Enzyme-free Isothermal Signal Amplification Nanoprobe For MiRNA Imaging Analysis

MicroRNA(miRNA)are short non-coding RNA which play an important role in post-transcriptional expression.As an important cancer marker,the abnormal expression of miRNA is often closely related to the occurrence and development of tumors.Therefore,it is important to detect miRNA sensitively and accurately for early diagnosis and treatment.Enzyme-free isothermal amplification technology is favo red by researchers because of the mild conditions,high efficiency and strong applicability.With the rapid development of nanotechnology and biomedicine,The theranostic nanoplatform(the integration of diagnosis and treatment in the same system)shows great potential in clinical diagnosis.The detection of cancer markers can provide positioning and guidance for subsequent precise treatment.Up to now,many integrated systems have been developed by researchers.In this paper,with the help of enzyme-free isothermal amplification technology,we constructed two diagnostic platforms for simultaneous detection multiple cancer-related miRNAs or miRNA imaging analysis to guide cancer treatment.The specific research work is as follows:(1)DNAzyme based Chemiluminescence Imaging(CL imaging)Sensor Array for the Simultaneous Measurement of Multiple Cancer-Associated miRNAs.The CL imaging sensor array with four different sensing units was constructed by modifying four different hairpins DNA(H1)onto detection wells.When the target miRNA is present,the corresponding hairpin H1 can be opened to undergo catalytic hairpin self-assembly(CHA)and hybridization chain reaction(HCR)reactions,and the complete DNAzyme structure of G-quadruplex DNAzyme can be restored,which can catalyze H₂O₂ oxidation of Luminol to generate luminescent signals.Combined the cascade amplification of CHA and HCR with outstanding catalytic activity of G-quadruplex DNAzyme,a novel visualized CL imaging assay platf orm was developed for simultaneous detection of four miRNAs.Possessing high specificity and generality,the sensor is easy easy operation and can be detected successfully by using imaging camera system or mobile phone camera,which meets with the detectio n requirements of POCT.The platform has an important role in simultaneous detection of miRNA and cancer screening due to its excellent detection performance for complex components,multiple detection capabilities,and simple and readable characteristics.(2)On the basis of the detection of miRNA in the previous project,The theranostic nanoplatform was developed.A tumor microenvironment responsive nanoplatform(MOF@hp-DNAzyme)was constructed for intracellular miRNA imaging analysis and synergetic therapy.First,The glutathione(GSH)-responsive metal-organic framework(MOF)was synthesized,severed as carrier of hairpin(hp1,hp2)to prepare MOF@hp-DNAzyme nanoprobe.There were partial fragments of deoxyribozyme(DNAzyme)at the ends of hp1,which was used to silence Survivin mRNA.After entering tumor microenvironment(TME),the high concentration of GSH can react with MOF@hp-DNAzyme nanoprobe,disintegrating the structure,releasing hp1,hp2 and abundant Mn²⁺.With the help of HCR isothermal amplification technique,the platform can realize ultrasensitive and accurate miRNA-21 imaging in living cells,guiding subsequent photodynamic therapy(PDT).Notably,due to the occurrence of HCR response,the intact structure of DNAzyme was restored,activating gene silencing.Moreover,Mn²⁺released by MOF can well alleviate the deficiency of intracellular cofactors to enhance the efficiency of Gene therapy.More importantly,the reaction between MOF and GSH not only consumes GSH in the cancer cells,but also enhances the fluorescence of MOF,and further enhances the therapeutic effect of PDT.This miRNA-21 and GSH-responsive MOF@hp-DNAzyme nanoplatform,can guide photodynamic therapy and gene silencing therapy,which provides a new approach for precise treatment of cancer and has potential biomedical application prospect.