Research On Biosensing Methods Based On Enzymatic Isothermal Exponential Amplification Reaction

Enzymatic isothermal exponential amplification reaction(IEXPAR),as an amplification technique that combines polymerase strand extension and single-strand nicking,has high amplification efficiency and rapid amplification kinetics.This method has the distinct advantages that it directly gets ssDNA from double-stranded target.Therefore,EXPAR has huge potential for dsDNA detection.Making use of both the specific recognition of nicking enzyme and the advantage of single-stranded DNA easy to be detected,we design two kinds of biosensors for nucleic acid and protein detection,respectively.1.An electrochemical biosensor based on enzymatic isothermal exponential amplification.We describe an electrochemical biosensor for double-stranded gene detection based on enzymatic isothermal exponential amplification.The natural state of DNA is double stranded,which makes it difficult to be directly detected.This method firstly used nicking enzyme for exploiting in the double-stranded DNA(dsDNA).Then,long single-stranded DNA(ss DNA)was generated from the cutting site through polymerase extension reaction.Whereafter,the long ssDNA triggered a hairpin self-assembly recycling reaction,which gave rise to another isothermal amplification reaction.Last,short ssDNA was formed after the this amplification process,which could hybridize with the capture probe immobilized on Au electrode and result in signal variation.This method showed excellent analytical performance for dsDNA,of which the linear range was 2 fM to 500 pM and the detection limit was 1.6 fM(S/N=3).2.A fluorescence biosensor based on DNA assembly structure switching and enzymatic isothermal exponential amplification.We develop a fluorescence biosensor for VEGF detection based on DNA assembly structure switching and isothermal exponential amplification.The design employs a DNA assembly made of a isothermal amplification template,a aptamer,a primer and a protector chain.The DNA assembly is unable to undergo isothermal amplification in the absence of target.The presence of the target,however,triggers a structure switching event that causes hybridization of primer with template to facilitate isothermal amplification.Whereafter,toehold-mediated DNA strand displacement reaction between the generated(single-stranded DNA)ssDNA and fluorescent/quencher labeled probe are performed.Then,the increase in fluorescence provides an analytical signal.This strategy opens up a sensitive,selective and simple sensing platform for detection of VEGF.The system was also implemented to analyze the VEGF in human serum samples with satisfactory results.