Fluorometric Determination Of Microrna By Using An Entropy-driven Three-dimensional DNA Walking Machine Based On A Catalytic Hairpin Assembly Reaction On Polystyrene Microspheres

ObjectiveBlood-based circular microRNAs(miRNAs)are a kind of small,en-dogenous non-coding RNAs which typically has a length of 19-24 bases.These short sequences mediate post-transcriptional gene regulation by base pairing to the 3'untranslated region of messenger RNAs.Increasing re-search evidences have indicated that miRNAs are involved in crucial bio-logical processes,including development,differentiation,apoptosis and proliferation,through imperfect pairing with target messenger RNAs(mRNAs)of protein-coding genes and the transcrtional or post-transcriptional regulation of their expression.Dysregulation of the ex-pression levels of miRNAs are involved in the initiation and progression of human cancer.Therefore,it served as promising and minimally invasive cancer biomarker candidates for potential diagnostic.Regrettably,there remains a challenge to sense miRNAs due to its connate characteristics,involving the low expression level,and high homology.Consequently,in order to realize the highly specific and sensitive detection of microRNAs to provide important evidence for the early diagnosis of cancer,a new3D-DNA walking machine was constructed.MethodsAn entropy-driven 3D-DNA walking machine is presented which in-volves catalytic hairpin assembly(CHA)for detection of microRNA.A 3D-DNA walking machine was designed that uses streptavidin-coated polysty-rene microspheres as track carriers to obtain reproducibility.The method was applied to nicroRNA 21 as a model analyte.Continuous walking on the DNA tracks is achieved via entropy increase.This results in a disassembly of ternary DNA substrates on polystyrene microspheres and leads to cy-cling of nicroRNA 21.The release of massive auxiliary strands from ter-nary DNA substrates catalyze the CHA.This is accompanied by increase in fluorescence,best measured at excitation/emission wavelengths of 480/520nm.Results1 Characterization of polystyrene microspheres:The scanning electron microscope(SEM)was used to characterize the streptavidin-coated poly-styrene microspheres.It can be observed that the polystyrene microspheres are highly uniform and well-dispersed.The average size is about 800 nm.The high quality of polystyrene microspheres insure the good performance of our 3D-DNA walking machine.2 The feasibility of this biosensor:Agarose gel electrophoresis and fluorescence spectrophotometer verified that the entropy-driven 3D-DNA walking machine coupled catalytic hairpin self-assembly reaction was fea-sible to detect microRNA.And the detectable fluorescence value in the presence of the target was significantly higher than that the signal of back-ground.3 Analytical performance of the method:With the optimal experimental conditions,in the range of 50 pM-20 nM,the fluorescence signal was line-arly correlated with the logarithm of the target concentration(R~2=0.997),and the minimum detection limit was calculated to be 41 pM(S/N=3).On ac-count of entropy-driven reaction,the assay is remarkably selective.It can differentiate nicroRNA 21 from homologous microRNAs in giving a signal that is less than 5%of the signal for nicroRNA 21 except for mi-croRNA-200b.4 Standard recovery results:In the 10-fold diluted serum samples,the recovery rates of the three samples with different concentrations(200 pM,1 nM and 5 nM)were 99.5%,102.0%and 104.8%,respectively.ConclusionThe proposed biosensor was successfully used for nicroRNA 21 de-tection at picomolar level,which manifested several attractive features.Firstly,it is an enzyme-free system without rigorous reaction conditions.Furthermore,we use streptavidin-coated polystyrene microspheres as track carrier with promising reproducibility.Finally,we directly design the target as random DNA walker,which is different from the design that makes the walker fixed on the track.This either does not need to optimize the ratio of the walker and the substrate nucleic acid on track to make the structure of3D-DNA walking machine relatively simple.In summary,we suggest that this integrated enzyme-free 3D-DNA walking machine coupled CHA reac-tion has potential application prospect in real-time detection and diagnosis.