A Biosensor Based On ExoⅢ-assisted Signal Amplification Detection Of MiRNA-122

miRNA is an endogenous,non-protein-coding single stranded RNA with a length of 18 to25 nucleotides.It regulates gene expression by inhibiting messenger RNA（m RNA）translation or degrading m RNA.miRNA was found to be closely related to cell proliferation,differentiation,metabolism and some physiological diseases.miRNA-122 is reported to be associated with diseases such as the human liver,which urgently requires the establishment of a sensitive biosensor for detection.ExoⅢcan efficiently identify 3’blunt end of double-stranded nucieic acid substrate,which provides a common platform for signal amplification for detection of various disease.It is noteworthy that SG I fluorescent dyes exhibits the fluorescence stability.moreover,DNA-AgNCs has a good biocompatibility,high fluorescence quantum yield,and the advantages of fluorescence emission is adjustable,making it widely used in biosensor and intracellular imaging,and successfully detect many kinds of cancer cells,proteins,nucleic acids,small molecules,metal ions,and so on.This paper aims at using SG I fluorescence dyes or DNA-AgNCs as fluorescence probe to develop three lable-free,high-specificity,sensitive fluorescent biosensors based on ExoⅢ-assisted signal amplification technology for the detection of miRNA-122.The major contents are outlined as follows:1.Based on the SG I fluorescent dyes and ExoⅢ,a lable-free,high sensitivity,high specificity of signal amplification method was constructed for the miRNA-122 detection.This method design two hairpin probes,its 3’stand out 6-7 bases for ExoⅢactivity.Target miRNA-122 with hairpin probe HP1 can form 3’blunt end,which can be cut by ExoⅢ.The product of hydrolysis is released,and partly hybrids with hairpin probe HP2 to form 3’blunt end,which can be digested by ExoⅢ;the other one is the target,and then conbines with the remaining HP1.This builds a double-cycle process.Then,SG I fluorescent dye was added.Since the dye can only embed the DNA double helix structure,the fluorescence intensity of the whole system was greatly reduced,and its fluorescence intensity was F.However,when the target miRNA-122 is not included in the system,the SG I fluorescent dye can be embedded into two hairpin probes（HP1 and HP2）,thus producing a strong background signal（F₀）.The fluorescence signal changes（F₀-F）and the concentration of miRNA-122 had a linear relationship between 0.2-2.4 nmol/L.The detection limit was 43 pmol/L（S/N=3）,and the recoveries were 92.6%to 103%.Based on ExoⅢ,we established a kind of signal amplification method.This method has universality and can be used to detect other miRNAs by changing nucleic acid sequences.This method has good selectivity.It can effectively distinguish the miRNA analogues of 10 times of the concentration.Besides,this method is easy to operate,low cost and has potential application value.2.Based on DNA-AgNCs and ExoⅢ,a dual-cycle lable-free fluorescence quenching method was established for sensitive and selective detection of miRNA-122.In the experiment design,the cytosine（-CCCCCC-）was used as the fluorescent output signal,and the other hairpin probe（HP1）was designed to hybrid with the target.When the target miRNA-122 dose not exist,the two hairpins can stabilize in the system,and then Ag⁺and Na BH₄ are added,thus a strong fluorescence signal（F₀）produces.When the system contains the miRNA-122,the target can be recognized and cut by ExoⅢ,which releasing the taget is into cycling one and the residual can hybrid with HP2 to form a 3’bilunt end.Then,fluorescence probe HP2 is cut by ExoⅢ,so that the fluorescence value of the system is greatly reduced（F）.The signal amplification can be achieved through the double-cycle recycling.The fluorescence signal changes（F₀-F）and miRNA concentrations were linear relationship ranged from 0.5-6 nmol/L.The detection limit is 104pmol/L（S/N=3）.The method has the following advantages:firstly,the DNA is lable-free,the reagent is easy to get,the operation is simple,and the cost is low.Secondly,the method has good sensitivity and specificity,and can distinguish the miRNA analogue of 10 times of the concentration.In addition,complex biological samples can be tested and the recoveries are 96%to 104%.Owing to versatility,this method is also vailable for detecting of other miRNAs,DNAs,proteins,small molecules and metal ions.3.A DNA-AgNCs fluorescence enhancing sensor is established for detection of miRNA based on ExoⅢ-assised DNA-RNA hybridization recycling.When the target miRNA-122 exists in the system,two hairpin probes（HP1 and HP2）are designed to cut by ExoⅢ.HP1 can be hybrid with miRNA-122,and HP2 contains the enclosed silver cluster synthesis template（-CCCCCCCCCC-）.When the target miRNA-122 is not included,the silver cluster template is closed and can produce a weak fluorescence（F₀）.When contains the miRNA-122,the target and HP1 can form a 3’blunt end,which can be digested by ExoⅢ.Then the target and a residual DNA is relased.Next,HP2 can recognize the residual DNA to form double-stranded DNA cut by ExoⅢ.Finally,the fluorescence of DNA-AgNCs can be generated and providing strong readout signal（F）.There was a good linear relationship between F/F₀ and the concentration of miRNA-122 ranged from 0.3-3.4 nmol/L.The detection limit is 265 pmol/L（S/N=3）,with good sensitivity.The recoveries are 94.2%and 106%,and the method can be used to detect the complex biologic samples.This method has good selectivity for miRNA analogs,which can obviously distinguish between the target and the analogs of 10 times higher concentration.In addition,this method has universality,in other words,it can be applied for other disease-related biomakers by replacing target recognition sequence.Finally,the method is simple,saving time,low cost,and the synthetic DNA-AgNCs fluorescence is stable and has good application prospect in biosensor design.