electrode (GCE). Oligonucleotides with amino groups at the 5' end were covalently linked onto carboxylic groups of MWCNTs on the electrode. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement using methylene blue (MB) as an indicator. Due to the ability of carbon nanotubes to promote electron-transfer and the high catalytic activities of palladium nanoparticles for electrochemical reaction of methylene blue, the sensitivity of presented electrochemical DNA biosensors was remarkably improved. The detection limit of the method for target DNA was 1.2×10^-13M.Chapter 4: Multilayer membranes via layer-by-Layer deposition of PDDA and DNA with Au nanoparticles as tags for DNA biosensingA novel Au nanoparticles (Au-NPs)-based protocol for DNA hybridization detection based on assembly of alternating DNA and poly(dimethyldiallylammonium chloride) (PDDA) multilayer films by layer-by-layer (LBL) electrostatic adsorption has been studied. Electrochemical impedance spectroscopy (EIS) and UV-vis absorbance measurements were used to study the film assembly. All the results indicate that the uniform multilayer can be obtained on the polypyrrole (PPy) coated electrode surface and the hybridization reaction can be amplified by the layer-by-layer progress. The hybridization was detected the reductive signal of Au-NPs by direct electrochemical method and nonspecific adsorption was greatly eliminated by an irrelated DNA sequence to the target DNA. Under optimum conditions, a significant sensitivity enhancement had been obtained, and the detection limit was down to 3.20×10^-14 M when 6 layers assembled. The DNA biosensor has good stability and reproducibility.Chapter 5: Electrochemical detection of DNA sequence with host-guest recognition onβ-cyclodextrin modified electrodeThe electrochemical sensing of DNA is accomplished firstly by the host-guest recognition system according to the very strong inclusion betweenβ-Cyclodextrin (β-CD) and m-toluic acid (mTA).β-CD, as the host molecular, was electropolymorized on the poly(N-acetylaniline) modified glassy carbon electrode by potential sweeping, and mTA, as the guest, was labled on the oligonucleotides with amino groups at the 5' end. The inclusion between P-CD modified electrode and mTA labeled DNA was investigated by CV and EIS, which turned out a strong inclusion interaction between them. Based on the host-guest recognition, introducing Au nanopaticles as tags and MB as indicator to detect DNA hybridization, respectively, has excellent detection limit and reproducibility.Chapter 6: Bifunctional biosensor based on aggregation methylene blue by cross-linked Au nanoparticles and the functionalized aptamer segmentsWe report here on the construction of a biosensor can detect thrombin or DNA molecules by two functional aptamer segments, which will form Au-NPs/analyte aggregation by adding the analytes, and the redox indicator, MB, that is used to interact specifically with the guanine bases on the aptamer segments. Hybridization for DNA or incubation for thrombin not only result in the binding multi-Au-NPs labeled probe to the analyte for formation of an extended polymeric network, but also in aggregation abundant MB molecules on the aptamer segments which can govern the signals for the analytes. The novel bifunctional apatmer-based biosensing capability is coupled to the enormous amplification feature of the activation for MB through the Au-NPs conductive matrix to yield remarkably low (fmole) detection limits.