CN112342277A - MicroRNA biosensor based on isothermal index amplification - Google Patents
MicroRNA biosensor based on isothermal index amplification Download PDFInfo
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Abstract
This patent has established a simple, sensitive and can short-term test microRNA's biosensor based on nucleic acid amplification strategy, and its preparation method is through target microRNA with the help of catalysis hairpin self-assembly with hairpin probe HP1 hybridization after, produce a large amount of DNA single strands after enzyme-mediated isothermal index amplification again, DNA single strand and fluorescence hairpin probe hybridization to produce the fluorescence signal that can detect, thereby realize carrying out quantitative determination to target microRNA fast sensitively. The linear range of the detection method is 100fmol/L to 10nmol/L, with a detection limit of 5.67 fmol/L. The result shows that the method has potential application value in disease diagnosis.
Description
Technical Field
The invention relates to a biosensor, in particular to a microRNA biosensor based on isothermal index amplification.
Background
The abnormal expression of miRNA is closely related to the occurrence and development of various human diseases (liver cancer, lung cancer, gastric cancer, colon cancer, etc.), and gradually becomes a potential tumor marker. Therefore, the analytical expression and efficient detection of mirnas are of great importance for biomedical research to understand their role in cancer cells and to further verify their function. The traditional methods comprise real-time polymerase chain reaction, RNA blotting, microarray and second-generation sequencing, and are four main methods for detecting the expression level of microRNA at present. However, these methods have some inherent limitations. Therefore, in order to rapidly and conveniently detect microRNA, various strategies such as electrochemical-based assays, surface enhanced raman spectroscopy-based assays, nanoparticle-based colorimetric assays and fluorescent assays have been developed in succession. However, each method has its drawbacks and limitations.
In order to improve the sensitivity and adaptability of detection, various nucleic acid sequence-based amplification strategies have been developed and widely applied to sensing platforms such as fluorescence, surface plasmon resonance, colorimetry, and electrochemistry, for example, strand displacement amplification, rolling circle amplification, loop-mediated isothermal amplification, enzymatic repair amplification techniques, catalytic hairpin self-assembly, hybrid chain reaction, and the like. Isothermal exponential amplification reactions have been used as an alternative amplification technique for detecting mirnas because of their high sensitivity, low cost and good tolerance to inhibitory components in clinical samples. SYBR Green I is commonly used as a label in isothermal exponential amplification assays. However, preferential binding of SYBR Green I to GC-rich sequences limits its further use in multiplex PCR. The catalytic hairpin self-assembly is a stable and efficient enzyme-free signal amplification technology. Under the condition of the existence of the target nucleic acid, the nucleic acid hybridization is used as energy to drive the DNA catalysis self-assembly of two hairpin structures, and stable double-stranded DNA is continuously formed, thereby realizing the signal amplification of the enzyme-free nucleic acid. Overcomes the limitation of SYBR Green I, combines catalytic hairpin self-assembly with molecular beacon and is successfully applied to the detection of various nucleic acids with high sensitivity and excellent specificity
Inspired by the above work, in the research, a novel miRNA detection method is constructed by integrating various signal amplification strategies, and a biosensor which is simple, sensitive and capable of rapidly detecting microRNA is developed by combining strand displacement amplification, catalysis hairpin self-assembly and isothermal index amplification reaction.
Disclosure of Invention
Aims to develop a simple and sensitive biosensor capable of rapidly detecting microRNA.
The specific technical scheme is as follows:
a microRNA biosensor based on isothermal index amplification and hairpin probes is prepared by the following steps:
(1) preparation of hairpin probes EP tubes containing HPLC purified HP1, HP2 and lyophilized powder of fluorescent probe nucleic acid strands, respectively, were centrifuged at 12000rpm for 5 minutes, and 180. mu.L, 180. mu.L and 300. mu.L of DEPC water were added to the EP tubes to prepare 10uM solutions. And then placing the EP tube in a water bath box at 95 ℃ for incubation for 5 minutes, closing the water bath box, slowly lowering the temperature to room temperature, thus preparing the hairpin structure probe, wrapping the EP tube filled with the hairpin probe by using tin foil paper, and then placing the EP tube at-20 ℃ for storage.
(2) And performing exponential isothermal amplification, mixing 0.1 muL, 0.2 muL and 2 muL of the prepared HP1, HP2 and fluorescent hairpin probe solution with a buffer solution respectively to obtain 50 muL of reaction solution, adding the microRNA to be detected into the reaction solution, reacting at 37 ℃ for 90 minutes, and then incubating at 80 ℃ for 20 minutes.
(3) And signal detection the reacted liquid was added to a 100 μ L quartz cuvette, the cuvette was placed in a spectrofluorometer, the slit width for excitation and emission was set to 5nm, the PMT detector high voltage was set to 600v, the sample was excited with 495nm light, and fluorescence emission was detected by scanning from 510nm to 650nm in 1nm steps. Thus, the microRNA biosensor based on exponential isothermal amplification is established
The ratio of the HP1, the HP2 and the fluorescent hairpin probe in the reaction solution in the step (2) is preferably 20:40: 400.
The Klenow DNA polymerase content in step (2) is preferably 1.25 units.
The nb. bbvci cleaving enzyme content in said step (2) is preferably 5 units.
The temperature for incubating the reaction solution in the step (2) is preferably 37 ℃.
The incubation time of the reaction solution in step (2) at 37 ℃ is preferably 90 minutes.
The invention develops a simple and sensitive biosensor capable of rapidly detecting microRNA by utilizing strand displacement amplification, catalytic hairpin self-assembly and isothermal index amplification reaction. The detection principle is that a microRNA is detected through a fluorescent signal generated after a DNA single strand generated by two cycles of isothermal exponential amplification is combined with a fluorescent hairpin probe as shown in figure 1. In cycle 1, HP1 contains three functional regions and two recognition sites for the nicking endonuclease nb. HP1 hybridizes to miR-21 and is extended under catalysis of Klenow fragment (exo-) DNA polymerase to form double-stranded DNA. Nicking enzymes can recognize and cleave recognition sites of double-stranded DNA. At the same time, by repeated extension, cleavage and release, a large number of products I (trigger strand) and II are produced, the product I (trigger strand) being the primer for initiating a new extension reaction. In cycle 2, HP2 contained four functional regions and three recognition sites for the nicking endonuclease nb. The product I (trigger strand) released in cycle 1 hybridizes with HP2 to form double-stranded DNA under the catalysis of Klenow fragment (exo-) DNA polymerase. Nicking enzymes produce a large amount of product I (trigger strand) and product II by recognizing and cleaving the recognition site of double-stranded DNA, repeatedly extending, cleaving, and releasing. The product II generated in the cycle 1 and the cycle 2 can be combined with a fluorescent hairpin probe so as to separate a fluorescent group from a quenching group and generate a fluorescent signal which can be detected in a spectrophotometer, thereby realizing simple, sensitive and rapid detection of microRNA.
The successful implementation of isothermal index amplification plays an important role in the preparation of the sensor, and the invention adopts fluorescence emission spectrum, polyacrylamide gel electrophoresis and melting curve to carry out characterization and feasibility analysis on the sensor. As shown in FIG. 2, the fluorescence emission spectrum results show that in the presence of miR-21(1nmol/L), if the reaction system does not contain any enzyme (FIG. 1, curve a), or only contains Klenow fragment (exo-) DNA polymerase (FIG. 2, curve b), or only contains endonuclease Nb. BbvCI (FIG. 1, curve c), almost no fluorescence signal is obtained, which indicates that neither polymerase nor nicking endonuclease can catalyze isothermal exponential amplification alone. In the absence of miR-21, there is only a weak fluorescence signal (FIG. 1, Curve d). In the presence of miR-21 and HP1, there was a slight fluorescence signal (FIG. 2, Curve e). After addition of HP2, a more pronounced fluorescence signal was observed (FIG. 2, curve f). Simultaneously, isothermal index amplification reactions were characterized by polyacrylamide gel electrophoresis (FIG. 3). After incubation of miR-21 and HP1, a bright band with lower electrophoretic mobility relative to lanes 1 and 2 appeared due to hybridization of miR-21 and HP1 (FIG. 3, lane 5). The Klenow fragment (exo-) DNA polymerase can polymerize into double strands after addition to the reaction system in lane 5, but the electrophoretic mobility in lane 6 is faster because double strands move faster than single strands. When HP1 was hybridized to its fully complementary strand, the speed of the double strand was indeed lower than the speed of the single strand (lane 7 in FIG. 3), further demonstrating the faster electrophoretic mobility of the double strand in the polymerase 6 lane. After nicking by adding Klenow fragment (exo-) DNA polymerase and endonuclease Nb. BbvCI to the reaction system in lane 5, new bands with molecular weights of 12bp (product I, i.e., trigger strand) and 22bp (product II) can be generated (FIG. 3, lane 8). After adding the fluorogenic probe in lane 8, a new band with lower electrophoretic mobility appears in lane 9 due to the hybridization of product II and the fluorogenic probe. After addition of HP2 in lane 8, a band with slower electrophoretic mobility appears in lane 10 relative to lane 3 due to double stranded DNA produced by the polymerase after hybridization of product I and HPII. Meanwhile, it can generate a new band with 17bp in lane 10 by catalysis of Klenow Fragmen and nb. A fluorescent hairpin probe was added in lane 10, and a new band corresponding to the position in lane 9 appeared since the product II could hybridize with the fluorescent hairpin probe (FIG. 3, lane 11). In lane 12, in the absence of miR-21, only 3 strands are equivalent to HP1, HP2, and the fluorescent hairpin probe. These results all demonstrate that isothermal exponential amplification can be performed successfully.
Meanwhile, in order to prove that products after isothermal index amplification can be combined with the hairpin fluorescent probe, melting curve analysis is carried out on different reaction systems. As shown in FIGS. 4 and 5, the fluorescent hairpin probe undergoes three stages of variation, with the Tm of the fluorescent probe being 66 degrees Celsius (FIG. 4, curve a of 5). When the Klenow fragment (exo-) DNA polymerase and/or nicking endonuclease Nb. BbvCI were not added to the reaction system, it underwent the same three stages of change as the fluorescent probe (FIG. 4, 5, curves b, c, d). When no miR-21 is added into the reaction system, the reaction system also goes through the same three change stages as the fluorescent probe (FIGS. 4 and 5, curve e). However, after the addition of HP1 to the reaction system, the melting curve undergoes three different stages of change, wherein the Tm value is 60 ℃ (FIG. 4, curve f in FIG. 5), because the addition of HP1 can generate a product II, which generates a fluorescent signal after hybridization with the fluorescent hairpin probe, but as the temperature is increased from 40 to 66 ℃, the product II and the fluorescent hairpin probe are separated, and the fluorescence intensity is reduced; then, as the temperature is increased from 66 to 95 ℃, the free fluorescent probe gradually spreads and the fluorescence intensity gradually increases. While both HP1 and HP2 were added to the reaction system, the melting curve trend was similar to curve f, but two Tm values were obtained, 50 and 60 degrees Celsius respectively (FIG. 4, curve g of 5). The fluorescence emission spectra, polyacrylamide gel electrophoresis and melting curve results above all illustrate the feasibility of the sensor invention.
The ratio of HP1, HP2 and fluorescent hairpin probe plays an important role in the detection performance of the method, because HP1 or HP2 are at too high a concentration, and non-specific amplification and shearing are generated under the action of Klenow fragment (exo-) DNA polymerase and nicking endonuclease nb. And the excessively high concentration of the fluorescent hairpin probe can increase the background signal, interfere the detection result and increase the detection cost. It is therefore particularly necessary to study the ratios of HP1, HP2 and fluorescent hairpin probes. As shown in fig. 6, 20:40:400 is the optimal ratio of HP1, HP2, and fluorescent hairpin probe.
Because Klenow fragment (exo-) DNA polymerase and nicking endonuclease Nb.BbvCI are used in the sensor, the efficiency of isothermal exponential amplification reaction is low because polymerase and nicking enzyme are too few, and the corresponding generated signal is low; if too much polymerase and nickase are used, non-specific polymerization and cleavage will occur in the isothermal exponential amplification reaction, and the corresponding signal-to-noise ratio will be low. It is therefore necessary to study the effect of these two enzymes on isothermal exponential amplification reactions. As shown in FIG. 7, as the amount of Klenow fragment (exo-) DNA polymerase increases, the signal-to-noise ratio increases, reaches a maximum at 1.25 units, and decreases beyond this level, so that 1.25 units is the optimal amount of Klenow fragment (exo-) DNA polymerase. As shown in fig. 8, as the content of the nicking endonuclease nb. bbvci increases, the signal/noise ratio increases, reaches the maximum at 5 units, and decreases beyond this content, so that 5 units are the optimum amount of the nicking endonuclease nb. bbvci.
Since the enzyme-catalyzed reaction requires appropriate temperature and time, it is necessary to study the effect of incubation temperature and time on the signal/noise ratio of isothermal exponential amplification reactions. As can be seen in FIG. 9, as the incubation temperature increases, the catalytic activity of the enzyme increases and the signal/noise ratio increases, reaching a maximum at 37 degrees Celsius, beyond which the catalytic activity of the enzyme decreases and the signal/noise ratio decreases, thus leading to an optimal incubation temperature for isothermal exponential amplification at 37 degrees Celsius. As shown in FIG. 10, the signal/noise ratio increased in the first 90 minutes of the incubation time at 37 degrees Celsius, and reached the maximum when the incubation time reached 90 minutes, and decreased as the incubation time increased, so we incubated at 37 degrees Celsius for 90 minutes as the reaction condition for isothermal exponential amplification.
The invention tests other microRNAs in the same family as the target microRNA. As shown in FIG. 11, compared with the fluorescence signal generated in the presence of the target microRNA, lower fluorescence signals were observed for other microRNAs in the same family
To evaluate the analytical performance of the sensing strategy on the target microRNA, we studied the strategy with a series of target micrornas of different concentrations. According to the results detected by target microRNA with different concentrations of 10fmol/L to 10nmol/L, as shown in FIG. 12, the signal increases with the increase of the concentration of the target microRNA. As can be seen from fig. 13, when the target microRNA concentration is in the range of 100fmol/L to 10nmol/L, the signal increase has a relatively good linear relationship with the logarithm of the target DNA concentration, and the regression equation is Y (a.u.) -71.788+95.653logC (fmol/L), the correlation coefficient is 0.9915, the detection limit is 5.67fM, Y represents the fluorescence signal, and C represents the target microRNA concentration.
To evaluate the feasibility of this sensing strategy in the analysis of actual samples, we studied this strategy using a sample recovery assay. We diluted the total RNA sample extracted from A549 cells to 400 ng/. mu.L with DEPC water and detected the amount of target microRNA in 1. mu.L of the diluted total RNA sample solution using the sensing strategy. Meanwhile, 1pmol/L of target microRNA is added into 1 mu L of diluted total RNA sample solution, and then the sensing strategy is used for detecting the amount of the target microRNA in the solution, and the steps are repeated for six times. As shown in figure 14, the amount of the target microRNA in the diluted total RNA sample solution is 361.8fmol/L, the amount of the target microRNA in the labeled sample is 1360.5fmol/L, the recovery rate is 98.98%, and the RSD is 4.46%. Therefore, the method can be used for quantitative analysis of the target microRNA in the actual sample.
The biosensor which is simple, sensitive and capable of rapidly detecting microRNA is developed by combining strand displacement amplification, catalysis hairpin self-assembly and isothermal index amplification reaction. Isothermal exponential amplification can generate a large number of DNA single strands to combine with the fluorescent hairpin probe to generate a fluorescent signal, so that signal amplification is realized. The sensing strategy is simple and portable in detection, high in sensitivity, low in detection limit of 5.67fmol/L and wide in detection range of 100fmol/L to 10 nmol/L. Meanwhile, the detection strategy provided by the inventor can realize the detection of other biomolecules by only changing the sequences of HP1 and HP 2. The result shows that the microRNA biosensor has potential application value in the aspect of disease diagnosis.
Drawings
FIG. 1 is a schematic diagram of isothermal exponential amplification
FIG. 2 shows fluorescence emission spectra of reaction systems with different components
FIG. 3 is a 10% polyacrylamide gel electrophoresis image
FIG. 4 shows melting curves for different reaction systems
FIG. 5 is a melting curve after integration
FIG. 6 is a graph of the effect of HP1: HP2: fluorescent hairpin probe ratio on the signal/noise ratio
FIG. 7 is a graph showing the effect of the amount of Klenow fragment (exo-) DNA polymerase on the signal/noise ratio
FIG. 8 is a graph of the effect of the amount of nicking endonuclease Nb. BbvCI on the signal/noise ratio
FIG. 9 is the effect of incubation temperature on the signal/noise ratio
FIG. 10 is the effect of incubation time on the signal/noise ratio
FIG. 11 is a specificity test of a sensing strategy
FIG. 12 shows fluorescence signals of different concentrations of target microRNAs
FIG. 13 is a linear relationship graph of target microRNA and fluorescence signal
FIG. 14 is a feasibility verification diagram of the sensing strategy for detecting the target microRNA in the actual sample
Detailed Description
The preparation method of the exponential isothermal amplification-based microRNA biosensor comprises the following steps:
(1) preparation of hairpin probes EP tubes containing HPLC purified HP1, HP2 and lyophilized powder of fluorescent probe nucleic acid strands, respectively, were centrifuged at 12000rpm for 5 minutes, and 180. mu.L, 180. mu.L and 300. mu.L of DEPC water were added to the EP tubes to prepare 10uM solutions. Then, the EP tube is placed in a water bath tank at 95 ℃ for incubation for 5 minutes, then the water bath tank is closed to slowly reduce the temperature to room temperature, so that the hairpin structure probe is prepared, and the EP tube filled with the hairpin probe is wrapped by tin foil paper and then is stored at-20 ℃.
(2) And performing exponential isothermal amplification, mixing 0.1 muL, 0.2 muL and 2 muL of the prepared HP1, HP2 and fluorescent hairpin probe solution with a buffer solution respectively to obtain 50 muL of reaction solution, adding the microRNA to be detected into the reaction solution, reacting at 37 ℃ for 90 minutes, and then incubating at 80 ℃ for 20 minutes.
(3) And signal detection the reacted liquid was added to a 100 μ L quartz cuvette, the cuvette was placed in a spectrofluorometer, the slit width for excitation and emission was set to 5nm, the PMT detector high voltage was set to 600v, the sample was excited with 495nm light, and fluorescence emission was detected by scanning from 510nm to 650nm in 1nm steps. Thus, the microRNA biosensor based on exponential isothermal amplification is established.
Claims (8)
1. A microRNA biosensor based on isothermal exponential amplification is prepared by the following steps:
(1) preparation of hairpin probes EP tubes containing HPLC purified HP1, HP2 and lyophilized powder of fluorescent probe nucleic acid strands, respectively, were centrifuged at 12000rpm for 5 minutes, and 180. mu.L, 180. mu.L and 300. mu.L of DEPC water were added to the EP tubes to prepare 10. mu. mol/L solutions. And then placing the EP tube in a water bath box at 95 ℃ for incubation for 5 minutes, closing the water bath box, slowly lowering the temperature to room temperature to prepare the hairpin structure probe, wrapping the EP tube filled with the hairpin structure probe by using tin foil paper, and then placing the EP tube at-20 ℃ for storage.
(2) And performing exponential isothermal amplification, mixing 0.1 muL, 0.2 muL and 2 muL of the prepared HP1, HP2 and fluorescent hairpin probe solution with a buffer solution respectively to obtain 50 muL of reaction solution, adding the microRNA to be detected into the reaction solution, reacting at 37 ℃ for 90 minutes, and then incubating at 80 ℃ for 20 minutes.
(3) And signal detection the reacted liquid was added to a 100 μ L quartz cuvette, the cuvette was placed in a spectrofluorometer, the slit width for excitation and emission was set to 5nm, the PMT detector high voltage was set to 600v, the sample was excited with 495nm light, and fluorescence emission was detected by scanning from 510nm to 650nm in 1nm steps. Thus, the microRNA biosensor based on isothermal exponential amplification is established.
2. The isothermal-index-amplification-based microRNA biosensor as claimed in claim 1, wherein the nucleic acid probes used in step 1 are purified by HPLC.
3. The exponential isothermal amplification based microRNA biosensor of claim 1, which is prepared by adding DEPC water in 180. mu.L, 180. mu.L and 300. mu.L respectively to the tubes containing HP1, HP2 and hairpin probe EP in step 1 to prepare a 10uM solution.
4. The exponential isothermal amplification based microRNA biosensor according to claim 1, wherein the preparation method comprises the step 1 of slowly cooling the hairpin probe to room temperature after incubation at 95 ℃ for 5 minutes.
5. The microRNA biosensor based on exponential isothermal amplification of claim 1, wherein in step 2, the HP1, the HP2 and the hairpin probe are respectively taken from 0.1. mu.L, 0.2. mu.L and 2. mu.L to 50. mu.L of reaction solution, and the concentrations of the HP1, the HP2 and the hairpin probe in the reaction solution are respectively 20nmol/L, 40nmol/L and 400 nmol/L.
6. The exponential isothermal amplification based microRNA biosensor of claim 1, wherein the reaction solution composition in step 2 of the preparation method comprises 1.25 units of U Klenow fragment DNA polymerase, 5 units of Nb.BbvCI endonuclease, 500umol/L dNTPs mixture, 20nmol/L HP1, 40nmol/L HP2, 400nmol/L fluorescent hairpin probe, NEBuffer 2(pH 7.9, 10mM Tris-HCl, 10mM magnesium chloride, 50mM NaCl and 1mM DTT), and CutSmart (pH 7.9, 20mM Tris-acetate, 500mM potassium acetate, 100 μ g/mL BSA and 10mM magnesium acetate).
7. The isothermal-index-amplification-based microRNA biosensor according to claim 1, wherein the reaction time of the reaction solution in step 2 is 90 minutes at 37 ℃ and then 20 minutes at 80 ℃.
8. The microRNA biosensor based on exponential isothermal amplification of claim 1, wherein the detection conditions in step 3 of the preparation method are set as 5nm for both excitation and emission slit width, 600v for PMT detector high voltage, 495nm for excitation wavelength, and fluorescence emission is scanned from 510nm to 650nm in 1nm step size.
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| CN113073132B (en) * | 2021-03-30 | 2024-01-19 | 安徽工业大学 | ECL biosensor and application thereof in preparation of detection system for detecting myocardial infarction miRNA |
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| CN113073132B (en) * | 2021-03-30 | 2024-01-19 | 安徽工业大学 | ECL biosensor and application thereof in preparation of detection system for detecting myocardial infarction miRNA |
| CN113981047A (en) * | 2021-11-08 | 2022-01-28 | 中国科学院合肥物质科学研究院 | Reverse transcription-strand displacement amplification method for miRNA detection and application |
| CN113981047B (en) * | 2021-11-08 | 2023-11-07 | 中国科学院合肥物质科学研究院 | Reverse transcription-strand displacement amplification method for miRNA detection and application thereof |
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