An "Off-on" Fluorescent Switch Assay For MicroRNA Using Nonenzymatic Ligation-rolling Circle Amplification

Objective:MicroRNA?miRNA?,a group of evolutionally conserved,small noncoding RNA involves in the regulation of gene expression,which affect a variety of biological processes including cellular differentiation,immunological response,tumor development,and so on.Recently,miRNA has been identified as a non-invasive biomarker to monitor certain clinical diseases.MiRNA-21 and mi RNA-141 have been found highly elevated in the advanced prostate cancer patients,miRNA-451 and miRNA-486-5p levels in plasma can suggest gastric cancer to some extent,miRNA-124-5p functions as a tumor suppressor and serves as a molecular marker for glioma diagnosis.Therefore,the simple,sensitive and specific detection of cancer-associated mi RNA provides a powerful basis for the early stages of cancer.In this work,we have fabricated a hybridization and adsorption fluorescent switch-on platform using nonenzymatic ligation-rolling circle amplification.This fluorescent sensing system that provided a useful alternative for target miRNA-21 analysis with a detection limit down to0.75 fM.At the same time,this method also provides a promising platform for point-of-care?POC?diagnostics and individualized treatment of cancer.Methods:1.Preparation of a fluorescent switch-on sensing system:A highly sensitive fluorescent biosensor was constructed based on a click chemistry-mediated nonenzymatic ligation-rolling circle amplification?NELRCA?strategy.We utilized agarose gel electrophoresis,atomic force microscopy?AFM?and fluorescence spectrophotometry to characterize the graphene oxide?GO?materials used in this study and verified the feasibility of the sensing system.2.Validation of fluorescent switch-on sensing system and optimization of experimental conditions:this sensing system was characterized by methods of agarose gel electrophoresis and fluorescent analysis;optimize the concentration ratio of Cu²⁺and ascorbic acid,Phi29 DNA polymerase,NELRCA reaction time and incubation temperature,relative concentration ratio of signal probe to GO and other experimental conditions.3.Investigation of the analytical performance of this sensing system and detection of the target:reasonable evaluation of the analytical performance of the proposed method,such as the linear range,the detection limit and specificity,etc.;Using miRNA-21 as a model target,the amplified signal have been measured.Samples were excited at 490 nm,and collected fluorescence from 500 nm to 600 nm.Results:1.Using different methods to characterize and verify the fluorescent switch-on sensing system based on NELRCA strategy.Those results showed that the modified circular template?MCT?can be successfully used for the specific recognition and amplification of target.2.The performance of the fluorescent switch-on sensing system based on NELRCA strategy:the detection of target mi RNA-21 with low abundance and high sensitivity;the detection linear range is 1 fM-1?M with a detection limit as low as 0.75 fM;the analysis time can be controlled within 3 hours;good specificity;this sensing system has great advantages over other miRNA detection methods with respect to the analytical performance.Conclusions:In the present thesis,an off-on fluorescent switch system has been reported with the assistance of NELRCA strategy to achieve the highly sensitive and specific detection of miRNA.We used CuAAC reaction,a nonenzymatic ligation reaction,to develop a MCT with 1,4-connect triazole linkage for identified and amplified agent for the target mi RNA-21.Meanwhile,a variety of techniques were used to characterize as-proposed MCT and NELRCA sensing system.These results showed that the amplified element was successfully prepared.The hybridization and adsorption switch-on sensing system can significantly simplify the target analysis process.More importantly,by rational design of the MCTs and multiple signal probes with different fluorophores,simultaneous monitoring of multiple cancer-associated biomarkers is expectable in the future study.It will also provide a potential platform for the early clinical diagnosis of cancer.