Construction And Evaluation Of MiRNA Detection Method Based On Isothermal Strand Displacement Polymerase Kaction Coupled With Electrochemical Sensing Technology

BackgroundMicroRNAs(miRNAs), a kind of endogenous, non-coding RNAs (19-24 nucleotides), play vital roles in post-transcriptional regulation. It has been demonstrated that miRNAs have been involved in various biological process, such asimmune defense, gene expression, hematopoiesis, organogenesis, proliferation and apoptosis of cell, fat metabolism. Since miRNAs are highly specific for gene regulation, altered expression patterns of these molecules indicate dysregulation of important biological processes, which is often the cause of disease. In the past decade, aberrant miRNA expression has been demonstrated in several diseases, including several types of cancer, cardiovascular and rheumatic diseases, as well as neurological disorders.Since recent research demonstrates that unique miRNA expression patterns was a very important role in the development of the disease, indicating that miRNAs have been served as novel diagnostic and prognostic biomarkers. Therefore, there is a crucial need to design reliable methods to quantify miRNAs with high specificity and sensitivity.Currently, several methods have been developed for quantitative measurement of miRNA expression, such as northern blotting、gene chip、in situ hybridization and real-time quantitative PCR. Nevertheless, these methods are time-consuming with high charge due to the reverse transcription. In addition, the temperature cycling in RT-PCR suggests that it need precise control what imposes instrumentation constraints on its a much wider variety of applications. If developed the suitable methods of miRNA detection for clinical laboratories, the miRNA assays should fulfill the following requirements:i. sensitive enough to provide quantitative measurements even with small amounts of targets; ii. specific enough for reproductive quantification of miRNAs to distinguish homologous sequences; iii. easy to perform and no need for expensive equipments or reagents.In recent years, isothermal strand-displacement polymerase reaction based on nuclease have also been reported to improve the application of various miRNA biosensors broken away from the dependence on sophisticated equipmenin actual operation and equipment requirements. This technique uses lengthening of a new strand to replace the target sequence, and thus releases the target to initiate a new polymerization reaction. Because isothermal strand-displacement polymerase reaction does not require a specific recognition site, it can be conveniently used for designing miRNA biosensors. However, at present isothermal amplification reaction was more likely to use optical detection instrument which requires an excitation light source, may cause interference of background light and fluorescence quenching in the external environment. Furthermore, lower sensitivity of optical detection method is not suitable for clinical widely used inparticularly primary hospital. How to solve these problems is becoming a key issue in the field of isothermal amplification.Electrochemical enzyme biosensors are emerging as excellent analytical tools for efficient, sensitive, specific, small, and inexpensive genetic detection. The sensitivity of an enzyme sensor stems from its extraordinary biocatalysis power, which in turn provides a great amplification of signals. Compared with optical detection methods, electrochemical sensor technology has many advantages such as easy miniaturization, good selectivity, low cost, low sample consumption, easy automation which has become a hot topic about nucleic acid detection. Nevertheless, sensitivity of the traditional electrochemical sensor is limited without involving amplicons. How to solve this problem is becoming a key issue in the field of electrochemical enzyme biosensors.From the above, the electrochemical sensing technology can effectively make up for easy fluorescence quenching, high background and large instrument requirement of isothermal amplification optical detection, and ISDPR can carry on the process of cycle-after-cycle of the hybridization, polymerization reaction and displacement to constantly produce new DNA sequences increase the sensitivity of electrochemical sensor. Herein, we designed an electrochemical sensor method for amplified detection of miRNA based on electrochemical sensing technology and isothermal strand-displacement property of polymerase reaction (ISDPR). The molecular beacon (MB) acted as a template of polymerization reaction, while the target miRNA acted as a trigger of polymerization reaction. The miRNA detection system was activated by the conformational change of the MB, which was induced by the hybridization between the MB and the target miRNA. Then, the primer annealed with the exposed 3’terminus of the opened probe and triggered a polymerase replication in the presence of dNTPs (dATP、dTTP、dGTP、bio-dCTP) and phi29 DNA polymerase. At the same time, the target miRNA was displaced by the phi29 DNA polymerase with strand-displacement activity during primer extension after which a complementary DNA (cDNA) was synthesized, forming a probe-cDNA complex. And the complex products were labeled with biotin molecules by the mixture of biotinylated dCTP in the reaction. Therefore, the hybridization, polymerization reaction and displacement occurred cycle-after-cycle, constantly producing DNA sequence. Finally, to renew the cycle, the displaced target hybridized with another probe, which triggered yet another polymerization reaction, resulting in the multiplication of the biotin-modified DNA on the surface of MMCs. And then, Sa-ALPs were linked to the surface of the MMCs through biotin-streptavidin conjugation after incubation with composite nanoparticles. Since ascorbic acid was the product of the enzymatic hydrolysis reaction, the miRNA could be detected by measuring the oxidation current of ascorbic acid (AA) on a SPCE. By monitoring the increase of current intensities, the target can be detected with high sensitivity.ObjectiveTo establish a fast, efficient, specific and easy method of miRNA expression detection based on isothermal strand-displacement property of polymerase reaction and electrochemical sensing technology and to complete a certain amount of microRNA samples test and evaluate the clinical application. The finalobjectives of this experiment is to provide a new electrochemical assay for the detection of miRNA.Methods1. Verification of combination of isothermal strand-displacement property of polymerase reaction and electrochemical detection1.1 Beacon designer software was used to design specific probes based on miRbase published sequences. The above results were confirmed with 3% agarose gel analysis.1.2 We built the fluorescence isothermal amplification reaction system. A model system in a solution was created by hybridizing the 5’-FAM,3’-Dabcyl labeled MB probe and miR-21 produced during the recycling reaction. The above results were confirmed with multiscan spectrum.1.3 The morphology of the composite nanoparticles was characterized by confocal microscope to verify the probes successfully connected to the surface of MMCs through ammonia carboxy complexation reaction.1.4 Cyclic voltammetry was used for investigating the interface features on conductive surfaces.1.5 Biotinylated dCTP was mix in the isothermal amplication reaction. To verify the effects of the amplification products by different proportions of biotin-dCTP. Agarose gel electrophoresis were carried out for separation, identification the isothermal amplication products and select the ideal ratio with biotin-dCTP.2. Construction and preliminary evaluation of combination of isothermal strand-displacement property of polymerase reaction/enzymatic electrochemical sensor system2.1 The optimum reaction conditions were optimized by comparing the changes in the electrochemical signal under different probe concentration, amount of enzyme, dilution ratio, incubating time etc.2.2 Under these reaction conditions, we can calculate a series of hybrid kinetic constants and get the mathematical model by the sensor equivalent circuit analysis through the real-time monitoring of the liquid phase miRNA and depthly analysis the response mechanisms of biological molecules at the sensor’s liquid phase.2.3 To verify the specificity of the gene sensor.2.4 It is necessary to make an evaluation for the clinical performance of the constructed system on the basis of relevant documents, and use the most commonly used method (real time PCR) as the reference method to detect to get the evaluation of its clinical application.Results1. The products amplified from target miRNA demonstrated corresponding bands in the gel, whereas those from the various types of mismatches did not show any bands, indicating the selective amplification with high specificity. It were confirmed that only phi29 DNA polymerase can start the circular strand-displacement reaction bymeasurement of multiscan spectrum. The morphology of the composite nanoparticles was characterized by confocal microscope to verify the probes successfully connected to the surface of MMCs by ammonia carboxy complexation reaction. Cyclic voltammetry is used for investigating the interface features on conductive surfaces-these results were consistent with confocal microscope images, indicating the successful conjugation of oligonucleotide and proteins on the MMCs. Finally, the biotin incorporation methods was successfully verified that it can’t affect processessmoothly in spite of the reaction rate through adding different proportions of bio-dCTP.2. Constructed on section one, when the concentration of miR-21 between 10-14mol/L to 10-8mol/L, there was a linear relationship between the oxidation peak current and the concentration of the target sequence. The limit of detection was 9fmol/L. The proposed method for miRNA assay suggest good selectivity even though there is only a single base mismatch. Our measurement results of miR-21 are consistent with PCR, further confirming the accuracy of our method.ConclusionWe have developed a highly sensitive and selective miRNA detection platform based on functionalized multienzyme magnetic microcarrier assisted isothermal strand-displacement polymerase reaction. As we know, miRNAs could be amplified by ISDPR using molecular beacons with high specificity. For another, the incorporation of enzymes (alkaline phosphatases) to generate the output electrochemical signals provided the capability for signal amplification, which will further increase the sensitivity. Under optimized conditions, the peak currents showed a good relationship with the logarithm of target oligonucleotide concentrations, with a detection limit of 9fM owning to the dual amplification of the assay. This method doesn’t need reverse transcription and large equipment and have a shorter time to suit widely application in primary hospital. And the development of electrochemical detection has some theoretical significance and potential practical value at low levels of miRNA chip. Our measurement results of clinical specimens are consistent with PCR, further confirming the accuracy of our method and provide a sensitive, rapid, economic and convenient miRNA diagnostics common platform.