| As the human genome project has uncovered the full sequence of human genomes, the analysis of the variations among individual genomes has attracted considerable interest in a wide range of areas including molecular diagnostics, environmental monitoring, and antibioterrorism.Protein is a type of important biomarkers, which plays a key role in life sciences, medical diagnostics. Many detection techniques have been developed relying upon target hybridization with radioactive, which create disposal problems, require specially trained personnel and have a short shelf life. There is a considerable requirement for rapid, low-cost, and sensitive detection of specific DNA and protein for the clinical diagnosis of genetic and pathogenic diseased. DNA biosensor, a new conception of genome detection based on Watson-Crick base match principle, is of great advantages such as easy handling, quick detection,favorable selectivity and no pollution.Nowadays;it has become one of the most important methods in DNA detection.One recent development is molecular beacons (MBs) that are highly selective in their recognition of oligonucleotides.Traditional MBs are doubly end-labeled olignucleotides that exist in solutions as stable stem-loop structures and utilize fluorescence of a reporter dye combined with a proximate quencher attached to 3'end. MBs are now extensively emploted in areas such as genetic screening, biosensor development and the detection of single-nucleotide polymorphisms (SNPs).To achieve the desired simplicity, low-cost and sensitivity, colorimetric detection using gold nanoparticles (AuNPs) has been attracting considerable interests, because AuNPs aggregation accompanied by the surface plasmon shift can be clearly recognized with the naked eye. The ease of synthesizing of AuNPs and the possibility to couple of different functionalities make AuNPs as a convenient tool for the sensing of metal ions, proteins and oligonucleotides.Aptamers are synthetic DNA/RNA oligonucleotides isolated for their ability to selectively bind to various biomolecules through SELEX. As alternative to antibodies, aptamers have many advantages including high binding affinity, convenient automated-synthesis, ease-of labeling, and high stability. Up to now, many aptasensors have been developed include optical and electrochemical aptasensors.Electrochemical aptasensors have attracted substantial attention because of their high sensitivity, simple instrumentation, low production cost, fast response and portability in the development of aptasensors.Since the first report by Sen and his co-workers in 1998, G-quadruplex-based DNAzymes which have also been identified by SELEX has received great attention.The folded guanine-rich oligonucleotides that serve as an aptamer for hemin could self assemble to form G-quadruplex structure.After binding with hemin, the resulting supramolecular complex mimics horseradish peroxidase(HRP) and catalyzes the H2O2-mediated oxidation of 2,2-azinobis(3-ethylbenzothiazoline)-6-sulfome acid (ABTS) and luminal.These G-quadruplex-based DNAzymes have shown great potential as a novel kind of catalytic label for the development of various biosensors owing to its high catalytic activity, easy synthesis and construction.The goal of present study is to design novel techniques with high sensitivity and selectivity. This paper has combines the excellent characteristics of AuNPs and DNAzymes to provide alternative candidates for DNA and protein detection.The dissertation includes three parts:Chapter One:introductionAptamers are artificial oligonucleic acids which are selected through SELEX to bind specific target molecules.Guanine-rich oligonucleotides are one kind of these aptamers which can bind hemin to form G-quadruplex-based DNAzymes. In the beginning of this dissertation, the importance of the DNA and protein detection was introduced, together with the development of DNA biosensor. Secondly, the concepts and applications of AuNPs-based colorimetric biosensing events, molecular beacon and electrochemical aptasensors were described in detail to provide the theoretical principle for this paper. At last the purpose and innovation of the dissertation were pointed out.Chapter Two:Label-Free Molecular Beacon-Functionalized Gold Nanoparticles as Colorimetric Probes for Single-nucleotide polymorphisms (SNPs) detectionCompared with the traditional linear DNA probe, molecular beacon shows higher selectivity and specificity due to its conformation variations between stem-loop and stretched structure in detection of mutations in oligonucleotides. We reported a new colorimetric SNPs detection method by combining optical properties of gold nanoparticles and molecular beacon with the high specificity and sensitivity. The thiolated molecular beacons were attached to AuNPs via well-established self-assembled monolayer chemistry. A perfectly matched target and the single-base mismatched target can be completely discriminated by causing the change of entropic and steric conformation at certain concentration of MgCl2.The color of the gold nanoparticle solution containing perfectly matched target is blue and the color of the gold nanoparticle solution containing single base mutation target is red. At last, we applied this method in P53 gene detection. The results indicated that it was possible to accurately determine SNPs as low as 5%.The proposed approach has a great potential for realizing an accurate, sensitive, rapid and low cost method of SNPs detection.Chapter Three:G-Quadruplex-Based DNAzymes aptasensor for the Amplified Electrochemical Detection of ThrombinA novel G-quadruplex-based DNAzymes aptasensor for the amplified electrochemical detection of thrombin has been described. The aptasensor utilized a combination of hemin and guanine-rich thrombin-binding aptamer (TBA) to form the horseradish peroxidase (HRP)-mimicking DNAzymes with peroxidase catalytic activity. In the presence of thrombin, the enzyme activity could be extensively promoted, thereby providing the amplified electrochemical readout signals for detecting thrombin. This aptasensor exhibited high sensitivity and selectivity for thrombin determination, which enabled the analysis of thrombin with a detection limit of 6×10-11M. On the basis of results, this method will serve as a versatile tool for proteins and other biomolecule detection. |
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