Enzyme-Free Fluorescent Assay For The Detection Of MicroRNA Based On Catalytic Hairpin Assembly

Background and objectiveMicroRNAs(MiRNAs)are a group of endogenous,non-coding and single-stranded RNA molecules,which are existed widely in eukaryotic cells.It has been regarded as a new type of potential biomarkers,which is useful for early diagnosis and prognosis of diseases.However,because of the restriction factors about mature miRNA such as short sequence,low abundance and highly homologous,detection technology for miRNA still remains challenges.Currently,conventional strategies for detection of miRNA including Northern blotting,microarray and real-time PCR have been reported.However,these strategies cannot meet the current demands due to the limitations like poor sensitivity,unsatisfactory specificity and complicated operation.In recent years,enzyme-assisted signal amplification methods have been reported to quantify miRNAs,which have improved the sensitivity of miRNA detection compared to the conventional strategies.But enzymes are usually costly and susceptible to environmental interference,which limit their application.Compared to that,enzyme-free amplification method based on DNA nanotechnology has attracted researchers’ attention and become research focus,which provide new ideas for miRNA detection.Here,a novel and simple quencher-free fluorescent biosensor based on target-triggered recycling catalytic hairpin assembly(CHA)was developed for rapid and sensitive detection of target miRNA.In this study,2-aminopurine(2-AP)fluorophore was used as the signal reporter.We evaluated the detection performance analysis of our proposed strategy and provided a new idea and research foundation for sensitive miRNA detection in clinical samples.Methods1.Construction of catalytic hairpin assembly reaction system and optimization of experimental conditions1.1 All kinds of nucleotide sequences and hairpin probes were designed and synthesized,and the physical parameters of the hairpin structure were calculated and assessed.1.2 The analyses of were performed through nondenaturating polyacrylamide gel electrophoresis(PAGE)analysis to analyze the reaction producct of CHA reaction,and thus verifying the feasibility of the proposed strategy for miRNA assay,1.3 Experimental conditions were optimized including the concentration ratio of P1 to P2,the concentration of P2,the reaction temperature and the incubating time by comparing the fluorescence intensity with each other.2.Analytical performance of enzyme-free and quencher-free fluorescent assay for miRNA detection and real sample analysis2.1 To evaluate the sensitivity of this proposed biosensor,assays were applied to detect miRNA-21 at various concentrations under the optimal conditions.After that,we calculate the detection limit and the linear range of the proposed method.2.2 To investigate the specificity of our proposed strategy,we measured five different kinds of miRNA sequences,including miRNA-21(target miRNA),M1(one-base mismatched),M2(two-base mismatched),M3(three-base mismatched),and miR-122 as a random sequence under the same condition.2.3 To demonstrate the potential capability of the proposed strategy in real sample analysis,recovery test was carried out in clinical human serum samples.The serum samples were spiked with miRNA-21 at various concentration levels and analyzed with our proposed method.Then we calculate the recovery rate and the RSD value.Result1.The result of the PAGE analysis shows that miRNA-21 could trigger the CHA reaction and produce a lot of stable P1-P2 deplex,which indicated that the CHA reaction system was established successfully and the cycle of CHA reaction was conducted.2.In order to achieve the optimal sensing performance,the key experimental conditions were optimized.The results indicate that the optimal ratio of P1 to P2 was 1.5:1.The optimized concentration of P2 was 500 nM.37℃ was chosen as the optimal temperature in subsequent research.30 min was selected as the most effective incubating time for all subsequent assays.3.Under the optimal conditions,the sensitivity of our proposed method has been evaluated.A limit of detection as low as 1 nM and a wide linear range from 1 to 800 nM are achieved by this assay.The proposed strategy shows high specificity in discriminating single-base mismatched miRNAs.The recovery test for clinical serum samples shows that the recovery rate was in the range of 98.48 to 103.16%.The satisfactory recovery rates demonstrate that our system could be applicable for miRNA analysis in real biological fluids.ConclusionIn summary,a novel quencher-free and enzyme-free fluorescent sensing strategy for rapid detection of miRNA with straightforward operations was established and evaluated using 2-AP probe based on CHA reaction with high efficiency and high specificity.A limit of detection as low as 1 nM was achieved by this method.The proposed biosensor shows high specificity in discriminating single-base mismatched miRNAs.At the same time,the method also shows a good performance in real sample analysis and has potential application ability in quantitative analysis of clinical serum samples.