A Label-free Fluorescent Quenching Sensor Based On DNA-scaffolded Silver Nanoclusters In The Application Of The Determination For Nuclease And DNA

With the continuous deepening research of nanotechnology,nano-materials have been widely applied in the field of biochemical analysis chemistry.DNA scaffolded silver nanoclusters(DNA-AgNCs)act as a new class of fluorescent nano-material,they have attracted many science researchers' attention due to their unique electronic structures and the excellent physical and chemical properties,such as ultra-small size,excellent brightness,high fluorescence quantum yield,tunable fluorescence emission,good biocompatibility and so on.Meanwhile,the superiority like facile synthesis,cost-effective and low toxicity help DNA-AgNCs more and more use in cell imaging,biomarker and biochemical sensor system,and an increasing number of fluorescence biosensors utilize DNA-AgNCs as fluorescence signal probe to successfully detect many kinds of ions,small-molecule,nucleic acid,protein,cancer cell and so forth.This paper aims at using DNA-AgNCs as fluorescence probe to develop novel biosensors for the detection of endonuclease EcoRI,human hemochromatosis gene,and human immunodeficiency virus gene,respectively.The brief contents are outlined as following:1.Summary the synthetic methods of DNA-AgNCs by different templates and the biological sensing platform of the DNA-AgNCs that apply in the fluorescence analysis.2.Different fluorescent property of DNA-AgNCs with different DNAs that contains different numbers of cytosine bases is diverse,based on this principle,we had developed a new label-free platform by DNA-AgNCs for detection EcoRI and its inhibitors.We design two kinds of especial DNA sequences,a template DNA named C-rich DNA,it is modified with two sequences of EcoRI recognition site GAATTC and three sequences of AgNC nucleic acid sequences(each nucleic acid sequence of AgNC is six cytosine bases sequence)between the EcoRI recognition site,a G-rich DNA can complete complement with the C-rich DNA.When the C-rich DNA mixes with G-rich DNA,they hybridize and form double-strand DNA(dsDNA)as the substrate of EcoRI.DNA-AgNCs is synthesized by the long chain C-rich DNA exhibite strong fluorescence in the absence of EcoRI.Once EcoRI appeare,it can cut the recognition site of sequence of the dsDNA;the fluorescence intensity of the system became weak.With the increasing of EcoRI concentrations,the fluorescence intensity of DNA-AgNCs gradually decrease,there are two linear relationships in the range from 4.34×10-4 U·?pL-1?1.74×10-3 U·?L-1and 2.17×10-3U·?L-1?1.09×10-2 U·?L-1,the limit of detection of EcoRI is 2.1×10-4 U·?L-1(S/N=3).This method exhibits excellent specificity that is able to distinguish the non-target endonuclease,this strategy is also able to reserch pyrophosphate and 5-fluorouracil which are the inhibitors of EcoRI,the IC50 value of them are approximately 2.33 mM and 64.06?M,respectively.Moreover,the assay can detect the target endonuclease in real human serum samples,the recoveries ranged from 96%to 102%for the six samples.It can clearly prove that the proposed menthod would have the great potential in drug screening and disease diagnostic.3.This system taking advantage of exonuclease III(Exo III)-assisted double recycling amplification and label-free fluorescent DNA-templated silver nanoclusters(DNA-AgNCs)to detecte nucleic acid.Taking human hemochromatosis gene as a model(T-DNA),a MB and a C-rich probe(C-DNA)are designed,one part of the MB can hybridize with T-DNA to form dsDNA with a blunt 3'-hydroxyl termini,another part of the MB is able to complement with the C-DNA to form another dsDNA with a recessed 3'-hydroxyl termini,they are both the substrates of Exo III.Once the T-DNA appears,MB will be opened and cut by Exo III,so that T-DNA and a residual sequence(R-DNA)are released,the releasing T-DNA hybridizes with unbroken MB again and R-DNA hybridizes with C-DNA to trigger digestion of C-DNA by Exo III,R-DNA is released again.Finally,there is no template to synthesize DNA-AgNCs and fluorescence response signal can not be output.However,in the absent of target DNA,the intact C-DNA act as template to synthesize DNA-AgNCs with strong fluorescent.So T-DNA enable to be detect based on the fluorescent of the system,a linear correlation with the concentration of T-DNA ranged from 200 pM to 40 nM and the limit of detection is as low as 120 pM(S/N=3).Meanwhile,this strategy exhibits excellent specificity that is capable of discriminating mismatched DNA from complete matched T-DNA,our method also can apply in real biological samples without label,the recoveries are 90?96.7%.These indicate that our assay have a potential application in a wide filed such as clinical diagnostics,gene therapy and biological process researches.4.This section describe a label-free and sensitive fluorescence biosensing platform for human immunodeficiency virus gene(HIV-DNA)detection based on luminescent DNA-scaffolded silver nanoclusters(DNA-AgNCs)and autonomous exonuclease III(Exo III)-assisted recycling signal amplification.In this assay,we design three kinds of DNA specially,long-chain DNA(X-DNA),assistant DNA(F-DNA)and a template DNA(P-DNA).One X-DNA enables to hybridize to one HIV-DNA and two F-DNA simultaneously,P-DNA is served as template of DNA-AgNCs,it also enable to complement with F-DNA.After HIV-DNA is introduced,HIV-DNA,F-DNA and P-DNA can hybridize to become a big double-stranded DNA(dsDNA)with blunt 3'-hydroxyl termini,so X-DNA is digested by Exo ?,F-DNA and HIV-DNA are released.The F-DNA will go to hybridize with P-DNA,and Exo ? cut P-DNA to stepwise remove mononucleotides,HIV-DNA and F-DNA enable to cyclic reaction.Finally,P-DNA is digested completely,the output of fluorescence response signal is prohibited.Oppositely,in the absent of HIV-DNA,the fluorescence of DNA-AgNCs can be generated and providing strong readout signal.This assay can quantitative detect HIV-DNA from 50 pM to 5nM,the detection limit was 35 pM.The specificity of this method is excellent,the approach is also capable to detect HIV-DNA in real human serum samples,the obtained recoveries were from 96.9%to 104%.The new method is really simple and nontoxic,it opens a promising approach for construction of sensitive and label-free biosensor.