CN109856217B - Method for detecting miRNA-21 based on electrochemical alternating current impedance - Google Patents
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Abstract
The invention discloses a method for detecting miRNA-21 based on electrochemical alternating current impedance, which comprises the following steps: step one, polishing and drying a gold electrode; soaking the surface of the gold electrode in miRNA-21 aptamer solution, sequentially performing electron beam irradiation, soaking in glucose aqueous solution, washing and drying; soaking in magnetic field, washing, and blow-drying; dropwise adding a mercaptoethanol solution for reaction, washing and drying to obtain an activated gold electrode; step three, preparing a plurality of activated gold electrodes, dripping miRNA-21 solutions with different concentrations, washing and drying after reaction to obtain a plurality of detection gold electrodes; step four, detecting the impedance value corresponding to the miRNA-21 solution with each concentration; calculating to obtain a linear relation between the difference value of the impedance values and the concentration; and step six, detecting the impedance value of the sample liquid to be detected, and calculating by adopting a linear relation to obtain the concentration of the miRNA-21 in the sample liquid to be detected. The invention has the advantage of high detection sensitivity.
Description
Technical Field
The present invention relates to the field of electrochemical detection. More specifically, the invention relates to a method for detecting miRNA-21 based on electrochemical alternating current impedance.
Background
Micrornas (mirnas) are a class of small, non-coding RNA molecules of about 22 nucleotides that function as transcriptional and post-transcriptional regulation of gene expression. According to some recent reports, some kinds of miRNA are closely related to human cancers, and miRNA-21 is one of the miRNA. Among these different mirnas, microRNA-21 is considered as a potential biomarker because it is overexpressed in many cancer tissues, such as lung adenocarcinoma, glioblastoma, breast cancer, cervical cancer. In addition, by delivering inhibitors to modulate specific microRNA expression, they may also be novel therapeutic targets for a number of diseases. Therefore, a sensitive microRNA quantitative method with high sensitivity and high selectivity is urgently needed. However, the short length, high sequence similarity and low abundance of mirnas (down to a few molecules per cell) make it difficult to develop a fast, simple, sensitive and selective electrochemical method for detecting mirnas. To overcome these disadvantages, different biosensing strategies for miRNA detection have been developed, such as fluorescence, electrochemiluminescence, surface plasmon resonance, colorimetry, electrochemistry and among these methods, electrochemical biosensors are a particularly promising class of efficient analytical techniques due to their outstanding aspects of high sensitivity and accuracy, fast response, low cost, user friendliness, simplicity of the instrument, ease of miniaturization and the like.
Aiming at the problems, a method for detecting the concentration of miRNA-21 in a solution by using an alternating current impedance method based on a miRNA-21 aptamer modified electrode is researched, the method is simple to operate, rapid in detection and high in sensitivity, and the miRNA-21 in a mixed sample solution can be identified with high sensitivity.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
Still another object of the present invention is to provide a method for detecting miRNA-21 based on electrochemical AC impedance, which can accurately detect the concentration of miRNA-21 in a mixed sample solution.
To achieve these objects and other advantages in accordance with the present invention, there is provided a method for electrochemical ac impedance-based detection of miRNA-21, comprising:
step one, grinding, polishing, washing and drying a gold electrode;
step two, activation treatment of the gold electrode:
a. soaking the surface of the gold electrode in the step one in miRNA-21 aptamer solution with the concentration of 2 mu M for 1min, then irradiating for 3min by adopting an electron beam with the intensity of 15kGy, soaking in glucose aqueous solution with the concentration of 10 mu M at the temperature of 4 ℃ for 4h, flushing for 30s by PBS solution with the pH of 6.8, and drying by using nitrogen;
b. soaking the surface of the gold electrode treated in the step a in PBS (phosphate buffer solution) with the pH value of 6.8, placing the gold electrode in a magnetic field with the magnetic field intensity of 1T while soaking the gold electrode for 5min, taking out the gold electrode, washing the gold electrode for 15min by using an ethanol solution of boric acid with the concentration of 5 mu M, then washing the gold electrode for 30s by using the PBS with the pH value of 6.8, and drying the gold electrode by using nitrogen;
c. dropwise adding a mercaptoethanol solution with the concentration of 1mM onto the surface of the gold electrode treated in the step b until the mercaptoethanol solution is fully distributed on the surface of the gold electrode, placing the gold electrode in an environment with the temperature of 4 ℃ for reaction for 1 hour, then flushing the gold electrode with a PBS solution with the pH value of 6.8 for 30 seconds, and drying the gold electrode with nitrogen to obtain an activated gold electrode;
step three, repeating the step one and the step two to prepare a plurality of activated gold electrodes, respectively dripping miRNA-21 solutions with different concentrations on the surfaces of the plurality of activated gold electrodes, placing the activated gold electrodes in an environment at 37 ℃ for 60min, then flushing the activated gold electrodes with a PBS solution with the pH value of 6.8 for 30s, and drying the activated gold electrodes with nitrogen to obtain a plurality of detection gold electrodes;
step four, respectively taking a silver-silver chloride electrode and a platinum electrode as a reference electrode and a counter electrode, taking a gold detection electrode in the step three as a working electrode, building a plurality of three-electrode systems, scanning by adopting an alternating-current impedance method to obtain a plurality of alternating-current impedance curve graphs, and fitting according to the alternating-current impedance curve graphs to obtain impedance values corresponding to the miRNA-21 solution with each concentration;
step five, calculating to obtain a linear relation between the difference of the impedance values of the miRNA-21 solutions and the concentration by taking the difference of the impedance values of the miRNA-21 solutions with the concentration not being 0 minus the impedance value of the miRNA-21 solution with the concentration being 0 as the ordinate and the concentration of the miRNA-21 solution as the abscissa;
step six, preparing an activated gold electrode according to the step one and the step two, dripping 10 mu L of a sample solution to be detected containing miRNA-21 with unknown concentration on the surface of the activated gold electrode, placing the activated gold electrode in an environment at 37 ℃ for 60min, flushing the activated gold electrode with a PBS (phosphate buffer solution) solution with the pH value of 6.8 for 30s, and drying the activated gold electrode with nitrogen to obtain a detected gold electrode;
and step seven, respectively taking the silver chloride electrode and the platinum electrode as a reference electrode and a counter electrode, taking the gold detection electrode in the step six as a working electrode, building a three-electrode system, scanning by adopting an alternating-current impedance method to obtain an alternating-current impedance curve graph, fitting according to the alternating-current impedance curve graph to obtain an impedance value corresponding to the sample liquid to be detected, subtracting the impedance value of the miRNA-21 solution with the concentration of 0 from the impedance value to obtain a difference value, substituting the difference value into the linear relation in the step five, and calculating to obtain the concentration of the miRNA-21 in the sample liquid to be detected.
Preferably, the first step is specifically:
placing the surface of the gold electrode on NaHCO3Soaking in 10 μ M ultrapure water solution with concentration of 5 μ M, KCl at 65 deg.C for 40min, washing with ultrapure water, soaking in 2 μ M ultrapure water solution with concentration of acetic acid of 5 μ M and silicic acid of 80 deg.C for 60min, washing with ultrapure water, polishing with 0.05 μ M polishing powder, and washing with ultrapure water.
Preferably, the miRNA-21 solution in step three has a concentration of 0mol/L and 2X 10-12mol/L、5×10-12mol/L、1×10-11mol/L、2×10-11mol/L, the volume of the solution added to the surface of the activated gold electrode is 10 mu L.
Preferably, the nucleotide sequence of miRNA-21 is: 5'-UAGCUUAUCAGACUGAUGUUGA-3', respectively; the nucleotide sequence of the miRNA-21 aptamer is 5' -TCAACATCAGTCTGATAAGCTATTT- (CH2)6-SH-3'。
Preferably, the method for preparing the aqueous glucose solution in step a comprises: freezing ultrapure water at-10 deg.C for 30min, adding acetic acid to obtain acetic acid concentration of 1 μ M, standing at 4 deg.C for 10min, freezing at-10 deg.C for 10min, adding glucose, and ultrasonic treating at 4 deg.C with ultrasonic frequency of 80KHz for 10 min.
Preferably, the scanning parameters in the fourth step and the seventh step are set as follows: an amplitude of 0.005V and a frequency of 0.1 to 1000Hz, in a reaction medium liquid containing [ Fe (CN)6]3-And Fe (CN)6 4-Wherein [ Fe (CN) ]6]3-And Fe (CN)6 4-The total concentration of (2) was 5 mmol.
The invention at least comprises the following beneficial effects: can accurately detect that the solution only contains 10-12The concentration of miRNA-21 in mol/L is high, the anti-interference performance is high, and the concentration of miRNA-21 in a mixed sample containing an interference solution can be accurately detected.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a graph of the AC impedance curves of miRNA-21 solutions of different concentrations in example 1 of the present invention;
FIG. 2 is a linear relationship graph of the concentration of miRNA-21 and the difference between impedance values in example 1 of the present invention.
Detailed Description
The present invention is further described in detail below with reference to the drawings and examples so that those skilled in the art can practice the invention with reference to the description.
It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.
< example 1>
The method for detecting miRNA-21 based on electrochemical alternating current impedance comprises the following steps:
step one, grinding, polishing, washing and drying a gold electrode;
step two, activation treatment of the gold electrode:
a. soaking the surface of the gold electrode in the step one in miRNA-21 aptamer solution with the concentration of 2 mu M for 1min, then irradiating for 3min by adopting an electron beam with the intensity of 15kGy, soaking in glucose aqueous solution with the concentration of 10 mu M at the temperature of 4 ℃ for 4h, flushing for 30s by PBS solution with the pH of 6.8, and drying by using nitrogen;
b. soaking the surface of the gold electrode treated in the step a in PBS (phosphate buffer solution) with the pH value of 6.8, placing the gold electrode in a magnetic field with the magnetic field intensity of 1T while soaking the gold electrode for 5min, taking out the gold electrode, washing the gold electrode for 15min by using an ethanol solution of boric acid with the concentration of 5 mu M, then washing the gold electrode for 30s by using the PBS with the pH value of 6.8, and drying the gold electrode by using nitrogen;
c. dropwise adding a mercaptoethanol solution with the concentration of 1mM onto the surface of the gold electrode treated in the step b until the mercaptoethanol solution is fully distributed on the surface of the gold electrode, placing the gold electrode in an environment with the temperature of 4 ℃ for reaction for 1 hour, then flushing the gold electrode with a PBS solution with the pH value of 6.8 for 30 seconds, and drying the gold electrode with nitrogen to obtain an activated gold electrode;
step three, repeating the step one and the step two to prepare a plurality of activated gold electrodes, respectively dripping miRNA-21 solutions with different concentrations on the surfaces of the plurality of activated gold electrodes, placing the activated gold electrodes in an environment at 37 ℃ for 60min, then flushing the activated gold electrodes with a PBS solution with the pH value of 6.8 for 30s, and drying the activated gold electrodes with nitrogen to obtain a plurality of detection gold electrodes;
step four, respectively taking a silver-silver chloride electrode and a platinum electrode as a reference electrode and a counter electrode, taking a gold detection electrode in the step three as a working electrode, building a plurality of three-electrode systems, scanning by adopting an alternating-current impedance method to obtain a plurality of alternating-current impedance curve graphs, and fitting according to the alternating-current impedance curve graphs to obtain impedance values corresponding to the miRNA-21 solution with each concentration;
step five, calculating to obtain a linear relation between the difference of the impedance values of the miRNA-21 solutions and the concentration by taking the difference of the impedance values of the miRNA-21 solutions with the concentration not being 0 minus the impedance value of the miRNA-21 solution with the concentration being 0 as the ordinate and the concentration of the miRNA-21 solution as the abscissa;
step six, preparing an activated gold electrode according to the step one and the step two, dripping 10 mu L of a sample solution to be detected containing miRNA-21 with unknown concentration on the surface of the activated gold electrode, placing the activated gold electrode in an environment at 37 ℃ for 60min, flushing the activated gold electrode with a PBS (phosphate buffer solution) solution with the pH value of 6.8 for 30s, and drying the activated gold electrode with nitrogen to obtain a detected gold electrode;
and step seven, respectively taking the silver chloride electrode and the platinum electrode as a reference electrode and a counter electrode, taking the gold detection electrode in the step six as a working electrode, building a three-electrode system, scanning by adopting an alternating-current impedance method to obtain an alternating-current impedance curve graph, fitting according to the alternating-current impedance curve graph to obtain an impedance value corresponding to the sample liquid to be detected, subtracting the impedance value of the miRNA-21 solution with the concentration of 0 from the impedance value to obtain a difference value, substituting the difference value into the linear relation in the step five, and calculating to obtain the concentration of the miRNA-21 in the sample liquid to be detected.
Wherein the concentration of miRNA-21 solution in step III is 0mol/L and 2 × 10 respectively-12mol/L、5×10- 12mol/L、1×10-11mol/L、2×10-11mol/L, the volume of the solution added to the surface of the activated gold electrode is 10 mu L.
The nucleotide sequence of miRNA-21 is: 5'-UAGCUUAUCAGACUGAUGUUGA-3', respectively;
the nucleotide sequence of the miRNA-21 aptamer is as follows: 5'-TCAACATCAGTCTGATAAGCTATTT- (CH2) 6-SH-3'.
The scanning parameters in the fourth step and the seventh step are set as follows: the amplitude is 0.005V, the frequency is 0.1-1000 Hz, and the detection is carried out in the reaction medium liquid which is the solution containing [ Fe (CN)6] 3-and Fe (CN)64-, wherein, the total concentration of [ Fe (CN)6] 3-and Fe (CN) 64-is 5 mmol.
The miRNA-21 aptamer solution is prepared by taking TE buffer as a solvent, wherein the TE buffer is 10mM Tris-HCl and contains 1mM EDTA, and the pH value is 7.4.
< example 2>
The method for detecting miRNA-21 based on electrochemical alternating current impedance is the same as in embodiment 1, wherein the first step specifically comprises the following steps:
placing the surface of the gold electrode on NaHCO3Soaking in 10 μ M ultrapure water solution with concentration of 5 μ M, KCl at 65 deg.C for 40min, washing with ultrapure water, soaking in 2 μ M ultrapure water solution with concentration of acetic acid of 5 μ M and silicic acid of 80 deg.C for 60min, washing with ultrapure water, polishing with 0.05 μ M polishing powder, and washing with ultrapure water.
< example 3>
The method for detecting miRNA-21 based on electrochemical ac impedance is the same as in example 1, wherein the method for preparing the aqueous glucose solution in step a comprises: freezing ultrapure water at-10 deg.C for 30min, adding acetic acid to obtain acetic acid concentration of 1 μ M, standing at 4 deg.C for 10min, freezing at-10 deg.C for 10min, adding glucose, and ultrasonic treating at 4 deg.C with ultrasonic frequency of 80KHz for 10 min.
< comparative example 1>
The method for detecting miRNA-21 based on electrochemical alternating current impedance is the same as in example 1, wherein the step two is different and specifically comprises the following steps:
a. soaking the surface of the gold electrode in the step one in miRNA-21 aptamer solution with the concentration of 2 mu M for 1min, then flushing the gold electrode with PBS solution with the pH value of 6.8 for 30s, and drying the gold electrode with nitrogen;
b. soaking the surface of the gold electrode treated in the step a in PBS (phosphate buffer solution) with the pH value of 6.8, taking out after 5min, flushing the surface for 30s with PBS with the pH value of 6.8, and drying the surface with nitrogen;
c. dropwise adding a mercaptoethanol solution with the concentration of 1mM onto the surface of the gold electrode treated in the step b until the mercaptoethanol solution is fully distributed on the surface of the gold electrode, placing the gold electrode in an environment with the temperature of 4 ℃ for reaction for 1 hour, then flushing the gold electrode with a PBS solution with the pH value of 6.8 for 30 seconds, and drying the gold electrode with nitrogen to obtain an activated gold electrode;
< gold electrode detection test >
1. As shown in FIG. 1, the concentration of miRNA-21 in each of curves a, b, c, d, and e is 0mol/L and 2X 10-12mol/L、5×10-12mol/L、1×10-11mol/L、2×10-11mol/L。
As shown in fig. 2, the linear relationship between the concentration and the impedance value of the miRNA-21 solution detected by the method of example 1 is as follows:
Y＝89.17+28.93X，R2＝0.9951
wherein Y represents the difference value between the impedance value of the sample solution to be detected and the impedance value of miRNA-21 with the concentration of 0;
x represents the concentration of miRNA-21 in the sample solution to be detected.
2. Preparing a detection interference liquid: respectively adding ultrapure water into lung adenocarcinoma cells, glioblastoma cells, breast cancer cells and cervical cancer cells, crushing, centrifuging and taking supernate to obtain 4 interference solutions;
preparing 4 groups of miRNA-21 solutions, wherein each group has 4 miRNA-21 solutions with different concentrations, and adding one interference solution into each group of miRNA-21 solutions to make the concentration of the interference solution 5 × 10-11mol/L, miRNA-21 concentration is 2 x 10- 12mol/L、5×10-12mol/L、1×10-11mol/L、2×10-11Obtaining 16 sample liquids to be detected in mol/L;
the 16 sample solutions to be tested were respectively tested by the method of example 1, and the concentration of the miRNA-21 obtained by the test is shown in Table 1, and the unit is mol/L:
TABLE 1
As can be seen from table 1, in the presence of an interfering liquid, compared with comparative example 1, the detection accuracy of the method of example 1 can still be ensured, which is significantly higher than that of comparative example 1, and it is described that the method of activating an electrode of example 1 is helpful to improve the anti-interference capability during detection, thereby improving the detection accuracy.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
SEQUENCE LISTING
<110> Guangxi college of learning
<120> method for detecting miRNA-21 based on electrochemical alternating current impedance
<130> 2
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> RNA
<213> Artificial Synthesis
<400> 1
uagcuuauca gacugauguu ga 22
<210> 2
<211> 25
<212> RNA
<213> Artificial Synthesis
<400> 2
tcaacatcag tctgataagc tattt 25
Claims (6)
1. The method for detecting miRNA-21 based on electrochemical alternating current impedance is characterized by comprising the following steps:
step one, grinding, polishing, washing and drying a gold electrode;
step two, activation treatment of the gold electrode:
a. soaking the surface of the gold electrode in the step one in miRNA-21 aptamer solution with the concentration of 2 mu M for 1min, then irradiating for 3min by adopting an electron beam with the intensity of 15kGy, soaking in glucose aqueous solution with the concentration of 10 mu M at the temperature of 4 ℃ for 4h, flushing for 30s by PBS solution with the pH of 6.8, and drying by using nitrogen;
b. soaking the surface of the gold electrode treated in the step a in PBS (phosphate buffer solution) with the pH value of 6.8, placing the gold electrode in a magnetic field with the magnetic field intensity of 1T while soaking the gold electrode for 5min, taking out the gold electrode, washing the gold electrode for 15min by using an ethanol solution of boric acid with the concentration of 5 mu M, then washing the gold electrode for 30s by using the PBS with the pH value of 6.8, and drying the gold electrode by using nitrogen;
c. dropwise adding a mercaptoethanol solution with the concentration of 1mM onto the surface of the gold electrode treated in the step b until the mercaptoethanol solution is fully distributed on the surface of the gold electrode, placing the gold electrode in an environment with the temperature of 4 ℃ for reaction for 1 hour, then flushing the gold electrode with a PBS solution with the pH value of 6.8 for 30 seconds, and drying the gold electrode with nitrogen to obtain an activated gold electrode;
step three, repeating the step one and the step two to prepare a plurality of activated gold electrodes, respectively dripping miRNA-21 solutions with different concentrations on the surfaces of the plurality of activated gold electrodes, placing the activated gold electrodes in an environment at 37 ℃ for 60min, then flushing the activated gold electrodes with a PBS solution with the pH value of 6.8 for 30s, and drying the activated gold electrodes with nitrogen to obtain a plurality of detection gold electrodes;
step four, respectively taking a silver-silver chloride electrode and a platinum electrode as a reference electrode and a counter electrode, taking a gold detection electrode in the step three as a working electrode, building a plurality of three-electrode systems, scanning by adopting an alternating-current impedance method to obtain a plurality of alternating-current impedance curve graphs, and fitting according to the alternating-current impedance curve graphs to obtain impedance values corresponding to the miRNA-21 solution with each concentration;
step five, calculating to obtain a linear relation between the difference of the impedance values of the miRNA-21 solutions and the concentration by taking the difference of the impedance values of the miRNA-21 solutions with the concentration not being 0 minus the impedance value of the miRNA-21 solution with the concentration being 0 as the ordinate and the concentration of the miRNA-21 solution as the abscissa;
step six, preparing an activated gold electrode according to the step one and the step two, dripping 10 mu L of a sample solution to be detected containing miRNA-21 with unknown concentration on the surface of the activated gold electrode, placing the activated gold electrode in an environment at 37 ℃ for 60min, flushing the activated gold electrode with a PBS (phosphate buffer solution) solution with the pH value of 6.8 for 30s, and drying the activated gold electrode with nitrogen to obtain a detected gold electrode;
and step seven, respectively taking the silver chloride electrode and the platinum electrode as a reference electrode and a counter electrode, taking the gold detection electrode in the step six as a working electrode, building a three-electrode system, scanning by adopting an alternating-current impedance method to obtain an alternating-current impedance curve graph, fitting according to the alternating-current impedance curve graph to obtain an impedance value corresponding to the sample liquid to be detected, subtracting the impedance value of the miRNA-21 solution with the concentration of 0 from the impedance value to obtain a difference value, substituting the difference value into the linear relation in the step five, and calculating to obtain the concentration of the miRNA-21 in the sample liquid to be detected.
2. The method for detecting miRNA-21 based on electrochemical alternating current impedance of claim 1, wherein step one is specifically:
placing the surface of the gold electrode on NaHCO3Soaking 5 μ M, KCl ultra pure water solution with concentration of 10 μ M in 65 deg.C water bath for 40min, washing with ultra pure water, placing in 2 μ M ultra pure water solution with concentration of acetic acid of 5 μ M and concentration of silicic acid, and soaking in 80 deg.C water bath for 60miAnd n, washing with ultrapure water, polishing with 0.05 mu m polishing powder on a polishing cloth, and washing with ultrapure water.
3. The method for detecting miRNA-21 based on electrochemical alternating current impedance of claim 1, wherein the miRNA-21 solution in step three has a concentration of 0mol/L and a concentration of 2 x 10-12mol/L、5×10-12mol/L、1×10-11mol/L、2×10-11mol/L, the volume of the solution added to the surface of the activated gold electrode is 10 mu L.
4. The method for detecting miRNA-21 based on electrochemical AC impedance of claim 1,
the nucleotide sequence of miRNA-21 is: 5'-UAGCUUAUCAGACUGAUGUUGA-3', respectively;
the nucleotide sequence of the miRNA-21 aptamer is 5' -TCAACATCAGTCTGATAAGCTATTT- (CH2)6-SH-3'。
5. The method for detecting miRNA-21 based on electrochemical alternating current impedance of claim 1, wherein the aqueous glucose solution in step a is prepared by: freezing ultrapure water at-10 deg.C for 30min, adding acetic acid to obtain acetic acid concentration of 1 μ M, standing at 4 deg.C for 10min, freezing at-10 deg.C for 10min, adding glucose, and ultrasonic treating at 4 deg.C with ultrasonic frequency of 80KHz for 10 min.
6. The method for detecting miRNA-21 based on electrochemical alternating current impedance of claim 1, wherein the scanning parameters in step four and step seven are set as follows: an amplitude of 0.005V and a frequency of 0.1 to 1000Hz, in a reaction medium liquid containing [ Fe (CN)6]3-And Fe (CN)6 4-Wherein [ Fe (CN) ]6]3-And Fe (CN)6 4-The total concentration of (2) was 5 mmol.
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