High Sensitive Nucleic Acid Fluorescent Sensor New Methods And Performance Analysis

In this thesis,we have constructed three highly sensitive DNA fluorescent biosensors based on nuclease-assisted nucleic acid amplification cascade DNAzyme-assisted,target recycling,isothermal strand displacement amplification signal amplification strategy,accomplished detection of nucleic acid.?1?An autonomous target recycling and cascade circular exponential amplification?TR-CEA?strategy was proposed to construct an autocatalytic DNA machine,which afforded one-pot,isothermal and ultrasensitive detection of nucleic acids.The whole sensing system matrix consists of two ingeniously designed hairpin-like DNA strands and two enzymes.The MB is used as the fluorescence reporter probe,it also contains a Nb.BbvCI recognition domain in the loop region and a 3'-protruding domain,which has mutual base complementarities with domain a of the HP.Then,polymerization from the 3'-end of MB using Bst 2.0 polymerase induces target recycling for participation in the next round of the hybridization process,which constitutes the target recognition and recycling module.Simultaneously,duplex DNA with an integrated recognition site for the nicking endonuclease is produced,which can then be nicked by Nb.BbvCI and reverse polymerization from the nick proceeds for the strand displacement amplification?SDA?process.Subsequently,the hairpin structure of the MB is opened and the recognition site for the nicking endonuclease in the MB is activated,followed by initiation of the nicking and SDA process in the opposite direction.The cleaved MB contributes to the generation of a fluorescence response.A low detection limit of 0.61 fM toward the target DNA with an excellent selectivity could be obtained.?2?We have constructed a double signal amplification fluorescent biosensor based on entropy beacon cascade DNAzyme-assisted strategy to detect DNA.In process of entropy beacon target recycling,the target DNA binds to the terminal toehold region of the probes,displaces the DNA strands PP through toehold-mediated strand displacement reactions,formed the intermediate I3.And the intermediate I3 hybridization with the DNA fuel strand?Fs?,which further displaces both the target and the DNA strand R.Ebeacon is driven forward by entropy increase of the system instead of free energy released from new base-pair formation.In recycle ?,the product of recycle I includes Mg²⁺-DNAzyme,DNAzyme cleavage fluorescent substrate MB.This sensor based on enzyme-free,have low backgroud signal,high stability and specificity.?3?We have constructed another highly sensitive biosensor,based on strand displacement amplification reaction?CNDP?and Pb²⁺-DNAzyme signal amplification,detection of p53 gene.The target DNA can recognize HP to promote the HP hybridization with PT,displaces the target DNA and PP through the extending of PT,induces target recycling for participation in the next round of the hybridization process.Simultaneously,duplex domin of product with an integrated recognition site for the nicking endonuclease is produced,which can then be nicked by Nt.BbvCI.The product of CNDP includes Pb²⁺-DNAzyme,it also serve as target analogue promote target recycling.Polymerase enzyme and nick enzyme achieve synergies,repeated the process of the polymerization-cleavage-replacement,accumulation of Pb²⁺-DNAzyme function structure,cleavage MB then produce fluorescent signal.The sensor amplified signal independently and effectively,simple operation,and improve the sensitivity.