Construction And Application Of A Novel Aptamer Fluorescent Probe Based On DNA Strand Displacement Reaction

With the development of genetic diagnosis,disease research,environmental pollution,and food safety monitoring,researchers have constructed a variety of fluorescent bio-sensing strategies.Among them,fluorescence biosensors based on the toehold-mediated strand displacement reaction(TSDR)have been widely used in the analysis and detection of various targets because of their advantages such as no enzyme,simple operation and rapid response.However,this strategy still has challenges in the activation of the toehold,and its sequence-dependency makes it impossible to design a universal fluorescent biosensor.In addition,the fluorescence anisotropy(FA)analysis and detection method has been widely used,because it only requires a single label to overcome photobleaching and can be used in complex samples.Most fluorescence anisotropy biosensors require special design of secondary structures or use of nanomaterials,and have problems such as intricate designs and incompatibility with enzymatic amplification strategies.In this paper,we focus on the existing issues and construct fluorescence biosensors for analysis and detection.The study is as follows:(1)Biosensor based on the mechanism of cooperative toehold-activated strand displacement reaction:We constructed a "cooperative toehold" to activate strand displacement reactions.The cooperative toehold constructed of two completely independent DNA fragments,and the toehold-mediated strand displacement reaction is activated by the stacking between adjacent bases.We verified the feasibility of this principle by real-time fluorescence detection and non-denaturing gel electrophoresis experiments,with a detection limit of 0.5 nM,with a linear range of 0-10 nM,R2=0.9979.The remote toehold and reversibility experiments confirmed that the system is compatible with other strategies and can be applied in complex systems.(2)Construction of a universal DNA fluorescence sensor and its application in bioassay:The fluorescence biosensor based on the toehold-mediated strand displacement reaction is not versatile due to the sequence dependence.According to our proposed "cooperative toehold" activation mechanism,we constructed a general-purpose fluorescent biosensor for miRNA,ATP analysis and PCR amplification monitoring.The miRNA detection limit was calculated as 2 nM with a linear range of 0-20 nM and R2 = 0.990.ATP fluorescence signal changes linearly from 0 to 50 ?M,and the detection limit was estimated to be 5 ?M.The plot of the ogarithmic of pUC18 concentrations and fluorescence intensity showed a linear relation in the presence of 400 aM-400 pM pUC18 and this universal biosensor has the potential for DNA quantitative and real-time analysis with aM detection limit.(3)Cleavable DNA-protein hybrid molecular beacon:A novel efficient signal translator for sensitive fluorescence anisotropy bioassay:we provided a novel signal translating strategy for sensitive FA bioassay based on a cleavable DNA-protein hybrid molecular beacon,using DNAzyme-Pb2+ as a model system.The design strategy of DNA-protein hybrid molecular beacon took advantage of both molecular beacon's intramolecular stability of the molecular beacon and the large molecular weight of the streptavidin,which can effectively amplify the fluorescence anisotropy signal.The two strategies combined together to afford this FA assay with a high sensitivity,and also to monitoring Pb2+ ions in real water samples with good recovery.