The Studies On Application Of T4 DNA Ligase And Graphene Oxide For Fluorescence Biological Sensors

Enzymes and nanomaterials play important roles in biotechnology.Biological sensors based on enzymes and nanomaterials have successfully applied in many fields across clinical diagnosis,drug screening,environmental monitoring and biochemical research.It is one of cutting-edge research projects in the field of biochemical analysis to develop new biological sensors,which is based on the previous studies.Due to the advantages such as universal,stabile,simple and fast,fluorescence polarization is very suitable for homogeneous assay.Though fluorescence polarization have showed the advantages in the field of biochemical analysis,integrating enzymes and nanomaterials with fluorescence polarization to build more sensitive and efficientive analytical methods for the further development of fluorescence polarization in the field of biochemical analysis,have still remained a challenge and more studies need to be done.The aim of this thesis is to further expand the application of enzymes and nanomaterials in the fluorescent biochemical analysis.Based on the previous researchs,some kinds of new fluorescence biological sensors based on T4 DNA ligase and graphene oxide were developed for detection of adenosine triphosphate(ATP)and oligonucleotide.The main contents are outlined as following:Part I:Based on T4 DNA ligase with ATP-dependent ligation activity and exonuclease III(Exo Ⅲ)with specific exodeoxyribonuclease activity for double-stranded DNA(dsDNA)from blunt or sticky 3’-terminus to 5’-terminus,using graphene oxide as signal enhancement factor,we developed a fluorescence polarization analysis platform for sensitive detection of ATP.Firstly,a DNA probe with a modified phosphate group at the 5 ’terminal(P-DNA)and a DNA probe with a modified fluorescein derivative(FAM)at the 3 ’terminal(F-DNA)were hybridized with a long complementary strand DNA(C-DNA)simultaneously to form a nicked duplex.In the absence of ATP,P-DNA and F-DNA could not be ligated together,F-DNA with sticky 3’-terminus and C-DNA with blunt 3’-terminus were digested by Exo Ⅲ from dsDNA subsequently,resulting in the release of a short DNA fragment carrying the FAM from the GO surface.Due to the slower rotation of short DNA fragment with small size,the fluorescence polarization value small.In the presence of ATP,P-DNA and F-DNA were ligated by the T4 DNA ligase,resulting in the form of a new long strand DNA with a 6-nucleotide single-strand overhanging domain.ExoⅢ can only digested C-DNA,the new long strand DNA remains and then adsorbed on the surface of GO,leading to a significantly increase in fluorescence polarization value owing to amplified volume of fluorescent unit.With increasing of the ATP concentration,the fluorescence polarization value increased gradually.The ΔP value versus ATP concentration in the range of 0.1～70nmol/L showed good linear relationships with a detection limit of 0.20nmol/L.PartⅡ:We have developed a graphene oxide-aided signal amplifying fluorescence polarization strategy for detection of ATP using ATP-dependent ligation reaction and toehold-mediated strand displacement reaction.In this assay,four DNA oligonucleotides were employed:single-stranded DNA probe(P-DNA)with a modified phosphate group at the 5’terminal,assistant single-stranded DNA(A-DNA),complementary DNA template(C-DNA)and FAM-labeled DNA(F-DNA).P-DNA and A-DNA both hybridized with C-DNA to form a nicked duplex with a 7-nucleotide single-strand overhanging domain(known as the toehold).In the absence of ATP,the ligation reaction could not occur between P-DNA and A-DNA.Then,F-DNA was introduced to displace P-DNA from the nicked duplex by toehold-mediated strand displacement reaction.Due to hybridize with C-DNA,F-DNA could not adsorbed on the surface of GO,the fluorescence polarization value weak due to the faster rotation of dsDNA with small size.In the presence of ATP,P-DNA and A-DNA were ligated by the T4 DNA ligase to form a new long strand DNA,the toehold-mediated strand displacement reaction could not occur between F-DNA and P-DNA,F-DNA was adsorbed on the surface of GO,resulting in an significantly increase in fluorescence polarization value owing to slower rotation of fluorescent unit with larger volume.A linear range between the ΔP value and ATP concentration was from 0.8 to 80nM,and the detection limit was 0.78nmol/L.The reported method is expected to provide a new strategy for specific and sensitive detection of ATP.Part Ⅲ:In this work,we developed a T4 DNA ligase-assisted label-free fluorescence method for quantitative detection of Hepatitis B virus DNA(HBV DNA),introducing exonuclease Ⅰ(Exo Ⅰ)and exonuclease Ⅲ(Exo Ⅲ)to minimize the background fluorescence signal and SYBR green I(SG I)as a readout fluorescence signal.In this strategy,we designed a dumbbell DNA probe consists of two loop regions and a stem region with a nick.In the absence of HBV DNA,the dumbbell DNA probe was sealed by the T4 DNA ligase,and the sealed loop resisted digestion by Exo I and Exo Ⅲ.The SGI binds to the stem region duplex and exhibits strong fluorescence.On the contrary,upon the addition of the HBV DNA,the HBV DNA hybridized and opens the the dumbbell DNA,the HBV DNA-dumbbell DNA duplex was completely digested by ExoⅠ and ExoⅢ,and the fluorescence of free SGI is very weak.The fluorescence intensity reduce efficiency shows a good linear correlation with the concentrations of HBV DNA from 1nmol/L to 35nmol/L.The detection limit was estimated to be 4.26nmol/L.This detection scheme can also be expanded to detect other DNA and RNA.