Signal Amplification For Nucleic Acids Analysis Based On Molecular Probes

Detection of nucleic acids, involving in disease diagnosis, genetic screening, environmental testing and other fields, is closely related to human health and social stability. Traditional nucleic acid detection methods are limited due to low sensitivity, complicated operation, time-consuming. Therefore, the development of new nucleic acid detection methods attracts extensive attention. To achieve sensitive, accurate, convenient and economical detection has become increasingly urgent demanding in various fields. In recent years, the development of signal amplification detection technologies based on various enzymes and nanoparticles, offered a new opportunity to achieve simple, fast, and highly sensitive detection of nucleic acids. In this thesis, to address these important scientific issues, a series of signal amplifying detection methods based on deoxyribozyme and the enzyme-mediated polymerase chain replacement for nucleic acid, the very important component of life, were developed, combining nucleic acid molecular probes and the sequence specific recognition and amplification of nucleic acid enzymes, with electrochemical, colorimetric, and fluorescence as detection means. The main content was summarized as follows:1. Signal amplified electrochemical detection for DNA based on peroxidase-like DNAzymeA novel electrochemical DNA detection assay was developed based on a hairpin structure probe with chronoamperometric as detection technique. Upon hybridization with the target, the hairpin structure was opened, and the G-quadruplex-based DNAzyme was generated, triggering the electrochemical oxidization of hydroquinone by H2O2, and realizing quantitative measure for target DNA. This method doesn’t need direct conjugation of redox-active element, easy to manipulate, fast, and specific for target DNA, with a detection limit of 0.6 nmol/L.2. Signal amplified colorimetric detection for miRNA based on peroxidase-like DNAzyme and strand-displacement polymerase reactionCombining peroxidase-like DNAzyme and strand-displacement polymerase reaction, a new miRNA assay was developed. In the presence of target miRNA, strand-displacement polymerase reaction was triggered with target miRNA as the primer and the DNA-RNA chimerical probe as template, producing numerous peroxidase-like DNAzyme sequences and then catalyzing the oxidation reaction which in turn leaded detective color changes. This assay is simple, convenience, selective, with no need for separation or complex manipulation. The detection limit for miRNA is as low as 2 fmol/L.3. Signal amplified fluorescence detection for miRNA based on molecular beacons and strand-displacement polymerase reactionWith the aid of RnaseH and nicking enzyme mediated circular strand-displacement polymerase reaction, a new isothermal circular amplification miRNA detection assay was developed with a DNA-RNA chimerical probe as the probe for target miRNA and the template of strand-displacement polymerase reaction. This assay avoided the false positive signal and pollution resulting from regular nucleic acid amplification. Furthermore, it can be expected to provide a simple, fast, specific detection method for miRNA with a detection limit of 0.5 fmol/L.