Construction And Performance Study Of Novel Fluorescent Nucleic Acid Probes

As a carrier of biological information,DNA is closely related to human life activities.Many major diseases are related to DNA replication,transcription and mutation processes in the body.Therefore,accurate analysis of DNA can achieve early diagnosis of diseases such as cancer and improve patient survival.In addition,DNA can also be modified as a bio-probe to achieve rapid and accurate monitoring of each target molecule,and has broad application prospects.This thesis studied the design of DNA molecular beacon sequences and the construction of molecular beacon signal amplification system while studying the DNA conformation characterization.The specific content is mainly composed of the following two aspects:Firstly,an accurate analysis of the interaction between nanoporphyrins and DNAs with different configurations was achieved using the reversible"off-on"mode of quantum dot fluorescence.The quenching effect of nanoporphyrins on quantum dots with different morphologies was investigated.The interaction between nanoporphyrins and different bases,lengths,single and double strands and G-quadruplexes with secondary configuration was also investigated.Finally,the partial least squares discriminant analysis model was introduced to carry out rapid quasi-deterministic quantitative analysis of DNA of different configurations at different concentrations,and the accuracy rate reached 95.1%.Meanwhile,this part of the experiment synthesized and characterized a novel nanoporphyrin material,successfully established the powerful use of fluorescent"off-on"mode to identify various DNA and G-quadruplex structure of nanoporphyrin/CdTe quantum dots.It provides a new perspective and guidance for the water-soluble application of oil-soluble drugs and the rapid analysis of different DNA structures in vitro.It also provides basic and theoretical support for the subsequent molecular beacon sequence design and detection mechanism research.Secondly,based on the above studies,a new method for detecting mercury ions in a fluorescent nucleic acid probe signal amplification sensing system was established.By applying the property of mercury ions which can induce the mismatch of thymine,a nucleic acid probe for capturing mercury ions and a molecular beacon that hybridized with each other were designed.By using catalyzing hairpin self-assembly,a complex formed by the combination of mercury ions and capture probes hybridized with molecular beacon firstly,then was replaced by other nucleic acid probes to become free,finaly this complex hybridized with molecular beacon again,receiving a hybrid ratio between the target complex and the molecular beacon greater than 1:1,which improved the sensitivity of mercury ions.The sensitivity and selectivity of the method for mercury ion detection were investigated.The detection limit of this method is 1.7×10^-9 mol/L,which is lower than the maximum pollutant level of mercury ion in drinking water by the US Environmental Protection Agency.The reaction time is 30 min,which is far lower than the reaction time reported in other literatures,therefore mercury ions can be qualitatively and quantitatively detected more quickly.The signal amplification strategy of the sensing system also provides a direction for improving the detection sensitivity of other targets.