Research On Universal Fluorescent Biosensors Based On DNA Amplification

It is an important task for biochemical analyzers to improve the sensitivity of analytical methods and to establish biosensors which can be widely used and quickly detection.Universal biosensors are designed to detect different substances by changing individual elements using almost the same principle.And its outstanding advantage is the universality of application.The method to improve the sensitivity is usually multiple cycles of DNA signal amplification.DNAzyme is a catalytic DNA fragment consisting of binding sites and recognition sites.And DNAzyme can hybridize with substrate and cleave it.Compared with traditional protein enzymes,DNAzyme's inherent advantages are its multi-functional design,stable at room temperature and high temperature,low cost,and easy to synthesize and label.G-triplex structure is generally considered as one of the most reasonable intermediates in the folding process of G-quadruplex structure.At the same time,this structure also shows catalytic function similar to G-quadruplex in vitro.In addition,the G-triplex has fewer G bases,and the design of G-rich hairpin is more flexible because the G-C in the neck is less.Therefore,it is more flexible design.Nucleic acid dyes are used to construct the relationship between the fluorescence signal and the concentration of the target.In this paper,three different types of universal fluorescent biosensors are constructed by using G-triplex as a fluorescent signal probe,using catalytic cleavage of DNAzyme and nucleic acid cleavage enzyme to assist the target cycle.And according to the spectral characteristics of nucleic acid dyes,they enable detection of DNA,metal ions and small molecules.?1?Ultra-sensitive fluorescence detection of H5N1 based on Exo III and DNAzyme amplification.When H5N1 existed in the reaction system,it is complementary to the hairpin H1 forming Exo III effect of 3'end,under its hydrolytic released H5N1 and DNAzyme segments.So H5N1 can participate in the next cycle.And DNAzyme identify the substrate recognition sites on the hairpin H2,under the action of Mg²⁺catalytic shear,thereby releasing G-rich chain of H2,interacted with Thioflavin T?Th T?which can show strong fluorescent signal.Experimental results showed that the concentration range of H5N1 at 0.1-25 n M was linear with the fluorescence signal,and the detection limit was 35 p M.Good linearity was also shown in the actual sample?serum?.?2?The detection of Bt DNA,Hg²⁺and thrombin was realized by a universal biosensor based on DNAzyme cycle amplification.The target can combine the split DNAzyme fragments into a three-chain complex,which is a complete DNAzyme activated by Mg²⁺and catalyzes the shear of the substrate to release the G-rich chain.Thus,the detectable signal is generated by the interaction between the G-triplex and the nucleic acid dye?Th T or Hemin?.The results showed that the concentration of Bt DNA within the range of 0.06-50 n M showed a good linear relationship with the fluorescence signal,and the detection limit was 28 p M;Hg²⁺showed good linearity in the concentration range of 0.3-30 n M,and the detection limit was 10.2 p M.Thrombin concentration ranged from 0.5 nm to 30n M,with a detection limit of 11 p M.In addition,all have good selectivity.?3?Detection of DNA and thrombin based on a label-free universal biosensor with multiple cycles triggered by target.When DNA and subfractions are present,the strand replacement amplification between the H1 and H2 hairpins can be triggered to form double-stranded DNA.Then,with the help of Nt.Bbv CI,multiple amplification was realized to generate a large number of G-rich sequence fragments.Fluorescence analysis and ultraviolet colorimetry were performed by adding Th T or Hemin/ABTS.The fluorescence analysis method was more sensitive,and the target DNA showed a good linear relationship with the fluorescence signal within the range of 0.6-120 n M,and the detection limit was 0.359 n M.Thrombin showed a good linear relationship at the concentration of 1-150 n M,and the detection limit was 0.697 n M.