Studies On Construction Of Fluorescent Biosensor Based On Exonuclease ?-assisted DNA Amplification Strategy

Exonuclease III(Exo III)can selectively recognize and gradually hydrolyzes double-strand DNA sequence from the 3'-blunt or recessed 3'-termini without the need of a specific recognition sequence.Therefore,Exo III is widely used in the construction of DNA signal amplification biosensors.With the thorough study of the interaction between small molecules and proteins,the steric hindrance caused by the binding of small molecules and their aimed protein can protect single-stranded DNA(ss DNA).The simple operation and the freedom coding of DNA sequence combine with the DNA signal amplification detection strategy,which makes the labeled ss DNA an ideal choice to detect these proteins.G-rich DNA sequences can be folded into a highly ordered G-quadruplex or G-triplexes structure under the induction of Na~+,K~+,small molecules,or certain fluorescent dyes.The G-quadruplex or G-triplexes can combine with fluorescent dyes such as thionin T(Th T)to emit enhanced fluorescent signals and has extremely wide applications in the construction of fluorescent biosensors.The Y-shaped three-way structure can be conveniently used for specific recognition of target DNA,because of its simple structure and high stability.This paper constructs a series of biosensors mainly using the G-quadruplex or G-triplexes structure,Th T,and Exo III-assisted DNA signal amplification strategy based on Y-shaped three-way structure to detect streptavidin(SA),target DNA,K-ras gene and HBV DNA,the main research content can be divided into the following three parts:(1)Ultra-sensitive detection of streptavidin based on small molecule DNA terminal protection strategy.When SA is present,SA binded to the biotin labeled on the 3'-end of L DNA,which protected and released L DNA from the hydrolysis of Exo III.L DNA triggered Exo III-assisted circulation amplification and released a large number of G-rich sequences to folding into G-quadruplex structure under the induction of Th T and giving a strong fluorescent signal.The experimental results show a linear between the fluorescence difference of the system and the logarithm of the SA concentration within the range of 0.005-2000 ng/m L,and the detection limit is0.002 ng/m L,which appears a similar linear relationship in 1%serum samples.(2)Universal label-free ultra-sensitive detection of DNA based on exonuclease III-assisted dual-circulation amplification.When the target DNA was present,the target DNA combined with MB and triggered Exo III-assisted circulation amplification to release the target DNA and a G-rich trigger DNA(TG).With the help of Exo III,TG could hybrid with HPB and triggered the chain replacement circulation for releasing another G-rich sequence HPBG.In the induction of Th T,TG and HPBG folded into G-quadruplex structure and emitted a strong fluorescent signal.The experimental results show a linear between the fluorescence difference of the system and the logarithm of the target DNA concentration within the range of 20 f M-2 n M,and the detection limit is 15 f M;a linear between the fluorescence difference of the system and the logarithm of the K-ras gene concentration within the range of 200f M-20 n M with a detection limit of 88 f M,the spiked recovery of K-ras gene in 0.5%serum samples was 93.40%-108.1%.(3)Detection of HBV DNA based on multiplex-circulation amplification strategy of Y-shaped three-way structure.When HBV DNA was present,HBV combined with HP and triggered the Exo III-assisted circulation amplification to release HBV and T fragments which had the same triggering effect as HBV.Both HBV and T fragments could form Y-shaped three-way structure with PC and triggered Exo III-assisted circulation amplification.After such multiple cycles,a large number of G-rich sequences were released and folded to form G-triplexes structures under the induction of Th T,emitting a strong fluorescent signal.The experimental results show that a linear between the fluorescence difference of the system and the logarithm of the HBV concentration within the range of 50 f M-500 p M with the detection limit of 33f M,and the spiked recovery in the 0.5%serum sample was 94%?101.8%.