| Objective:MicroRNA (miRNA)is a type of endogenous non-coding RNA molecule with length of 18-25 nucleotides that plays an important role in many biological functions.A growing number of studies have proved that the disorder of miRNA is associated with many diseases,particularly cancer.The miRNA-141,for example,abnormal expression in prostate cancer,while the miRNA-21 common excessive expression in breast,pancreatic,and liver cancers.Therefore,accurate detection of miRNA has a significant meaning for clinical diagnosis and the study of pathogenesis.However,sensitive and selective detection of miRNA still faces great challenges due to low miRNA abundance and sequence homology among family members.Therefore,it is necessary to explore a convenient,sensitive and specific miRNA analysis platform.In this work,we design a fluorescent microRNA biosensor based on exponential amplification reaction(EXPAR)and nicking endonuclease-powered three-dimensional(3-D)bipedal DNA walkers(BDW).At the same time,our method has enormous potential for the application of BDW-related biosensors.Methods:1.Preparation of integrated fluorescence biosensor: a new fluorescence biosensor was constructed based on exponential amplification reaction-triggered three-dimensional bipedal DNA walkers for highly sensitive detection of microRNA.The non-denaturing polyacrylamide gel electrophoresis(PAGE),Real-Time quantitative PCR detection system,fluorescence spectrophotometer and other methods were used to verify the EXPAR and enzymatic cleavage reaction of BDW.2.Validation the feasibility of 3-D DNA walker integrated with EXPAR strategy and optimization of experimental conditions: we use fluorescence microscope and fluorescence spectrophotometer to characterize the integrated sensing system;the length of DNA template strand,the reaction time of EXPAR,the Nb.Bbv CI mediated DNA walker reaction time,the concentration of KF polymerase and the concentration of Nb.Bbv CI were optimized.3.Analytical performance evaluation and real-sample analysis:evaluation of the analytical performance of the detection system,like linear range,the detection limit,specificity and reproducibility;to analyze its detection ability in complex biological samples.Results:1.A variety of experimental methods were used to verify the constructed fluorescence biosensor based on EXPAR-triggered 3-D bipedal DNA walker strategy.The results show that the reaction system can realize the specific recognition and amplification of target molecules.2.Analytical performance and real-sample analysis of the walker sensor based on EXPAR: specific recognition and sensitive detection of the target miRNA-21;the linear range is 10 f M-5 n M with a detection limit down to 5.2 f M;the reaction time of the assay takes about 70 min;target miRNA-21 can be distinguished from other homologous miRNAs;reproducibility for five independent experiments is satisfactory;good detection capability is maintained in 10-fold diluted human serum samples;this method has great advantages over the existing fluorescence nucleic acid detection methods.Conclusions:In this work,we design a fluorescent microRNA biosensor based on EXPAR and nicking endonuclease-powered 3-D bipedal DNA walkers.Target microRNA initiates EXPAR with the help of polymerase and nicking endonuclease to generate the large number of BDW in solution.The newly generated BDW can be continuously assembled onto polystyrene microsphere track co-modified with fluorescence-labelled DNA strand.Thus,in the presence of nicking endonuclease,the walking machine is activated to produce enhanced fluorescent signal in the supernatant.The combination of enzyme-assisted EXPAR in solution and enzyme-powered BDW on particle significantly increases the signal amplification efficiency and improves the detection sensitivity.Therefore,our method has enormous potential for the application of BDW-related biosensors. |
