Biosensors Based On G-Rich Nucleic Acid Probe And Duplex-Specific Nuclease

Nucleic acids are important biological macromolecules essential for life,which are widely founded in all the organism.Due to its biological function,nucleic acids are a kind of disease markers,which have been widely applied in disease diagnosis and treatment.In addition,with the advantages of easy to synthesize,stable,biocompatible,simple design and flexible signal mechanism,nucleic acids have become an ideal molecule tools and play an important role in biochemical analysis.A series of novel biosensing methods have been designed for the detection of microRNA and heavy metal irons based on G-rich nucleic and duplex-specific nuclease.1)Chapter 2.In this study,beta nickel hydroxide??-Ni?OH?₂?nanosheets,one of the transition metal oxyhydroxides with two dimensional?2D?structures,were explored as a new fluorescent biosensor platform and applied in constructing optical sensors for bioanalysis.It was found that?-Ni?OH?₂ nanosheets displayed a high fluorescence quenching ability and different affinity toward single-versus double-stranded DNA.Moreover,the absorption properties of?-Ni?OH?₂ nanosheets can be well controlled by changing cations,solution pH and the length of DNA.In comparison with some reported 2D nanosheet platforms?e.g.graphene,metal chalcogenides?,the absorbed DNA can also be desorbed by degrading the?-Ni?OH?₂ nanosheets,which is a simple but effective DNA desorption method.Based on these findings,a sensitive and selective optical miRNA sensor with a detection limit of 1 pM was demonstrated by combining the fluorescence quenching ability of?-Ni?OH?₂ nanosheets and duplex-specific nuclease signal amplification.The presented sensor has been successfully used for miRNA analysis in samples containing cancer cells and shown great potential in multiplexed miRNA analysis for clinical diagnosis.2)Chapter 3.We developed a simple and highly selective amplification biosensor for miRNA detection basis of G4MB and DSN.G4MB with a G4 motif stem is used as recognition probe.In the present of target miRNAs,G4MB hybridizes with target miRNA perfectly and forms a G4MB-miRNA duplex.Then,DSN subsequently cleaves the G4MB of the G4MB-miRNA duplex to recycle the target miRNA,which leads to fluorescence signal amplification.In the absence of target miRNAs,DSN can not digest the stem of G4MB because of the protection of G4 motif,which eliminates the false positive signal,and produces low fluorescence background.Importantly,the powerful dis-criminating abilities of both G4MB and DSN make the novel sensor suitable for miRNAs detection with high single-base selectivity.Comparing with traditional linear ssDNA probe-DSN-based method,the signal response of similar miRNA sequences with one-base difference has been reduced from 24%to 6%by using this G4MB-DSN-based method.3)Chapter 4.The G-rich probe can self-assembled into a G-triplex structure.Herein,we found that the G-triplex can bind with fluorescent dye thioflavin T,leading to a high fluorescence enhancement of thioflavin T.However,in the presence of Hg²⁺ion,Hg²⁺ion influenced the formation of G-triplex structure and further inhibited the binding between thioflavin T and G-rich DNA probe,which resulting the fluorescence value of thioflavin T decreased.Based on the fluorescence signal change,the Hg²⁺ion can be detected in this new method.The feasibility of this method has been demonstrated,meanwhile,the experimental conditions were optimized.