| The key to the construction of fluorescent biosensor is the design of fluorescent probe.DNAzyme as a nuclease,non-protein,doesn’t require harsh reaction condition(such as temperature,pH),has high specific recognition and shear effect,and is not easy to cause immune response.It is very suitable for the construction of biosensor for the analysis and detection of biomolecules in vivo or in vitro.G-triplex is an intermediate formed by the folding process of G-quadruplex.It has similar chemical properties as G-quadruplex,and is easier to design than G-quadruplex.After binding with Th T,it can emit strong fluorescence under certain excitation and is more suitable for designing fluorescent probe.Exo III,as a commonly used protein enzyme,is widely used in the field of biological analysis.It can specifically cleave the double-stranded DNA with a blunt or a recessed 3’-terminus and gradually hydrolyze the phosphodiester bonds of single nucleotides.Generally it functions as a signal amplification and specific recognition strategy.MOF,as a porous metal nanomaterial,is formed by metal ions and organic ligands through coordination bonds.In recent ten years,the special properties(large specific surface area,adjustable pores,thermal,chemical stability,biodegradation,etc.)have attracted much attention.ZIF-8,as a member of the MOF branch ZIF family,has the advantages of both zeolite and MOF(stronger chemical stability,regulatory properties,etc.),biodegradability,easy to synthesize,and cheap raw materials.The research on ZIF-8 mainly focuses on catalysis,separation,imaging,drug delivery and so on,but the construction of fluorescent biological probes combining the adsorption of nucleic acid to ZIF-8 and the PET process was rarely reported.DNAAgNCs is synthesized by using DNA as a template,in which DNA plays a role of stabling AgNCs.As a kind of sub-nanometer-level metal nanomaterial,it is usually formed by several to dozens of Agatoms and has good fluorescence emission characteristics.And the stability was good,easy to synthesize.It can be used as a good material to construct fluorescent probe.This paper mainly combines DNAzyme,Gtriplex,ZIF-8,DNA-AgNCs their properties and design different fluorescent probes for nucleic acid detection.(1)Fluorescence detection of H5N1 based on Exo III assisted recycling and DNAzyme amplification.When target(H5N1)exists in the system,H5N1 complement with HP to form a 3’ flat end,which is recognized and hydrolyzed by exonuclease III,Target is released and involved in the next cycle,the DNAzyme sequence originally locked in the HP stem would be released.DANzyme further identify substrate MB,and cleave MB to release rich G sequences which were locked in MB stems,After that,rich G sequences fold into G-triplex and combine with Th T,which emits strong fluorescence under a certain exitation.Experimental results showed that it had a good linearity in the200 p M-20 n M range,the detection limit was 68 p M.The actual sample(serum)also showed a good linear relationship.(2)Fluorescence biosensor based on Zeolite Imidazole Framework(ZIF-8)for HIV DNA detection.This method is simple and easy to operate.The DNA probe labeled by fluorophore adsorbed on the ZIF-8 surface.Due to the process of PET(Photoinduced Electron Transfer),the fluorescence would be quenching and the fluorescence of the system would be very weak.When the target(HIV DNA)exists,it complements the Probe chain to form double strands and then Probe detaches from the ZIF-8 surface,thus probe fluorescence recover.The experiment showed that the concentration had a good linear relationship at 0-100 n M,the actual lowest detectable concentration was 1n M,and had good selectivity.The actual sample(0.5% serum)analysis was done and still showed a good linear relationship.(3)A biosensor was constructed based on target DNA-triggered catalytic hairpin assembly and fluorescence enhancement of DNA-silver nanoclusters.H1 is used as DNA template to synthesize H1-AgNCs as fluorescent probe.Target DNA as trigger chain,opens hairpin H1 and H2 in sequence and initiates CHA(Catalytic Hairpin Assembly)process,and generates H1-AgNCs-H2.Target DNA is replaced to participate in the next cycle.The G-rich sequence originally locked in the stem of the H2 place in close proximity and the red fluorescence emission of H1-AgNCs is significantly enhanced.When the target is not present,the fluorescence of the system is very weak and almost negligible.The fluorescence biosensor is constructed based on the change of fluorescence intensity and constructs a linear relationship between fluorescence intensity and concentration.The results showed that an excellent linear relationship existed in the range from 100 p M to100 n M between ΔF and target concentration,the actual lowest detectable concentration was 0.1 n M,which provided good selectivity. |
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