CN111187806A - MicroRNA detection method based on 3D DNA nano-net structure dual-signal amplification technology - Google Patents
MicroRNA detection method based on 3D DNA nano-net structure dual-signal amplification technology Download PDFInfo
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Abstract
The invention relates to a microRNA detection method based on a 3D DNA nano-net structure double-signal amplification technology, and an electrochemical biosensor based on a double-signal amplification technology is constructed based on integration of target self-circulation catalytic hairpin self-assembly and the 3D DNA nano-net structure. Once initiated by the target, the two hairpin sequences are designed to form a thermodynamically stable hybrid duplex structure, while releasing the target into the next cycle. The process successfully converts the target into a more stable structure, thereby achieving the purpose of signal amplification for the first time. Subsequently, a 3D DNA nano-net structure is introduced, and the structure is formed by connecting X-type DNA structures formed by hybridizing three DNA sequences through T4 ligase. The 3D DNA nanoweb structure can hybridize to the duplex structure formed by the above reaction, thus achieving a second signal amplification. Has the advantages of high detection sensitivity, good detection specificity and the like.
Description
Technical Field
The invention belongs to the technical field of microRNA detection, and relates to a microRNA detection method based on a 3D DNA nano-network structure double-signal amplification technology.
Background
Circulating micrornas (mirnas) are a class of small, non-coding RNAs that are secreted from living cells into the extracellular environment and remain relatively stable in various body fluids, such as blood, urine, bile, cerebrospinal fluid, and pancreatic fluid. Due to their tissue-specific origin, abundant information content and the advantage of easy collection, circulating mirnas have proven to be ideal non-invasive biomarkers for clinical diagnosis of a variety of diseases.
Currently, various emerging biosensing technologies such as fluorescence, microfluidic devices, electrochemiluminescence analysis, colorimetry, and electrochemical detection have been used for the detection of mirnas. Among these methods, electrochemical biosensors have high sensitivity, low sample demand, easy operation, and low cost, and thus are considered as promising strategies for detecting circulating mirnas.
Due to the low abundance of circulating mirnas in the extracellular environment, more efficient signal amplification strategies are needed for the construction of sensors. At present, the electrochemical sensor mainly amplifies signals through the change of the surface material of the electrode, and needs the complex material change of the surface of the electrode.
Disclosure of Invention
In view of the above, the present invention provides a microRNA detection method based on a 3D DNA nano-network double-signal amplification technology.
In order to achieve the purpose, the invention provides the following technical scheme:
1. a construction method of an electrochemical biosensor based on a 3D DNA nano-network structure dual-signal amplification technology comprises the following steps:
(A) target-assisted catalytic hairpin self-assembly (CHA): firstly fixing a hairpin DNA probe H1 on the surface of a gold electrode, then mixing the hairpin DNA probe H2 with targets with different concentrations, dropwise adding the mixture to the surface of the gold electrode, incubating, opening a hairpin structure of H1 by the targets to expose a region hybridized with H2, further opening the hairpin structure of H2 to form a stable H1-H2 hybridization duplex and generate a free tail, and simultaneously releasing the targets; after multiple cycles, generating a plurality of H1-H2 hybrid duplexes with free tails, and entering step (B);
(B) self-assembly of 3D DNA nanoweb structures: forming a 3D DNA reticular structure by a plurality of X-DNAs through T4 ligase, wherein the X-DNAs are formed by three ssDNAs (single-stranded DNAs) through hybridization annealing, and a large number of free tails generated in the step (A) form the fourth edge of the X-DNAs, so that the specific hybridization of an H1-H2 hybridization duplex and the DNA reticular structure is realized, the modification of a gold electrode is completed, and the electrochemical biosensor is further constructed;
wherein, the nucleotide sequence of H1 is shown as SEQ ID NO.1, and the nucleotide sequence of H2 is shown as SEQ ID NO. 2.
Preferably, the modified gold electrode is used as a working electrode, the saturated calomel electrode is used as a reference electrode, and the platinum electrode is used as a counter electrode to form a three-electrode system, so that the electrochemical sensor is obtained.
Preferably, in the step (A), the target is miR-21, and the nucleotide sequence of the target is shown in SEQ ID NO. 3.
Preferably, in step (A), H1 and H2 are prepared by annealing at 95 ℃ for 5 minutes and then slowly cooling to 25 ℃.
Preferably, the specific method of step (a) is: dissolving H1 in a fixing buffer solution, then dripping the solution on the surface of a gold electrode, and placing the gold electrode in a water bath box at 37 ℃ overnight (12 hours) to fix H1 on the surface of the gold electrode; h2 and various concentrations of target were then dissolved in hybridization buffer, added dropwise to the gold electrode surface, and incubated at 37 ℃ for 1.5 hours.
Further preferably, the pH of the fixation buffer is 7.4, comprising: 12.5mM magnesium acetate, 40mM Trisbase, 2mM EDTA (ethylenediaminetetraacetic acid), 20mM acetic acid.
Further preferably, after H1 is fixed on the surface of the gold electrode, MCH is dripped into the gold electrode, the gold electrode is incubated for 1 hour at 4 ℃, and the electrode is closed to avoid specific adsorption of the electrode.
Further preferably, the hybridization buffer has a pH of 7.4 and comprises: 50mM PBS (phosphate buffered saline), 0.5M sodium chloride.
Preferably, in step (B), the X-DNA is prepared by the following method: mixing the three ssDNAs with T4 Buffer and DEPC (diethyl pyrocarbonate) treatment water, heating at 95 ℃ for 2 minutes, then cooling to 65 ℃ for 5 minutes, then cooling to 60 ℃ for 3 minutes, and finally slowly cooling to 25 ℃ for 1 hour to obtain the X-DNA.
Preferably, in the step (B), the 3D DNA nano-net structure is prepared by the following method: adding T4 ligase into the X-DNA, and incubating for 30 minutes at 16 ℃ to obtain the 3D DNA nano-net structure.
Preferably, in the step (B), the three ssDNAs are respectively X1-P, X2-P, X3-P, and the nucleotide sequences are respectively shown in SEQ ID NO. 4-6.
2. The electrochemical biosensor based on the 3D DNA nano-network structure dual-signal amplification technology is obtained by the construction method.
3. The electrochemical biosensor is applied to microRNA detection.
4. The method for detecting the microRNA based on the 3D DNA nano-net structure dual-signal amplification technology is realized by utilizing the electrochemical biosensor, firstly, methylene blue solution is dripped on the surface of a modified gold electrode, the gold electrode is protected from light for 30 minutes at room temperature (25 ℃), then, the current value is measured in electrolyte containing 50mM sodium chloride and 10mM Tris-HCl by using a differential pulse voltammetry method, and then, the target concentration is calculated.
The invention has the beneficial effects that:
the invention constructs an electrochemical biosensor of a double-signal amplification technology to detect circulating microRNA by utilizing the integration of catalytic hairpin self-assembly based on target self-circulation and a 3D DNA nano-net structure.
The circulating miRNA is easy to degrade during detection, has a short sequence and is difficult to detect, and the target-assisted catalytic hairpin self-assembly process is designed for target identification and circulation. Once initiated by the target, the two hairpin sequences are designed to form a thermodynamically stable hybrid duplex structure, while releasing the target into the next cycle. The process successfully converts the target into a more stable structure, thereby achieving the purpose of signal amplification for the first time. Subsequently, a 3D DNA nano-net structure is introduced, and the structure is formed by connecting X-type DNA structures formed by hybridizing three DNA sequences through T4 ligase. The 3D DNA nanoweb structure can hybridize to the duplex structure formed by the above reaction, thus achieving a second signal amplification. The two-time signal amplification strategy improves the detection sensitivity, and the operation is simple and easy to realize.
The method does not need complex electrode surface material change, only uses the DNA nano material to amplify the secondary signal of the target, and has simple operation and good detection effect. The concrete effects are as follows:
1. the detection linear range is wide, and reaches 6 orders of magnitude, from 10fM to 1 nM.
2. The detection limit was low, only 3.0683 fM.
3. The specificity is good, and the detection of single base mismatch, multi-base mismatch and other different kinds of microRNAs can be recognized.
4. Clinical serum samples can be directly detected.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a schematic diagram of the assembly and testing process of a sensor;
FIG. 2 is a representation of the formation of a 3D DNA nanoweb structure; wherein, A is polyacrylamide gel electrophoresis picture, B is blue dextran solution experiment;
FIG. 3 is a representation of the sensor formation process, wherein A is a polyacrylamide gel electrophoresis plot, B is a differential pulse voltammogram, C is a cyclic voltammogram, and D is an electrical impedance plot;
FIG. 4 shows the specificity of the detection method of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
Example (b):
the method mainly comprises two signal amplification processes, namely a target-assisted catalytic hairpin self-assembly process and a 3D DNA nano-net structure self-assembly process. The assembly and detection process of the specific sensor is shown in fig. 1.
(1) Target-assisted catalytic hairpin self-assembly process:
this process is mainly achieved by two hairpin DNA probes (H1, H2). The H1 and H2 probes are prepared by annealing at 95 ℃ for 5min and then slowly cooling to 25 ℃. Subsequently, 2. mu. M H1 probe was dissolved with 1mM TCEP (tris (2-carboxyethyl) phosphine) in a fixation buffer (12.5mM magnesium acetate, 40mM Trisbase, 2mM EDTA, 20mM acetic acid, pH 7.4). Then, 4. mu.l of the solution was dropped onto the surface of the gold electrode, and the gold electrode was placed in a 37 ℃ water bath overnight to immobilize the H1 probe on the surface of the gold electrode. Then 1mM MCH (6-mercaptohexanol) was added dropwise to the gold electrode and incubated at 4 ℃ for 1 hour, the electrode was blocked to avoid specific adsorption to the electrode. Then, 2. mu. M H2 probe was prepared and incubated with different concentrations of target (miR-21) in hybridization buffer (50mM PBS, 0.5M sodium chloride, pH7.4) for 1.5 hours at 37 ℃ with 4. mu.l drop-wise addition to the surface of the gold electrode.
There is a certain complementary sequence between the H1 and H2 probes, when the target does not exist, the two will not be complementarily hybridized due to the stability of the hairpin structure; when the target is present, the target first opens the hairpin structure of H1, exposing the region of hybridization with H2, and then opens the H2 hairpin structure to form a stable hybrid duplex. Meanwhile, the target mir-21 is released to carry out the next cycle, and the purpose of signal amplification for the first time is achieved.
(2) Self-assembly process of 3D DNA nano-network structure:
the 3D DNA nano-net structure is prepared in a liquid phase environment in advance. 2 mul each of X1-P, X2-P, X3-P, 2 mul of 10 XT 4 Buffer, 11.5 mul of DEPC treated water in a 20 mul system, then metal bath at 95 ℃ for 2min, cooling to 65 ℃ for 5min, then maintaining at 60 ℃ for 3min, finally slowly cooling to 25 ℃ at the speed of 1 ℃/min to form X-DNA. Subsequently, 0.5. mu. l T4 ligase was added and incubated at 16 ℃ for 30 minutes to form a 3D DNA nano-network. Finally, the mixture was diluted to 0.5. mu.M with a hybridization buffer, and 4. mu.l of the diluted mixture was added dropwise to the electrode modified in the previous step, followed by incubation at 37 ℃ for 1.5 hours.
The 3D DNA network used in the present invention is a structure formed by a large amount of X-DNA by T4 ligase. The X-DNA is formed by three ssDNA hybridizations annealed with one less side than the conventional X-DNA, which is the key to the second signal amplification. The hybrid duplex of H1 and H2 produced a large number of free tails that made up the fourth side of the X-DNA during the catalytic hairpin self-assembly process, thus resulting in specific hybridization of the H1-H2 hybrid duplex to the DNA network, achieving two signal amplifications.
(3) And (3) electrochemical detection process:
the detection is carried out by using an electrochemical workstation of Chenghua CHI 760E model, wherein an electrochemical system consists of a traditional three-electrode system, namely a modified gold electrode is used as a working electrode, a saturated calomel electrode is used as a reference electrode, and a platinum electrode is used as a counter electrode. Cyclic voltammetry and electrical impedance were used for the characterization of the sensor formation process, both scanned in 10M potassium ferricyanide solution. The scanning range of the cyclic voltammetry is-0.2V-0.6V, the scanning rate is 100mV/s, and the frequency range of the electrical impedance method is 0.1-105 Hz. Differential pulse voltammetry was used to measure target concentration, scanning in electrolyte containing 50mM sodium chloride and 10mM Tris-HCL. The scanning range is-0.1V to-0.6V, the pulse amplitude is 10mV, the pulse width is 50ms, and the pulse period is 0.0167 s.
Mu.l of 1mM methylene blue solution (containing 10mM Tris-HCl and 50mM sodium chloride) was added dropwise to the surface of the gold electrode subjected to the two signal amplifications, and then the gold electrode was protected from light at room temperature for 30 minutes, followed by measuring the current value using the differential pulse voltammetry in an electrolyte containing 50mM sodium chloride and 10mM Tris-HCl. Because the methylene blue molecules can be specifically adsorbed on the DNA double strand, the electric signal of the methylene blue and the concentration of the target have a certain linear relation, and the concentration of the target can be quantitatively calculated by detecting the signal of the methylene blue.
The nucleotide sequences involved in the present invention are shown in Table 1.
TABLE 1 nucleotide sequences to which the invention relates
Test examples
1. Characterization of formation of 3D DNA nanoweb structures:
in FIG. 2, A is a polyacrylamide gel electrophoresis diagram, wherein lanes 1-4 represent one side, two sides, three sides, and 3D DNA nanoweb structures of X-DNA, respectively. As the structure formation gradually increases, the molecular weight increases, and the ribbon movement speed becomes slow, in line with the theory. In FIG. 2, B is a blue dextran solution experiment, and the formed DNA structure sample was gently added to a prepared blue dextran solution (2. mu.M blue dextran, 150mM sodium chloride). The molecular weight of the blue dextran molecule is large (2X 10)6). X-DNA is above the blue dextran solution due to its smaller molecular weight and corresponding lower density; in contrast, the 3D DNA nano-network structure sinks to the bottom due to its large molecular weight and high density.
2. Characterization of the sensor formation process:
in FIG. 3, A is the polyacrylamide gel electrophoresis diagram. Lanes 1-3 represent H1, H2, target miR-21, respectively; in the absence of target, there was only minimal hybridization of H1, H2 probes (lane 4); in contrast, with the aid of the target, a large number of hybridizations were generated (lane 5); lanes 6-8 represent one side of the X-DNA, forming a 3D DNA nanoweb structure, respectively; lane 9 represents the results after binding of the 3D DNA nanonet structure to the H1-H2 hybrid duplex formed above. In fig. 3, B is a differential pulse voltammetry, the meaning of each curve is shown in a graph, and the stronger the generated electric signal is with the continuous construction of the sensor. In fig. 3, C is cyclic voltammetry, and D in fig. 3 is electrical impedance method, wherein a curve a is an electrical signal generated by a bare gold electrode, b is after fixing an H1 probe, C is after MCH is closed, D is after adding an H2 probe and a target to form first signal amplification, and e is after adding a 3D DNA nano-mesh structure to start second signal amplification.
3. Study of the sensitivity of the present invention:
and (3) diluting the target miR-21 from 1nM to 10fM in a gradient manner, and detecting each concentration by using the method to obtain a corresponding current value. The results can be fitted to a linear regression equation: 0.6508log c +1.900 (R)20.9980), where I represents the measured current value and c represents the concentration of target miR-21. Therefore, the corresponding value can be calculated by measuring the current value through differential pulse voltammetryThe concentration of the target. The results of 5 blank samples were measured simultaneously, according to equation 3Sb/k(SbRepresenting the standard deviation of the current values for the blank samples and k representing the slope of the linear regression equation described above) to a detection limit of 3.6083 fM.
4. The study on the specificity of the invention:
FIG. 4 shows the specific detection result of the invention, when a target miR-21 and a blank sample, a single base (Miss-1), a double base (Miss-2), a three base mismatch (Miss-3) sample and the other three microRNA samples (miR-107, miR-145 and miR-25) are detected under the same condition, the electric signal generated by the target is significantly higher than that generated by other control samples. The nucleotide sequences of Miss-1, Miss-2, Miss-3, miR-107, miR-145 and miR-25 are shown in SEQ ID NO. 7-12, and are specifically shown in Table 1.
5. Clinical serum sample detection:
standing whole blood of healthy people for layering, and taking upper serum. To avoid the influence of complex components in serum, the serum was diluted ten-fold with hybridization buffer and used. Then, a quantitative amount of a target (100fM, 1pM, 10pM, 100pM) of known concentration was added to the diluted serum, and the detection concentration was calculated using the detection of the present invention and compared with the actual concentration. As shown in Table 2, the recovery rate is 93.30% -110.70%, and the relative standard deviation is 1.28% -11.17%, which shows that the invention can be applied to the detection of actual clinical specimens.
TABLE 2 comparison of the measured concentration and the actual concentration of the clinical simulation specimen in the method
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
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Claims (10)
1. A construction method of an electrochemical biosensor based on a 3D DNA nano-network structure dual-signal amplification technology is characterized by comprising the following steps:
(A) target-assisted catalytic hairpin self-assembly: firstly fixing a hairpin DNA probe H1 on the surface of a gold electrode, then mixing the hairpin DNA probe H2 with targets with different concentrations, dropwise adding the mixture to the surface of the gold electrode, incubating, opening a hairpin structure of H1 by the targets to expose a region hybridized with H2, further opening the hairpin structure of H2 to form a stable H1-H2 hybridization duplex and generate a free tail, and simultaneously releasing the targets; after multiple cycles, generating a plurality of H1-H2 hybrid duplexes with free tails, and entering step (B);
(B) self-assembly of 3D DNA nanoweb structures: forming a 3D DNA reticular structure by a plurality of X-DNAs through T4 ligase, wherein the X-DNAs are formed by three ssDNAs through hybridization annealing, and a large number of free tails generated in the step (A) form the fourth edge of the X-DNAs, so that the specific hybridization of an H1-H2 hybridization duplex and the DNA reticular structure is realized, the modification of a gold electrode is completed, and the electrochemical biosensor is further constructed;
wherein, the nucleotide sequence of H1 is shown as SEQ ID NO.1, and the nucleotide sequence of H2 is shown as SEQ ID NO. 2.
2. The construction method according to claim 1, wherein the electrochemical sensor is obtained by forming a three-electrode system by using a modified gold electrode as a working electrode, a saturated calomel electrode as a reference electrode and a platinum electrode as a counter electrode.
3. The method of claim 1, wherein in step (A), H1 and H2 are prepared by annealing at 95 ℃ for 5 minutes and then slowly cooling to 25 ℃.
4. The construction method according to claim 1, wherein the specific method of step (a) is: dissolving H1 in a fixing buffer solution, then dripping the solution on the surface of a gold electrode, and placing the gold electrode in a water bath box at 37 ℃ overnight to fix H1 on the surface of the gold electrode; h2 and various concentrations of target were then dissolved in hybridization buffer, added dropwise to the gold electrode surface, and incubated at 37 ℃ for 1.5 hours.
5. The method according to claim 1, wherein in step (B), the X-DNA is prepared as follows: mixing the three ssDNAs with T4 Buffer and DEPC treatment water, heating at 95 ℃ for 2 minutes, then cooling to 65 ℃ for 5 minutes, then cooling to 60 ℃ for 3 minutes, and finally slowly cooling to 25 ℃ for 1 hour to obtain the X-DNA.
6. The method for constructing a 3D DNA nano-network structure according to claim 1, wherein in the step (B), the 3D DNA nano-network structure is prepared by: adding T4 ligase into the X-DNA, and incubating for 30 minutes at 16 ℃ to obtain the 3D DNA nano-net structure.
7. The construction method according to claim 1, wherein in step (B), the three ssDNAs are X1-P, X2-P, X3-P, and their nucleotide sequences are shown in SEQ ID NO. 4-6.
8. An electrochemical biosensor based on a 3D DNA nano-network double-signal amplification technology, which is obtained by the construction method of any one of claims 1-7.
9. Use of the electrochemical biosensor according to claim 8 for the detection of microRNA.
10. The method for detecting the microRNA based on the 3D DNA nano-net structure dual-signal amplification technology and realized by the electrochemical biosensor of claim 8 comprises the steps of firstly dripping a methylene blue solution on the surface of a modified gold electrode, keeping the modified gold electrode away from light for 30 minutes at room temperature, then measuring the current value in an electrolyte containing 50mM sodium chloride and 10mM Tris-HCl by using a differential pulse voltammetry method, and further calculating to obtain the target concentration.
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