Studies Of Lable Free Fluorescent Biosensor Based On Exonuclease ?

Exonucleases are enzymes that can catalyze the hydrolysis of phosphodiester bonds from one end of a nucleic acid molecule to obtain a single nucleotide.They do not require DNA sequences,but some exonucleases have a certain degree of identity to the DNA ends,Thus there is a certain degree of flexibility in the design of bioluminescent sensors.Exonuclease III is a type of exonuclease that has the ability to efficiently catalyze hydrolysis,and it can specifically identify 3' double stranded DNA,and also be along the 3'-5' direction of the gradual catalytic resection of single nucleotide,so as to improve the sensitivity of the sensor.Metal Nano-cluster(NCS)are novel luminescent nanomaterials,Due to their simple synthesis,high stability and good biocompatibility,nanoclusters have become a promising new type of fluorescent nanocrystals in many fields,especially in biosensing applications.Nucleic acid aptamers are oligonucleotide fragments obtained by using in vitro screening technology and are a sequence of DNA or RNA,which can be highly specific and highly selective binding target,so Nucleic acid aptamers are widely used in biochemical analysis,environmental monitoring and many other aspects.Nucleic acid dyes are used to establish the relationship between the fluorescence signal and the concentration of the target,and the target can be quantitatively detected by utilizing the change of the fluorescence signal intensity.This paper mainly uses the spectral characteristics of metal nanoclusters and nucleic acid dyes to construct different biosensors to detect HIV DNA,p53,and ochratoxin A,which mainly includes the following three parts:(1)HIV was detected based on DNA strand replacement and nucleic acid extranase III by fluorescence.When HIV was not present,Exo III hydrolyzed the two hairpin DNA into single and partially single-stranded DNA structures.Upon addition of SGI,the system detected a weak fluorescence signal.When HIV is present,HIV and HP1,HP2 DNA strand occur displacement reaction,releasing the ends which are prominent long double-stranded DNA 3'end without being Exo III hydrolysis.After adding SGI,the system detects a strong fluorescent signal.The experimental results show that when the HIV concentration is in the range of 0.01-1 nM,the fluorescence intensity of the system is linear with the HIV concentration,and the detection limit is 2.14 pM.The spike recovery of the actual sample(serum)was 94%-98%.(2)The detection of p53 was based on gold-silver nanoclusters and exonuclease III label-free fluorescence method.When p53 is present,the G-DNA is released under the action of Exo ?.The G-DNA is complementary to the partially hybridized gold-silver nanoclusters of the DNA template,so that the fluorescence intensity of gold and silver nanoclusters increased significantly due to the close proximity of G rich sequences.When p53 is absent,there is no degradation after adding Exo ?,so the fluorescence signal intensity of the gold-silver nanoclusters is very weak without the proximity of the rich G sequence.The fluorescence signal of gold-silver nanoclusters is proportional to the concentration of p53.The fluorescence signal of gold-silver nanoclusters is proportional to the concentration of p53,the linear range is 0.5-15nm and the detection limit is 51.1pM.The actual sample(serum)of the standard recovery of 98.2%-102%.(3)Detection of ochratoxin A based on nucleic acid aptamers and exonuclease III unlabeled.When ochratoxin A is not present,the DNA double-stranded(S1-S2),HP1,and HP2 structures remain stable,and no hydrolysis occurs after Exo III,so G-DNA is not released,after adding ThT,it can not fold the formation of G quadruplex,the system detected a weak signal.When,ochratoxin A is present,ochratoxin A specifically binds to the aptamer chain(S2)in the long double strand of DNA(S1-S2),releasing S1 and S2 from the long double strand of DNA(S1-S2),they can hybridize with HP1 and HP2 to form long double-stranded DNAs,which was degraded after Exo III was added,releasing the free G-quadruplex sequence and detecting a strong signal.According to this,ochratoxin A can be detected by colorimetric method,UV-Vis,and fluorescence method.Among them,the sensitivity of fluorescence method is the highest,the detection concentration range is 3-150nM,and the detection limit is 323.5pM.The recovery of ochratoxin A in human serum by fluorescence detection was 97.7%-107.4%.