Activatable Fluorescent Probes Based On DNA-templated Silver Nanoclusters For Cell Nucleus Imaging And MicroRNA Detection

Due to its high sensitivity,good selectivity,convenient and fast characteristics,fluorescence imaging and analysis method has been an important research means in biochemical analysis.Fluorescent probe is the core and foundation.Conventional fluorescent probe mainly based on organic dye,which exists some drawbacks,such as poor photo-bleaching resistance and short fluorescence lifetimes,restricting its application range.In recent years,with the development of nanotechnology,a series of novel fluorescent probes based on fluorescent nanomaterials,such as semiconductor quantum dots,upconversion fluorescent nanomaterials,fluorescent metal nanoclusters have emerged.These novel fluorescent probes have not only overcomed the defects of organic dyes,but also have a series of excellent physical and chemical properties,such as adjustable size,controllable fluorescence spectrum and large surface-area,etc.DNA-Ag NCs with good fluorescence characteristicsare,but also have the advantages of good biocompatibility,water solubility,easy preparation,low cost and easy modification,thus arising great concern in biochemical analysis.However,the fluorescent probe based on DNA-Ag NCs also have some questions.On the one hand,the existing DNA-Ag NCs strategies for subcellular and cellular imaging are still rare,especially activatable fluorescent probes need to be urgently exploited.On the other hand,the fluorescence stability and quantum yield of existing DNA-Ag NCs are unable to meet the demand of complex biological samples and trace material.Aiming at the two aspects,choosing cell nucleus imaging and miRNA detection,we have constructed two kinds of novel DNA-Ag NCs activatable fluorescent probes and developed simple and convenient new specific imaging and detecting technology.The detailed description is listed as followed:1.DNA-Ag NCs activatable fluorescent probe for cell imaging based on cell nucleus in situ fluorescence activationA novel DNA-Ag NCs activatable fluorescent probe with in situ fluorescence enhancement effect was constructed to image cell nucleus.The fluorescent probe was based on the DNA-templated Ag NCs with G-rich sequence adjacent fluorescence enhancement phenomenon and the characteristic that the ubiquitous telomere in nuclei of eukaryotic cells containing repeated DNA sequence TTAGGG.Our research showed that the probe could be activated by telomere sequence in buffer,attaining a enhancement ratio of 12.7.Choosing immobilized CCRF-CEM cells as the main research subjects,we found fluorescence activation phenomenon in the cell,and activated efficiency was closely related to the working concentration,incubated time and template sequence of Ag NCs.It was also revealed through laser confocal microscopy that the position of luminescence was mainly in cell nucleus,and the mechanism of luminescence was due to the fluorescence of Ag NCs activated in nucleus.Then selecting live CCRF-CEM cells and three other cell lines Hela,SMMC-7721 and HBL-100,we found the labeling capability of Ag NCs was generic and could characterize target recognition ability of Aptamer AS 1411.Therefore,this DNA-Ag NCs activatable fluorescent probe might hold a great potential to be developed as a simple,convenient,label-free and versatile platform for specific cell nucleus imaging.2.DNA-Ag NCs activatable fluorescent probe for miRNA detection based on competitive hybridization-responsiveA novel DNA-Ag NCs activatable fluorescent probe with competitive hybridization-responsive was constructed to detect miRNA.The probe was based on the DNA-templated fluorescent Ag NCs with high brightness and stability,combined with the quenching molecular BHQ-1 labeled sealed probe and Fluorescence Resonance Energy Transfer(FRET).In the free state,the fluorescence of Ag NCs were quenched by BHQ-1.However,as the target miRNA was introduced,BHQ-1 was pushed out through competitive hybridization,resulting in fluorescence recovery.By selecting miRNA-21 as the model target,a series of factors,including the concentration of Ag NCs,the sequence of sealed probe and radio between two strands were optimized.Result displayed that the selective microRNA detection was successfully achieved with a good linearity between 100 pM and 20 nM and a detection limit of 100 pM.Therefore,the activatable DNA-Ag NCs fluorescent probe might hold a great potential to be developed as a simple,convenient,sensitive and selective platform for miRNA detection.