Studies On The Application Of Biosensor Based On The Signal Amplification Technique And G-quadruplex

Recently,sensitive and specific detection of nucleic acids finds fast growing applications in diagnostics of infectious diseases,microRNA analysis,food control,epigenetics,human identification in forensic investigations,as well as in biomedical research.While detection of trace amounts of nucleic acid is extremely important in gene therapy,mutation analysis and clinical diagnosis.They almost rely on several signal amplification technology,for example,enzyme assisted signal amplication,hybridization chain reaction(HCR),strand displacement amplification(SDA),and rolling circle amplification(RCA)et al.Among these signal amplification approaches,the enzyme-based signal amplification has recently become a research focus,because it improves the sensitivity of the biosensor and shows simple operation,rapid reaction and can be easily developed for homogenous assay.This paper aims to further extend the application of biosensor in nucleic acid and small molecule assay.Based on T7 exonuclease(T7 exo)aided signal amplification and G-quadruplex,respectively,we built new fluorescence and fluorescence polarization(FP)biosensor for DNA and ATP assay.The main results are outlined as follows:1.An overview of signal amplification technology and G-quadruplex in the application of biosensor was summarized.2.In this paper,a homogeneous enzyme-assisted signal amplification combined with graphene oxide(GO)acted as the FP signal amplifier was constructed for HIV DNA detection.T7 exo could stepwise hydrolytically digest mononucleotides from the 5'-terminus of a dsDNA with a blunt or recessed '-terminus,while it has no activity on ssDNA or dsDNA with a 5'-protruding terminus.Moreover,probes PI are designed in such a way that they are resistant to catalytic digestion by T7 exo in the absence of the target DNA.Target DNA hybridizes with P1 and opens its hairpin structure,then T7 exo cleaves them and liberate a lot of ssDNA,ssDNAs are absorbed on the surface of GO,which obtains a high FP value.This method takes advantages of T7 exo cleavage reaction and the strong FP enhancement function of GO.Due to the significant signal amplification,the proposed method exhibits a good linear response when the target DNA concentration ranges from 50 pmol·L-1 to 2000 pmol·L-l and 38.6 pmol·L-1 HIV DNA can be detected.Moreover,the proposed method can discriminate single-base mismatched DNA from target DNA.Compared with other methods,it is a simple and sensitive method for DNA detection.This strategy is done in homogeneous solution,and not requiring other troublesome procedures.Finally,the proposed FP sensing platform can be used to detect a wide range of DNA targets.3.In this work,a homogeneous enzyme-assisted quadratic amplification combined with GO as the FP signal amplifier was constructed for HIV DNA detection.In the absence of target,probe P1 and P2 are all absorbed onto the surface of GO,it has low FP values.While in the present of target,it hybridizes with 5'-protruding terminus of P1 to form partially dsDNA with 5'-blunt terminus at one side and 5'-protruding terminus at the other side,which can be cleaved by T7 exo to release the target and X-DNA.The released target again hybridizes with P1 to initiate the target recycling cycles to generate numerous X-DNA(recycle 1).The X-DNA hybridizes with P2 and opens the hairpin structure to form partially dsDNA,the same way as P1 with target DNA.Then T7 exo cleaves them and liberate a lot of ssDNA,the X-DNA can hybridize with P2 and start another recycling cycle(recycle 2).So a quadratic amplification strategy is achieved to generate a large number of ssDNA,these ssDNA are absorbed onto the surface of GO,which result in significantly high FP value of the solution.The method takes advantages of the T7 exo-assisted quadratic amplification and the strong FP enhancement function of GO.Because of significant signal amplification,9.12 pmol·L-1 HIV DNA can be detected.The biosensor shows excellent specificity to discriminate single base mismatched targets well,and was successfully applied to human serum sample.This strategy is carried out in homogeneous solution,and does not require separation and other troublesome procedures.Compared with other methods,it is a simple and sensitive method for DNA detection.Furthermore,this FP biosensor has a great potential for detection in vivo.4.In this work,a label free split G-quadruplex aided fluorescence biosensor for adenosine triphosphate(ATP)based on Exonuclease III(Exo ?I)and ligation reaction was constructed.In the absence of ATP,T4 DNA ligase has no effect on G1 DNA and 5'-p-G2 DNA,so the ligation reaction can not occur.G1 DNA and P-DNA are all digested by Exo?I,so G2 DNA can not form G-quadruplex structure.When we add NMM into the solution,the biosensor has low fluorescence intensity.However,in the presence of ATP,G1 DNA and 5'-p-G2 DNA are ligated by T4 DNA ligase,which forms dsDNA with 3'-protruding terminus at the one side and 3'-blunt terminus at the other side,so this dsDNA is digested by Exo III and liberites a long ssDNA(G1 DNA + G2 DNA).So ssDNA forms a G-quadruplex structure,the biosensor obtains high fluorescence intensity with NMM.The fluorescence intensity exhibits a linear relationship with ATP concentration from 0.5 nmol·L-1 to 200 nmol·L-1 and a detection limit was estimated to be 0.2 nmol·L-1.The biosensor shows excellent specificity to discriminate target,in addition,our method is convenient and simple,and demonstrates the great potential of G-quadruplex in biosensors development.