Exonuclease ?-assisted Signal Amplification Fluorescence Biosensor For Detection Of DNA

Exonuclease III has a specific hydrolysis effect on double-stranded DNA,and the single nucleotide in the 3'flat end of the double chain is gradually sheared along the 3'?5'direction,and the sensitivity of DNA sensor can be improved by its shearing characteristics.Deoxyribonucleotide(DNA)sequences are the main chemical constituents of DNA chromosomes,as a genetic instruction,it directs the development of biology and the operation of life function,Therefore,nucleic acid detection is particularly important,in biological research,clinical diagnosis and biological applications.In recent years,the developed DNA fluorescence biosensor has the advantage of easy operation and high sensitivity,so it becomes one of the most widely used detection methods.2-Aminopurine(2-AP)is a adenine analog which is the isomerism of adenine and has good fluorescence properties.As a new type of nanomaterials,gold nanocluster(AuNCs)has advantages of small size,good stability,large stokes displacement and good biocompatibility,and it has been widely used in analytical chemistry and other fields in recent years.The water-soluble nucleicacid dye thioflavinT(3,6-dimethyl-2-(4-dimethylaminophenyl)benzo-thiazolium cation ThT)is a fluorescent dye with excellent G-quadruplex recognition specificity against other structural DNAs.In this paper,several new fluorescent biosensors were constructed based on the hydrolysis of Exonuclease III and the spectral properties of 2-aminopurine,gold nanoparticlesand nucleic acid dye ThT.Which mainly include the following contents:(1)Rapid detection of p53 mutant genes based on Exonuclease III and 2-aminopurine fluorescence probes.Firstly,a 2-AP labeled probe(cDNA)and its partially complementary DNA(bDNA)are designed to form a cDNA/bDNA dual-chain sensor.when the target DNA(tDNA)was added,the strand displacement reaction formed a more stable cDNA/tDNA dsDNA with a blunt or recessed 3'terminus,which can be stepwise hydrolytically digest mononucleotides from the 3'-OH terminus by Exo III,releasing the target DNA and constantly producing free 2-AP.Then the released tDNA induced a new round of enzymatic hydrolysis reaction,the reaction system fluorescence signal constantly enhanced,so as to achieve the system fluorescence signal amplification.The p53 concentration has a good linear relationship with the variation value(?F)of the system fluorescence in the 1-850nm range and the detection limit is 104pM The method has the advantages of fast analysis,simple operation,high selectivity and low detection limit.(2)A DNA-stabilized gold nanoclusters as fluorescent sensors for HIV-1 detection based on exonuclease ?-assisting.By using DNA as template to synthesize gold nanoclusters,we constructed DNA-AuNCs fluorescent probes.Using the shear characteristics of Exo III to double strand DNA,on one hand,Exo III can hydrolytically digest double-strand DNA formed by HIV-1 and its partially complementary DNA,realizing the cycling of HIV-1 and achieving the aim of improving sensitivity;on the other hand,under the action of Exo ?,the DNA sequence(G-DNA)which is complementary to the DNA-AuNCs fluorescence probe was continuously produced.Therefore,the system fluorescence signal was amplified,the HIV-1 DNA was detected according to the change of the system fluorescence signal.The results showed that the AF has a linear relationship with the HIV-1 in the 1-400nm range,the detection limit is 154pM.The method is simple and low-cost,high sensitivity,good specificity,which can be used for real sample detection.(3)Exonuclease ?-assisted cascade signal amplification strategy for label-free and sensitive fluorescence and visual detection of HBV DNA.When the existence of HBV,HBV successively opened two hairpin DNAs(HP 1?HP2)to form the HBV-HP1-HP2 double chain structure with 3'-blunt terminus.Which can be cleaved by Exo III to release the HBV(tDNA),the partial complementary sequence of HP2(pDNA)and the G-quadruplex sequence(G-DNA).tDNA and pDNA are respectively involved in the cycle I and cycle II,on the basis of the spontaneous cleavage of cycles I and ?,achieved cyclic amplification.Upon completion of the ExoIII-assisted recycling,the released G-quadruplex sequence can bind ThT or Hemin to yield G-quadruplex complexes.The ThT/G-quadruplex resulted in obvious enhancement of the fluorescent signal;the Hemin/G-quadruplex can catalyze the oxidation of ABTS2-in the presence of hydrogen peroxide,the color of solution apparently changed and measured its ultraviolet visible absorption spectroscopy.Accordingly,we can use the fluorescence,colorimetric,and UV-Vis methods for the detection of HBV.Among them,the sensitivity of the fluorescence method is the highest.The ?F has a linear relationship with the concentration of HBV in 0.2-50nM range,the detection limit is 54 pM.