Detection Nucleic Acids In Complex Samples Via Magnetic Separation-assisted Multiple Amplifications

Nucleic acids are a type of biological macromolecules that play an important role in life activities.Some of short nucleic acids have attracted wide attentions in recent years,such as cell-free DNA(cfDNA)and microRNA(miRNA).Because these nucleic acids are often the disease markers or the targets for cancer treatment,the detection of these nucleic acids is of great value.However,they are often present at lower abundance in complex samples such as exosomes,blood or saliva,its detecion suffers from the problems of detection sensitivity and signal accuracy.Thus,in this thesis,magnetic beads-based separation strategy was used to eliminate the interferences of coexisting components and enrich the target.Then,using nucleic acid-based amplification technology,it was realized to detect nucleic acids in complex samples(such as serum and cell culture medium)with high sensitivity.Details are as follows:1.Magnetic microbead-based catalyzed hairpin assembly(CHA)and "DD-A" fluorescence energy resonance transfer(FRET)for the detection of nucleic acids in complex samples.This method used three DNA hairpin probes,namely Capture which was modified on magnetic microbeads,H1(labeled double FAM)and H2(labeled single TAMRA).First,the Capture on the magnetic microbeads bound with the targets in the samples,exposing a fragment that could trigger the CHA reaction.Then,the target could be separated and enriched by the function of magnetic field.Finally,the magnetic microbeads were resuspended in the buffer containing H1 and H2,and CHA was initiated to generate a large amount of H1/H2 complex.So that an apparent FRET signal was observed.However,in the absence of the target,CHA reaction could not be initiated,and the FRET signal was weak.By using magnetic microbeadassisted CHA and “DD-A” FRET,this method effectively eliminated the errors caused by background interferences and amplified the signal.The detection limits for miRNA-21 was as low as 34 pM.This method was also used to detect miRNA-21 in serum samples and cell culture media.It is expected to be applied to biomedical research and clinical testing.2.Detection of miRNAs in complex samples based on dye self-quenched exonuclease(Exo III)cleavage and DNA bio-barcode technology.In previous work,it was found that when two FAM dyes were labeled in adjacent nucleotides,fluorescence intensity was greatly quenched.When DNA was digested by the enzyme,fluorescence intensity could be increased up to 20 times.Based on this phenomenon,four DNA probes were designed,including the CP modified on the magnetic microbeads,the AP modified on the gold nanoparticles(AuNP),the HP modified on the AuNP and signal probe SP(labeld with adjacent double FAM).Firstly,the CP modified on the magnetic microbeads was partially complementary to miRNA target,and the target was separated and enriched by the assistance of magnetic field.Then,the magnetic microbeads were resuspended in the AuNP solution.The AP hybridized with the rest sequence of the target,resulting in a complex sanwich structure between magnetic microbeads and AuNP.Unbound AuNP were washed off by magnetic separation.Finally,the magnetic microbeads were dispersed in the DTT solution.Under the function of DTT,HP and SP modified on the surface of AuNP were substituted into the solution.The Exo III cleaved SP and made FAM-labeled nucleotides far away from each other to recover the fluorescence.This method had a detection limit of miRNA-141 as low as 69 pM,suggesting that it could be used to detect target in serum samples.This method has the advantages of easy design,high signal-background ratio and the exemption of quenching moieties.3.Detection of miRNAs in complex samples based on dyes self-quenched Exo III-cycle cutting and DNA bio-barcode technology.In previous two works,the detection in complex samples was realized but its sensitivity need be improved.Thus,Exo III-cycle cutting was introduced to further amplify signal.Four DNA probes were designed,including the CP modified on the magnetic microbeads,the AP modified on the gold nanoparticles(AuNP),the BP modified on the AuNP and hairpin probe SP(labeld with adjacent double FAM).Firstly,the CP immobilzed on magnetic microbeads partially hybridizeded with miRNA target,and enriched the target by the function of magnetic field.Then,the magnetic microbeads were resuspended in the AuNP solution.The AP modified on the surface of AuNP hybridized with the rest segment of the target,resulting in a complex structure between magnetic microbeads and AuNP.Unbound AuNP were washed off by magnetic separation.Finally,under the influence of DTT,BP modified on the surface of AuNP were released.After magnetic separation,BP could hybridize with SP so that the hairpin structure of SP was opened to initiate the Exo III cleavage reaction.The FAM fluorophore originally modified in two adjacent nucleotides was far away,recovering the fluorescence.Rereleased BP would participate in the next round of digestion.Due to the introduction of circular amplification,the detection limit for miRNA-21 was as low as 0.15 pM.This method could also be used to detect target in serum samples.