Construction And Application Of MicroRNA Upconversion Sensing Nanoplatforms Based On The Signal Amplification Strategy

MicroRNAs(miRNAs)are a class of endogenous non-coding RNAs(ncRNA)with the length of approximately 22 nt.The abnormal expression of miRNAs in humans is closely related to the occurrence and development of tumor diseases.Therefore,miRNAs are expected to become tumor diagnostic markers.However,two problems still present.First,the content of miRNAs in body fluids and cells is very low,which is difficult to monitor by the conventional methods;second,the relationship between miRNAs and tumor diseases is complicated,resulting in being difficult to accurately diagnose.These problems limit the practical applications of miRNAs as tumor markers in clinical diagnosis.To solve these problems,the first step is to develop high sensitive miRNA biosensors.However,the traditional biosensors always focus on detecting miRNAs in vitro,and the developed biosensors always use fluorescent dyes or quantum dots as fluorophores,which limit their application in living cells due to the drawbacks of fluorescent dyes or quantum dots.Therefore,it is very important to find new fluorescent donors and develop new sensitive miRNA biosensors for further studying miRNAs as tumor diagnostic markers.In this dissertation,lanthanide-doped upconversion nanomaterials(UCNPs)are used as fluorophores.The aim of this study is to construct new miRNA biosensors based on UCNPs coulping with the target cycling amplification technology.Our works focus on the assembly of the upconversion nanoprobe,the design of the DNA structures with signal amplification capacity,and imaging miRNA in living cells.The main research works are as following:1.A Universal Upconversion Sensing Platform for the Sensitive Detection of Tumour-Related ncRNA through an Exo ?-Assisted Cycling Amplification Strategy:A univisal ncRNA upconversion sensing nanoplatform was developed.Gold nanoparticles(AuNPs)and UCNPs(NaYF4:Yb,Er@NaYF4)were assemblized using hairpin DNA(HP).Owing to luminescence resonance energy transfer(LRET),upconversion luminescence(UCL)of UCNPs is quenched by AuNPs,and signal DNA(SDNA)can open HP making AuNPs to move away from UCNPs resulting in UCL recovery.To achieve univisality and sensitivity of the nanoprobe,exonuclease ?(Exo ?)is introduced into the sensing system,a hairpin DNA(mHp)containing ncRNA recognition sequence and SDNA sequence was designed to recognize ncRNA and trigger Exo ? gradually degraded mHp,releasing ncRNA and SDNA.The released ncRNA could be reused to release more SDNA,improving sensing sensitivity,and a variety of ncRNAs could be detected by changing the recognition portion of mHp.Using HOTAIR segment and miRNA-21 as a model to verify the method,results showed that the designed biosensor could high sensitively detect two kinds of ncRNAs and successfully measured the expression level of miRNA-21 in human breast cancer cells(MCF-7).2.Lighting up MicroRNA in Living Cells by the Disassembly of Lock-like DNA-Programmed UCNPs-AuNPs through the Target Cycling Amplification Strategy:A sensitive miRNA biosensor was developed and used for detecting miRNA in vitro and imaging miRNA in living cells.A lock-like DNA(LLD)with the target cycle amplification function was formed by hybridization of a hairpin DNA(H1)to a DNA sequence(bDNA).UCNPs(NaYF4@NaYF4:Yb,Er@NaYF4)and AuNPs were assemblized using LLD.Owing to the LRET,upconversion emission of UCNPs was quenched by AuNPs.With the aid of fuel hairpin DNA(H2),a single miRNA repeatedly opened the LLD,so that the AuNPs were far away from the UCNPs to trigger the upconversion emission recovery,resulting in a high detection signal gain.The proposed biosensor had low detection limit and can image miRNA-21 in different tumour cells,and could directly and accurately distinguish tumour cells from normal cells.3.Enhancing Intracellular MicroRNAs Imaging:A New Strategy by Combining the Double-Channel Exciting Single Colour Fluorescence with the Target Cycling Amplification Reaction:A miRNA nanoprobe was developed based on the synergistic effect of the double-channel excitating single colour fluorescence and the target cycle amplification strategy,which could enhance signal output for intracellucar miRNA imaging.FAM and-SH modified LLD(FAM-LLD-SH)were loaded to polydopamine-coated UCNPs(NaYF4@NaYF4:Yb,Er@NaYF4)(PDA-UCNPs)surfaces,AuNPs were assemblized on PDA surfaces by the Au-S bond.Due to the resonance energy transfer,AuNPs and PDA could quench the green fluorescence emission of UCNPs and FAM.With the aid of fuel hairpin DNA(H2),one copy of miRNA as a catalytic molecule could repeatedly open multiple LLDs,making AuNPs and FAM to move away from the PDA shell,lead to the emission recovery of UCNPs and FAM.Together with the 980 nm laser and the 488 nm excitation light,the nanoprobe could improve the signal output for intracellular miRNAs imaging.The method was applied to the imaging of miRNA-21 in HeLa cells with satisfactory results,and could directly and accurately distinguish tumour cells from normal cells.4.A High Plasmonic Enhancement of the Upconversion Luminescence Emission of Upconversion Nanocrystals by Gold Nanorods:AuNRs with different SiO2 shell thicknesses and different structures UCNPs were synthesized.The satellite nanostructures of AuNRs@SiO2-UCNPs were synthesized by electrostatic interaction.The UCL of UCNPs enhanced by AuNRs@SiO2 was studied.The results showed that the enhancement effect was the biggest about 50-fold,when the thickness of SiO2 shell was about 24 nm and UCNPs were NaYF4@NaYF4:Yb,Er@NaYF4(2.5 nm of outer shell).This research result would have potential application in the biosensing and bioimaging.