Micro RNA detection method based on one-step triggered branched DNA nanostructure
阅读说明：本技术 一种基于一步触发的分支dna纳米结构的微小rna检测方法 (Micro RNA detection method based on one-step triggered branched DNA nanostructure ) 是由 云雯 熊政委 哈霞 吴虹 李宁 于 2020-04-17 设计创作，主要内容包括：本发明基于目标微小RNA诱导的分支DNA纳米结构,开发了一种简单且超灵敏的微小RNA检测方法。双链体两侧的短DNA单链尾巴可以用发夹DNA4和发夹DNA5启动第二级HCR扩增,以在形成的长双螺旋DNA的两侧产生许多长双链体由此形成的支链DNA纳米结构可以携带大量的甲基蓝,可以有效增强电化学信号并实现低检测限。重要的是,该策略提供了简单的操作,良好的可靠性和超高灵敏度。它已成功应用于人体血清样品,显示出令人满意的结果和有希望的未来。重要的是,一站式方法可以极大地减少操作步骤并避免由于多个操作步骤而导致的系统错误。(The invention develops a simple and ultrasensitive micro RNA detection method based on a target micro RNA induced branched DNA nano structure. Short DNA single-stranded tails on both sides of the duplex can initiate second-stage HCR amplification with hairpin DNA4 and hairpin DNA5 to generate many long duplexes on both sides of the long double-helical DNA formed so that the branched DNA nanostructure formed can carry a large amount of methyl blue, can effectively enhance electrochemical signals and achieve low detection limits. Importantly, this strategy provides simple operation, good reliability and ultra-high sensitivity. It has been successfully applied to human serum samples, showing satisfactory results and promising future. Importantly, the one-stop approach can greatly reduce the number of operational steps and avoid system errors due to multiple operational steps.)
1. A micro RNA detection method based on a one-step triggered branched DNA nano structure comprises the following steps:
(1) preparing a hairpin DNA1-5 structure;
(2) preparing a gold electrode;
(3) fixing hairpin DNA1 on the surface of a gold electrode;
(4) adding a proper amount of the hairpin DNA2-5 structure prepared in the step (1) into a solution containing the target micro RNA to form a solution to be detected;
(5) immersing the gold electrode prepared in the step (3) into the solution to be tested obtained in the step (4) for incubation;
(6) immersing the electrode obtained in the step (5) into a methyl blue solution;
scanning the electrode obtained in the step (6) by using a square wave voltammetry method, and using the obtained current signal for calculating the concentration of the micro RNA; the calculation method comprises the following steps: calculating the concentration of the micro RNA in the solution to be detected by using a standard curve of which the current signal obtained by a square wave voltammetry method increases along with the increase of the concentration of the micro RNA,
wherein, the sequence of the micro RNA: AGCUGGUAAAAUGGAACCAAAU, hairpin DNA1 sequence:
GGTTTGGAACCAAATGCCTTAGCGATTTGGTTCCATTTTACCAGCTGCGGTGAAGCAGCGTG, hairpin DNA2 sequence:
TTAACCGGCATTTGGTTCCAAACCCAAAGTGGTTTGGAACCAAATGCCTCCTCTAATACACTCACTAT, hairpin DNA3 sequence:
GGTTTGGAACCAAATGCCTTAGCGGGCATTTGGTTCCAAACCACTTTGTCCTCTAATACACTCACTAT, hairpin DNA4 sequence:
AATACACTCACTATGAATGAATAGTGAGTGTATTAGAGGA, hairpin DNA5 sequence:
TCATTCATAGTGAGTGTATTTCCTCTAATACACTCACTAT。
2. the method of claim 1, wherein the hairpin DNA1-5 structure of step (1) is formed by annealing the sequence at 90 ℃ for 5 minutes and cooling to room temperature.
3. The method according to any of the preceding claims, wherein in step (2) the gold electrode is polished with 0.05 μm alumina powder and sonicated in water.
4. The method according to any one of the preceding claims, wherein in step (3), 10 μ M hairpin DNA1 in 10mM PBS containing 1M NaCl is dripped onto the gold electrode surface overnight, after which 1mM MCH is added and incubated for 2h to prevent non-specific adsorption.
5. The method according to any one of the preceding claims, wherein the solution to be tested in step (4) is: the hairpin DNA2-5 at 500nM and the microRNA of interest are dissolved in 50mM PBS, pH7.2, containing 0.5M NaCl.
6. The method according to any one of the preceding claims, wherein in step (5) the incubation time is 4 hours.
7. The method according to any one of the preceding claims, wherein in step (6), the electrode obtained in step (5) is immersed in 20 μ MMB and stirred for 10 minutes, after which the electrode is washed with PBS to remove non-specifically bound MB.
8. The method of any one of the preceding claims, wherein Square Wave Voltammetry (SWV) scans the electrode from-0.40 to 0.00V with an amplitude of 25mV in 10mM PBS solution (pH 7.2).
Technical Field
The invention relates to the field of micro RNA detection, in particular to the field of a micro RNA detection method based on a branched DNA nano structure triggered by one step.
Background
microRNAs are small non-coding RNAs, approximately 19-23nt in length. It has become a posttranscriptional regulator and plays an important role in every fundamental biological process (e.g., proliferation, differentiation and apoptosis). It has been reported that many human micrornas are expressed throughout the genome in the etiology of diseases, including, for example, cancer, cardiovascular disease and alzheimer's disease. The research on the expression of the microRNA has important value for the diagnosis and prognosis process of early cancer. Therefore, it is highly desirable to develop a method for microrna detection, especially for complex clinical samples and single cells. Some prior art has shown potential for use in clinical diagnosis of micrornas, including Northern blot techniques and microarrays. However, they have low sensitivity in clinical applications.
PCR is considered an efficient amplification strategy for nucleic acid detection. However, the short length of micrornas requires short primer designs for typical PCR techniques, thereby reducing PCR efficiency and increasing the chance of non-specific amplification. In contrast to PCR, the hybrid strand reaction (HCR) is a powerful nucleic acid quantitation method, requiring no precise temperature control cycles and expensive enzymes. Importantly, the DNA duplex formed by HCR is highly ordered. The density of signal molecules can be precisely controlled on these helices, providing higher amplification efficiency. Thus, it has been used for the detection of DNA, metal ions, small biological molecules and proteins by coupling with electrochemistry, colorimetry, fluorescence, electrochemiluminescence, enhanced Raman scattering or surfaces. At present, there is no precedent for using hybrid strand reaction (HCR) for the detection of microRNAs.
Disclosure of Invention
In order to solve the problems, the invention provides a micro RNA detection method based on a branched DNA nano structure triggered by one step.
The invention comprises the following steps:
a micro RNA detection method based on a one-step triggered branched DNA nano structure comprises the following steps:
(1) preparing a hairpin DNA1-5 structure;
(2) preparing a gold electrode;
(3) fixing hairpin DNA1 on the surface of a gold electrode;
(4) adding a proper amount of the hairpin DNA2-5 structure prepared in the step (1) into a solution containing the target micro RNA to form a solution to be detected;
(5) immersing the gold electrode prepared in the step (3) into the solution to be tested obtained in the step (4) for incubation;
(6) immersing the electrode obtained in the step (5) into a methyl blue solution;
scanning the electrodes obtained in the steps (2), (3), (5) and (6) by using a square wave voltammetry method and measuring electrochemical impedance spectrum signals of the electrodes, wherein the electrodes obtained in the step (6) are scanned by using the square wave voltammetry method, and the obtained current signals are used for calculating the concentration of the micro RNA; the calculation method comprises the following steps: calculating the concentration of the micro RNA in the solution to be detected by using a standard curve of which the current signal obtained by a square wave voltammetry method increases along with the increase of the concentration of the micro RNA,
wherein the sequence of the micro RNA and the sequence of the hairpin DNA1-5 are shown in the following table:
preferably, in step (1), the hairpin DNA1-5 structure is formed by annealing the sequence at 90 ℃ for 5 minutes and cooling to room temperature.
Preferably, in step (2), the gold electrode is polished with 0.05 μm alumina powder and sonicated in water.
Preferably, in step (3), 10. mu.M hairpin DNA1 in 10mM PBS containing 1M NaCl was dropped onto the gold electrode surface overnight, and then 1mM MCH was added and incubated for 2h to prevent nonspecific adsorption.
Preferably, the solution to be tested in step (4) is: hairpin DNA2-5 at 500nM and the microRNA of interest are dissolved in 50mM PBS, pH7.2, containing 0.5M NaCl.
Preferably, in step (5), the incubation time is 4 hours.
Preferably, in step (6), the electrode obtained in step (5) is immersed in 20 μ MMB and stirred for 10 minutes, after which the electrode is washed with PBS to remove non-specifically bound MB.
Preferably, Square Wave Voltammetry (SWV) scans the electrode from-0.40 to 0.00V in a 10mM PBS solution (pH 7.2) with an amplitude of 25 mV.
The invention develops a simple and ultrasensitive micro RNA detection method based on a target micro RNA induced branched DNA nano structure. The target microRNA can hybridize to hairpin DNA1(H1) on the surface of the gold electrode and induce opening of the hairpin. Subsequently, hairpin ends were exposed to initiate first-order HCR (HCR-1) amplification between two alternating hairpin DNAs (hairpin 2(H2), hairpin 3(H3)) and to produce a long double-helical DNA containing a large number of short DNA single-stranded tails protruding from the long double-helical DNA. Short DNA single-stranded tails on both sides of the duplex can initiate second-order HCR (HCR-2) amplification with hairpin DNA4(H4) and hairpin DNA5(H5) to generate many long duplexes on both sides of the long double-helix DNA formed so that the branched-chain DNA nanostructure formed can carry a large amount of Methyl Blue (MB), can effectively enhance electrochemical signals and achieve low detection limits. Importantly, this strategy provides simple operation, good reliability and ultra-high sensitivity. It has been successfully applied to human serum samples, showing satisfactory results and promising future. Importantly, the one-stop approach can greatly reduce the number of operational steps and avoid system errors due to multiple operational steps.
Drawings
Fig. 1 is a schematic diagram of the present invention.
FIGS. 2A and 2B are graphs of the entire process of the optimal detection process through Cyclic Voltammograms (CV) and Electrochemical Impedance Spectroscopy (EIS); FIG. 2C is a graph comparing the current intensity of different samples or detection processes.
Fig. 3A is a graph of current intensity versus incubation time, fig. 3B is a graph of current intensity versus concentration of H2 and H3, fig. 3C is a graph of current intensity versus concentration of H4 and H5, and fig. 3D is a graph of current intensity versus time of immersion of the electrode in methyl blue solution.
FIG. 4A is a graph of electrochemical signals of microRNAs at different concentrations, FIG. 4B is a linear graph of electrochemical signals- -the different concentrations of microRNAs, and FIG. 4C is a graph of comparison of electrochemical signals of blank, random DNA, random RNA, single base mismatch microRNA (smRNA) and microRNA.
Detailed Description
The present invention will be described in further detail with reference to embodiments.
As shown in fig. 1, the principle of the present invention is: h1 was immobilized on the gold electrode surface via the thiol group on the 5 initiating groups. MCH was then used to block the surface and reduce non-specific adsorption, thereby improving hybridization efficiency and accessibility of surface-bound hairpin DNA. The target microRNA can hybridize to H1, thereby exposing the end of H1. The ends of H1 were complementary to the toe and stem regions of H2, resulting in H2 opening and exposing the ends of H2. The end of H2 can unwind H3 and release the end of H3. The release end of H3 was in the same order as the active portion of H1. Thus, HCR-1 between H2 and H3 can be triggered to form a long nicked dsDNA flanked by capture probes. The capture probes on both sides of the duplex can then initiate HCR-2 between two alternating hairpin DNAs (H4 and H5). Thus, branched DNA nanostructures are formed, and many MBs can be inserted into the base pairs of the formed branched DNA nanostructures, resulting in a significant amplification of the electrochemical signal intensity.
The following experiments verify the feasibility of the detection method of the invention: the detection result of the optimal detection process is compared with the detection result obtained after the partial conditions of the optimal detection process are changed (as shown in fig. 2C), and the feasibility of the method is proved. The optimal detection process is as follows: hairpin DNA1-5 structure is formed by annealing the sequence at 90 ℃ for 5 minutes and cooling to room temperature. The gold electrode was polished with 0.05 μm alumina powder and sonicated in water. 10 μ M hairpin DNA1 in 10mM PBS containing 1M NaCl was dripped onto the gold electrode surface overnight. Then, 1mM MCH was added and incubated for 2h to prevent non-specific adsorption. The prepared electrode was incubated with test solutions (500nM H2 and H3, 2. mu.M H4 and H5, and the target microRNA in 50mM PBS containing 0.5M NaCl, pH 7.2) for 4 hours. The MB serves as an electron transfer mediator and is inserted into the base pair of the branched double-stranded DNA formed. Therefore, the electrode was immersed in 20 μ MMB and stirred for 10 minutes. Thereafter, the electrode was washed with PBS to remove non-specifically bound MB. Square wave voltammetry (from-0.40 to 0.00V, amplitude 25mV) was used. Scanning the electrode obtained in each step and measuring the electrochemical impedance spectrum signal. In the final step, the current intensity signal obtained by scanning the obtained electrode with square wave voltammetry is the signal of sample 5 in fig. 2C. The entire process of the optimal detection process is characterized by Cyclic Voltammograms (CV) and Electrochemical Impedance Spectroscopy (EIS). As shown in the figure2A and B, the bare gold electrodes show a pair of [ Fe (CN)6]-3/-4Reversible redox peaks and small half-circular domains (curve a), indicating good charge transfer properties. After H1 was immobilized on the gold surface, the redox peak current was significantly reduced and the semi-circle for EIS was increased (curve b), due to the negatively charged phosphate backbone of H1 and [ Fe (CN)6]-3/-4. After treatment with MCH, the oxidation reduction peak and electron transfer resistance of the electrode continued to deteriorate (curve c). After reaction with the target microRNA, there was little change in CV and EIS, which was caused by more negatively charged DNA on the electrode (curve d). Between H2 and H3, the current dropped significantly and the EIS increased significantly for HCR-1 due to the high electrostatic repulsion force caused by the long dsDNA and the resistance at the electrode interface (curve e). Finally, after HCR-2 was between H4 and H5 (curve f), the redox peak current further decreased and the electron transfer resistance increased (curve f), which means that branched DNA nanostructures were formed.
Sample 1 is a blank solution, i.e., the solution to be tested does not contain micro-RNA, the rest of the process is the same as the optimal detection process, and the blank sample without micro-RNA shows negligible electrochemical signal (signal of sample 1 in fig. 2C), indicating that if micro-RNA is not present, HCR amplification cannot be initiated.
Sample 2 was a test solution of 500nM H2 and H3 and the target microRNA in 50mM PBS pH7.2 containing 0.5M NaCl, i.e., the test solution did not contain H4 and H5, and the rest of the process was the same as the optimal detection process, and the signal showed weak current intensity (sample 2 signal in FIG. 2C), indicating that only HCR-1 was activated in the presence of H2 and H3.
Sample 3 was a test solution of 2. mu.M H4 and H5 and the target microRNA in 50mM PBS at pH7.2 containing 0.5M NaCl, i.e., the test solution contained no H2 and H3, the rest of the process was the same as the optimal detection process, and the measured current intensity was similar to that of the blank sample (signal of sample 3 in FIG. 2C), which indicates that the amplification of two HCRs could not be triggered by using only H4 and H5.
Sample 4 was incubated with the test solution for 2 hours for half the corresponding incubation time for the optimal detection procedure, and the rest of the procedure was identical to the optimal detection procedure, with the signal showing a relatively weak electrochemical signal (signal of sample 4 in fig. 2C) because HCR amplification was incomplete for only half the HCR incubation time.
Optimization of the Experimental conditions
Several key parameters were optimized to improve sensitivity, including HCR incubation time, hairpin DNA concentration and MB intercalation time. As shown in fig. 3A, the electrochemical signal gradually increased with the incubation time. The signal became stable around 4 hours, indicating that the reaction was complete within 4 hours. The concentrations of H2 and H3 affect the HCR-1 amplification reaction. The results showed that the signal levels tended to level off when the concentrations of H2 and H3 were about 500nM (FIG. 3B). In addition, the concentration of H4 and H5 greatly affected the HCR-2 amplification reaction (FIG. 3C). The results showed that the signal became stable when the concentration of H4 and H5 was 2. mu.M. The insertion time of the MB affects the number of MBs that insert the branched DNA nanostructure base pairs. The signal rises sharply with time and reaches a plateau after 10 minutes (fig. 3D). According to these results, the optimal incubation time was 4H, the optimal concentration of H2 and H3 was 500nM, the optimal concentration of H4 and H5 was 2. mu.M, and the optimal intercalation time of MB was 10 minutes.
To evaluate the analytical performance of the method, it was used to detect microRNAs at different concentrations. As shown in FIG. 4(A), the current signal obtained by square wave voltammetry gradually increases with the increase of the concentration of the microRNA. FIG. 4(B) shows that the micro RNA concentration from 10fM to 50pM shows a good linear relationship with the current signal obtained by square wave voltammetry. The linear regression equation is that I (mu A) is 0.376logCMicro RNA+6.003, linear correlation coefficient 0.991. Furthermore, the limit of detection (LOD) is estimated as 2fM, multiplied by 3 σ (three times the standard deviation of the blank value, n-6).
The selectivity of the method was assessed by different samples, including blank, random DNA, random RNA, single base mismatched microrna (smrna) and microrna (fig. 4C). The results show that the intensity of random DNA and random RNA was similar to that of the blank sample. The weaker current response for smRNA and the stronger current response for the target microRNA indicate that only the target microRNA is effective in causing both levels of HCR amplification strategy and formation of branched DNA nanostructures. These results indicate that the method has satisfactory selectivity for microRNA detection.
The performance of the electrochemical biosensor was evaluated by detecting micrornas in human serum samples using standard addition methods. Analytical samples were prepared by adding micrornas of different concentrations to serum of healthy humans at 10-fold dilutions. As shown in table 1, the recovery changed from 92.0% to 104.4%, and the RSD of the six replicates changed from 5.68% to 8.78%. These results show that the method has wide prospect in detecting the microRNA in the actual human serum.
TABLE 1 electrochemical signals generated by the addition of microRNAs of different concentrations to serum of healthy humans diluted 10-fold
In summary, the present invention achieves ultrasensitive microrna detection by a one-step procedure with two HCR amplifications to form branched DNA nanostructures. This strategy shows high sensitivity and reliability to the target microRNA. It has also been successfully used for detecting micro RNA in human serum samples, and shows great application prospect. The strategy can complete the formation of the branched DNA nano structure by only one-step operation, greatly simplifies the experimental procedure and reduces the influence of system errors.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.