The Studies On Application Of GO-based Nano-biosensor For Fluorescence Polarization System

Graphene oxide(GO)is a two-dimensional.carbon nano-material with an atomically thin structure.In recent years,GO has been drawn tremendously attention because of its unique electronic,strong mechanical strength,nice water solubility and biocompatibility,excellent photoluminescence,facile surface modification capability for bio-conjugation.GO has been demonstrated to be a excellent platform for the construction of desirable bio-electronic devices and promising biosensors.It has been reported that GO can strongly absorb single-DNA(ssDNA)via hydrophobic interaction,vander Waals forces,?-? stacking,hydrogen bonding between the hexagonal cells of GO and the nucleotide bases,but hardly interacts within double-DNA(dsDNA).Interestingly,GO has been demonstrated to be a superquencher of dyes via nonradiative dipole-dipole coupling or effective fluorescence resonance energy transfer.Meanwhile,recent studies propose that GO can be used as an effective fluorescence polarization enhancer(FP)thanks to its extraordinarily larger volume.This thesis aims to further stretch on the applications of GO in bioassay of fluorescencepolarization assay.Based on GO material,a variety of novel fluorescence polarization systems were designed for oligonucleotides determination.The main details are outlined as following:1.Based on splendid fluorescence quenching efficiency of GO,a new-fashioned sensitive,simple,not expensive detection strategy has been developed for oligonucleotides by fluorescence analysis.When single-stand probe DNA was mixed with GO,the probe DNA(FAM-DNA)was absorbed onto the honeycombed surface of GO,the fluorescence of probe was quenched by GO.Meanwhile,the interaction between SYBR Green I and FAM-DNA is too weak,leading to a low fluorescence intensity both FAM-DNA and SYBR Green I.DNA hybridization is formed when target DNA present,DNA hybridization is separated from GO,because of weak ferce between dsDNA and GO,FAM-DNA emits fluorescence.At the same time,SYBR Green I binds to DNA hybridizations,the fluorescence signals of SYBR Green I is tremendous enhanced.In this method,greatest emission wavelength and the greatest excitation wavelength of FAM-DNA and SYBR Green I are almost similar,when detecting target DNA via fluorescence analysis,the fluorescence peaks of FAM-DNA and SYBR Green I overlap completely,the fluorescence intensity of this study will be tremendous enhanced.Under the optimal conditions.the fluorescence signal of the SYBR Green I/GO system increases with the increasing of target DNA concentration.This method shows sensitivity for target with a linear range from 5.0 nmol/L to 50 nmol/L and a detection limit of 3.624 1.mol/L(S/N=3).2.Based on T7 exonuclease-assisted target cyclic amplification,we reported a new amplification method for fluorescent polarization biosensor detection of oligonucleotides adopting GO as a mass amplification factor.The probe used for target detection is a hairpin DNA with a 5'-sticky end consisting of 6 nucleotides act as a toehold domain,T7 exonuclease cannot digest hairpin DNA with a 5'-sticky end.When hairpin DNA was mixed with GO.GO binds hairpin DNA via the ?-? stacking interactions between the GO surface and the base rings of the loop of hairpin DNA,leading to a significantly enhance in the mass of the FAM,thus the fluorescent polarization value increases.In the presence of target DNA,the hairpin DNA can be opened by hybridizing with target DNA from toehold domain to form a duplex DNA with a blunt 5'-termini at one hand and a protruding 5'-termini at the other hand.T7 exonuclease can catalyze the stepwise removal of the mononucleotides from 5'-termini of hairpin DNA to release fluorophore FAM and target T-DNA.The released T-DNA can hybridize with next hairpin DNA to trigger a new cleavage process and the released FAM dye is kept away from GO.Due to the faster rotation of FAM with small size,the fluorescent polarization value dramatic decreases.Under the optimal conditions,with inereasing of the target T-DNA concentration,the fluorescent polarization change increased gradually.A good linear relationship between target T-DNA and the fluorescent polarization change is observed at the low target T-DNA concentration region(3.0× 10-10 mol/L?2.4× 10-9mol/L),the limit of detection(LOD)was 132.9 pmol/L(S/N=3).For detecting target DNA at a high concentration range(3.0×10-9 mol/L?5.0 X 10-9 mol/L),the limit of detection(LOD)was 688.4 pmol/L(S/N=3).3.In current study,we develop a novel quadratic signal amplification method for oligonucleotides detection based on exonuclease ?-assisted catalyze DNA recycling amplification.We specifically design molecular beacon(HP)with a short ssDNA at its 3'-end which resisting EAo III digestion,the short ssDNA can partly hybridize with target T1-DNA.In the absence of the target T1-DNA,X sequence which complete complementary with probe DNA(FAM-P)was incorporated into HP in an original locked format.As a single stranded,FAM-P could strong bind with GO,leading to FAM-P exhibits an enormous enhance fluorescent polarization due to GO-based amplified volume of FAM-P.However,upon addition of target T1-DNA,target T1-DNA would partly complementary hybridize with the short ssDNA near 3'-termini of HP to form a stem with blunt 3' terminus,which Exo ? can cleave to release the X sequence and the target DNA.The released target T1-DNA could hybridize with HP to start the target Tl-DNA recycling cycles aiming at accumulating lots of X sequence.Meanwhile,X sequence can partly hybridize with FAM-P to form partially complementary double DNA.Exo III could remove mononucleotide from 3'-blunt terminus to release X sequence and FAM dye.The released X sequence could hybridize with next FAM-P initiate another independent X sequence recycling cycle.Finally,a large number of FAM dye was form which cannot interaction with GO and keep away from GO,leading to a weakened fluorescent polarization signal.Under the optimal conditions,the fluorescent polarization change exhibits a good linear relationship with target T1-DNA concentration in the range from 3.0×10-10 to 7.0×10-8mol/L,the limit of detection(LOD)was 4.8 nmol/L(S/N=3).