Low Background Cascade Amplification Electrochemical RNA Sensing Based On Mesoporous Silica

RNA is a kind of ribonucleic acid molecule which could be closely related to various life activities of cells.Especially some kinds RNAs may often reflect the occurrence,development,and metastasis of tumors.RNA has received more and more attention in the field of cancer research as a new type of tumor marker for diagnosis and prognosis of tumors,as well as a potential target of medicament design.Traditional detection methods for RNA tumor biomarkers are often complex in operation,low sensitivity,false-positive results,vulnerable to pollution,radio-labeled,and so on.Electrochemical biosensing techniques have great advantages in the detection of RNA tumor markers because of their high sensitivity,easy miniaturization,low cost,high specificity,and good feasibility in complex samples.In addition,the combination of cascade amplification and mesoporous silica nanoparticles which load electroactive molecules and minimize the background signal are of significance for improving the sensitivity and specificity.Based on the literature and laboratory equipments available for electrochemical research,this dissertation is devoted to the research of the following two works:1.Low Background Cascade Signal Amplification Electrochemical Sensing Platform for mRNA Quantification by Target Activated Hybridization Chain Reaction and Electroactive Cargo ReleaseA low background cascade signal amplification electrochemical sensing platform has been proposed for the ultrasensitive detection of mRNA by coupling the target activated HCR and electroactive cargo release from mesoporous silica nanocontainers(MSNs).In this sensing platform,the 5'-phosphate terminated DNA(5'-PO4 cDNA)complement to mRNA target are hybridized with the trigger DNA and anchor DNA on the surface of MSNs,aiming at forming a double-stranded DNA gate molecules and sealing the methylene blue(MB)in the inner pores of MSNs,In the presence of mRNA,the 5'-PO4 cDNA is displaced from the MSNs and competitively hybridized with mRNA,which led to the liberation of the trigger DNA and the opening of the MSNs pore.The liberated trigger DNA can be then immobilized onto the electrode surface through hybridization with the capture DNA,triggering HCR on the electrode surface.In the meanwhile the MB released from the MSNs will intercalate into the HCR long dsDNA polymers,giving rise to significant electrochemical response.In addition,due to the ?-exonuclease(?-Exo)cleavage reaction assisted target recycling,more amounts of trigger DNA will be liberated and trigger HCR,as well as numerous MB are uncapped and intercalates into the HCR products.Featured with amplification efficiency,label-free capability,and low background signal,this strategy could quantitatively detect target of thymidine kinase 1(TK1)mRNA down to 2.0 aM with a linear calibration range from 0.1 fM to 1 pM.This proposed sensing platform may open up new avenues for highly sensitive quantification of biomarkers and thus should hold great potentials in clinical diagnosis.2.Enzyme-free Low Background Cascade Signal Amplification Electrochemical Sensing Platform for miRNA 21 Detection by Catalytic Hairpin Assembly Driven Electroactive Cargo Release and Hybridization Chain ReactionA enzyme-free low background cascade amplification electrochemical sensing platform has been constructed for the detection of miRNA by coupling catalytic hairpin assembly(CHA)driven hybridization chain reaction(HCR)and electroactive cargo release from mesoporous silica nanocontainers(MSNs).In this sensing system,the hairpin DNA probe H1 are hybridized with anchor DNA,forming hairpin-sharped DNA gate molecules which could seal the methylene blue molecules(MB)in the inner pores of MSNs.In the presence of miRNA,the hairpin DNA probe H1 will hybridize with miRNA and switch its strcture from hairpin to single-stranded linear DNA,which led to the release of MB from the openned MSNs pore.The single-stranded linear DNA H1 would be hybridized with another hairpin probe H2 competitively,resulting in the release of target miRNA.The miRNA target will recongnize DNA gate recurrently and induce CHA process between H1 and H2 probes.Then the released MB will intercalate into dsDNA chains,showing electrochemical signals for the CHA products H1-H2 being immobilized onto the electrode surface through hybridization with the SH-CP probes,Futhermore,the CHA products will trigger the HCR process between H3 and H4,forming long dsDNA polymers and giving rise to significant electrochemical response.It is worth mentioning that this strategy could quantitatively detect the target of model RNA tumor biomarker miRNA 21 down to 36 aM with a linear calibration range from 0.1 fM to 2 pM,benefiting from amplification efficiency,enzyme-free capability,and low background signal.Futhermore,this proposed sensing platform may have a better applicability in complex samples without the limitation of enzyme environment to specific temperature and concentrain of ions,which holds great promise in clinical diagnosis and provide warning for tumor proliferation.