CN106957908B - method for detecting miRNA and/or target molecule with aptamer and detection probe - Google Patents

method for detecting miRNA and/or target molecule with aptamer and detection probe Download PDF

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CN106957908B
CN106957908B CN201710024277.1A CN201710024277A CN106957908B CN 106957908 B CN106957908 B CN 106957908B CN 201710024277 A CN201710024277 A CN 201710024277A CN 106957908 B CN106957908 B CN 106957908B
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郭英姝
李双
尚鑫鑫
王玉洁
张书圣
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Abstract

The invention discloses a detection method and a detection probe for miRNA and/or target molecules with aptamers, wherein the detection method comprises the following steps: the gold nanoparticle-H1 complex, the gold nanoparticle-H2 complex and the target recognition complex; the gold nanoparticle-H1 compound and the gold nanoparticle-H2 compound are formed by combining neck ring DNA H1 and H2 with the gold nanoparticles respectively; the target recognition complex comprises: at least one of a carboxyl magnetic microsphere-DNA 1/2 complex for recognizing miRNA and DNA3 for recognizing target molecules. The detection method provided by the invention does not need chemical labeling, the color change can be seen by naked eyes, the method is simple, the application range is wide, and the method can be used for detecting miRNA and target molecules with nucleic acid aptamers.

Description

method for detecting miRNA and/or target molecule with aptamer and detection probe
Technical Field
The invention relates to the technical field of nucleic acid hybridization detection, in particular to a detection method and a detection probe for miRNA and/or target molecules with aptamers.
Background
miRNAs are a class of endogenous non-coding small molecules (18-22nt) which play an important regulatory role in gene expression or reverse transcription. It is associated with a wide range of biological processes such as cell proliferation, apoptosis and death. Abnormal expression of mirnas has been implicated in a variety of diseases, particularly human cancers, neurological diseases, viral infections and diabetes. The obvious regulation and control function of miRNA in the physiological and pathological processes makes miRNA used for clinical disease diagnosis, gene therapy and discovery of new anti-cancer drugs. For example, miRNA-203 is overexpressed in various malignancies, including breast, cervical, leukemia, and laryngeal carcinoma. Therefore, the expression of miRNA-203 is a promising biomarker for the diagnosis and prognosis of disease states, providing an attractive approach to genetic diseases and potential drug targets in gene therapy. The expression level of miRNA-21 is abnormally increased in various tumor specimens and cell lines, and the miRNA-21 is a recognized oncogenic small RNA.
However, due to the characteristics of low abundance, short size, high sequence homology and the like among miRNA family members, miRNA detection is a challenge, and a miRNA detection method needs to have good specificity, sensitivity and stability. The traditional detection methods of miRNA are as follows: northern blotting, real-time Polymerase Chain Reaction (PCR), and gene chip detection of miRNA. Among them, Northern blotting is considered as the gold standard method for detecting miRNA, but because of the disadvantages of complicated operation, time and labor waste, relatively low sensitivity and large sample amount (10 μ g sample), the Northern blotting method is not suitable for routine clinical diagnosis; PCR and gene chip detection methods have the disadvantages of low efficiency, low detection limit, time consumption, low economy and complex operation, and limit the biological and biomedical applications thereof, so that the development of a new miRNA detection strategy is of great significance.
The aptamer is a single-stranded oligonucleotide which is screened out through in vitro artificial synthesis and developed in recent years, can efficiently and specifically bind various biological target molecules, and the appearance of the aptamer provides a new research platform for the chemical biology and the biomedical community. The aptamer has the advantages of good self-stability, relatively simple and rapid preparation and synthesis, easy acquisition, easy functional modification and labeling and the like, and therefore, the aptamer is flexible and wide in application in biosensor design.
At present, a report of detecting ATP using a biosensor based on an ATP aptamer has been reported, but in the ATP detection process, the aptamer needs to be chemically labeled, and then detection is performed based on changes in the label itself or its catalytic substrate before and after the recognition reaction, which results in complicated operation and high cost.
The unique chemical stability, catalytic activity, processability and metallic properties of gold nanoparticles make them an attractive nanomaterial and have unique size-related optical and electronic properties, and therefore, gold nanoparticles are widely used in the technical fields of catalysis, nanoelectronics and biomedicine (sensing, diagnosis, imaging and labeling). However, no report has been found to combine gold nanoparticles with DNA hybridization chain reaction to detect mirnas and/or target molecules with aptamers.
Disclosure of Invention
In view of the above-mentioned deficiencies in the prior art, the present invention aims to provide a method and a probe for detecting miRNA and/or a target molecule having an aptamer. The detection method does not need chemical labeling, the color change can be seen by naked eyes, the method is simple, the application range is wide, and the method can be used for detecting miRNA and target molecules (such as ATP, thrombin, lysozyme, epidermal growth factor receptor and the like) with aptamers.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a probe comprising: the gold nanoparticle-H1 complex, the gold nanoparticle-H2 complex and the target recognition complex;
the gold nanoparticle-H1 compound and the gold nanoparticle-H2 compound are formed by combining neck ring DNA H1 and H2 with the gold nanoparticles respectively;
the target recognition complex comprises: at least one of a carboxyl magnetic microsphere-DNA 1/2 complex for recognizing miRNA and DNA3 for recognizing target molecules.
In the probe, the sequence of the neck ring DNA H1 is shown as SEQ ID NO.1, the sequence of H2 is shown as SEQ ID NO.2, and the probe specifically comprises the following components:
H1：5’-SH-GCG ATT CCT AGG TTG AGC CCA GGG TTT TTT CAC AGT CCC TGG GCT CAA CCT AGG-3’；(SEQ ID NO.1)
H2：5’-CCC TGG GCT CAA CCT AGG AAT CGC TTT TTT CCT AGG TTG AGC CCA GGG ACT GTG-SH-3’；(SEQ ID NO.2)。
in the probe, the carboxyl magnetic microsphere-DNA 1/2 complex is formed by linking two partially complementary DNAs 1 and 2 on a carboxyl modified magnetic microsphere.
The DNA1 contains sequences complementary and/or partially complementary to the miRNA to be detected.
The DNA3 contains an aptamer sequence that specifically binds to the target molecule to be detected.
Preferably, the particle size of the gold nanoparticles is 5 nm.
Preferably, the particle size of the carboxyl magnetic microsphere is 1 μm.
Preferably, the miRNA is miR-203 or miR-21.
When the miRNA to be detected is miR-203, in the probe, the DNA1 comprises a sequence partially complementary to miR-203, the sequence of the DNA1 is shown as SEQ ID NO.3, the sequence of the DNA2 is shown as SEQ ID NO.4, and the specific steps are as follows:
DNA1：5’-GGG C TAG TGG TCC TAA ACA TTT CAC-NH2-3’；(SEQ ID NO.3)
DNA2：5’-GGA CCA CTA G CCC TGG GCT CAA CCT AGG AAT CGC-3’；(SEQ ID NO.4)。
when the miRNA to be detected is miR-21, in the probe, the DNA1 ' comprises a sequence partially complementary to miR-21, the sequence of the DNA1 ' is shown as SEQ ID NO.5, the sequence of the DNA2 ' is shown as SEQ ID NO.6, and the specific steps are as follows:
DNA1’：5’-GGG T CAA CAT CAG TCT GAT AAG CTA-NH2-3’；(SEQ ID NO.5)
DNA2’：5’-CTG ATG TTG A CCC TGG GCT CAA CCT AGG AAT CGC-3’；(SEQ ID NO.6)。
preferably, the target molecule is ATP.
When the detected target molecule is ATP, the sequence of DNA3 in the probe is shown as SEQ ID NO.7, and the specific steps are as follows:
5’-CCC AGG T CCC TGG GCT CAA CCT AGG AAT CGC GGG ACC TGG GGG AGT ATT GCG GAG GAA GGT-3’；(SEQ ID NO.7)
the invention also provides application of the probe in detecting miRNA and/or target molecules with aptamers.
The application of the probe in the preparation of a kit for detecting miRNA and/or target molecules with aptamers is also the protection scope of the invention.
In a second aspect of the present invention, there is provided a method for preparing the above probe, comprising the steps of:
(1) preparing a gold nanoparticle-H1 complex and a gold nanoparticle-H2 complex: respectively incubating the neck ring DNA H1 and H2 and the gold nanoparticles in a buffer solution, and reacting for 5-7H; separating by agarose gel electrophoresis, and dialyzing the separated strip in a dialysis membrane to obtain the final product;
(2) preparation of carboxyl magnetic microsphere-DNA 1/2 complexes: activating the magnetic beads modified by carboxyl, adding DNA1 into the activated magnetic beads, reacting for 3-5h, and carrying out magnetic separation on the obtained compound to obtain a carboxyl magnetic microsphere-DNA 1 compound; and then adding DNA2, reacting for 1-3h, and performing magnetic separation to obtain the carboxyl magnetic microsphere-DNA 1/2 compound.
In the step (1), the preparation method of the gold nanoparticles comprises the following steps: adding ice sodium borohydride into a mixed solution of chloroauric acid and trisodium citrate, stirring until the solution becomes pink, and reacting for 2-5h to obtain the sodium hydrogen bromide.
Preferably, the mixed solution of chloroauric acid and trisodium citrate has a chloroauric acid concentration of 0.25mM, trisodium citrate concentration of 0.25mM and sodium borohydride concentration of ice of 0.1M.
In the step (1), the mole ratios of H1 and H2 to the gold nanoparticles are both 1: 2.
in the step (1), the buffer solution is 0.5 × TBE buffer solution, and the composition of the buffer solution is as follows: 89mM Tris, 89mM boric acid, 2mM EDTA, pH 8.0.
In the step (2), the activation method of the carboxyl modified magnetic beads comprises the following steps: adding N-hydroxysuccinimide sodium salt (NHS) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) into the carboxyl modified magnetic beads, reacting for 1h at room temperature, and carrying out magnetic separation on the mixture to obtain the magnetic bead.
In a third aspect of the present invention, there is provided a method for detecting miRNA and/or a target molecule having an aptamer, comprising the steps of:
(1) respectively mixing the target identification compound with target solutions to be detected with a series of concentration gradients, reacting, adding the gold nanoparticle-H1 compound and the gold nanoparticle-H2 compound into the reacted solution, incubating for 3-5H, measuring the ultraviolet absorption spectrum of the incubated solution, and constructing a working curve between the concentration of the target to be detected and the ultraviolet absorption peak;
(2) and (3) extracting a target object in the sample to be detected, measuring the ultraviolet absorption spectrum of the incubated solution by adopting the method in the step (1), and substituting the ultraviolet absorption spectrum into the working curve to obtain the concentration of the target object in the sample to be detected.
Specifically, the method for detecting miRNA comprises the following steps:
(1) respectively mixing the carboxyl magnetic microsphere-DNA 1/2 compound with miRNA solutions to be detected with a series of concentration gradients, reacting for 1-3h at 20-30 ℃, performing magnetic separation after reaction, and sucking supernatant; adding the gold nanoparticle-H1 complex and the gold nanoparticle-H2 complex into the supernatant, incubating for 3-5H, measuring the ultraviolet absorption spectrum of the incubated solution, and constructing a working curve between the miRNA concentration and the ultraviolet absorption peak;
(2) and (3) extracting RNA in the sample to be detected, measuring the ultraviolet absorption spectrum of the incubated solution by adopting the method in the step (1), and substituting the ultraviolet absorption spectrum into the working curve to obtain the concentration of miRNA in the sample to be detected.
The method for detecting a target molecule (for example ATP) with an aptamer is:
(1) mixing DNA3 with ATP solutions with series concentration gradients, reacting for 1-3H at 20-30 ℃, adding a gold nanoparticle-H1 compound and a gold nanoparticle-H2 compound into the reacted solution, incubating for 3-5H, measuring the ultraviolet absorption spectrum of the incubated solution, and constructing a working curve between the ATP concentration and the ultraviolet absorption peak;
(2) extracting ATP in the sample to be detected, measuring the ultraviolet absorption spectrum of the incubated solution by adopting the method in the step (1), and substituting the ultraviolet absorption spectrum into the working curve to obtain the concentration of ATP in the sample to be detected.
The principle of detecting miRNA and/or target molecule with aptamer is as follows:
for the detection of miRNA, the invention firstly constructs a probe based on gold nanoparticle-DNA hybrid chain reaction, and the probe comprises: the gold nanoparticle-H1 compound, the gold nanoparticle-H2 compound and the carboxyl magnetic microsphere-DNA 1/2 compound; the carboxyl magnetic microsphere-DNA 1/2 complex is formed by linking two partially complementary DNAs (namely DNA1 and DNA2) on a carboxyl modified magnetic microsphere, the DNA1 contains a sequence partially complementary to miRNA to be detected, and when a target miRNA exists, the target miRNA acts on the DNA1 to release the DNA 2. And adding a gold nanoparticle-H1 compound and a gold nanoparticle-H2 compound, opening neck ring structures of H1 and H2 under the induction of DNA2, so that the gold nanoparticle-H1 and the gold nanoparticle-H2 generate hybrid chain reaction, the gold nanoparticles are close to each other, the color is changed, and the identification and detection of miRNA can be realized by a colorimetric method, wherein the specific schematic diagram is shown in figure 1.
For the detection of target molecules with nucleic acid aptamers, the invention firstly constructs DNA3 containing a nucleic acid aptamer sequence capable of being specifically combined with target molecules to be detected, the DNA3 is of a neck ring structure in a normal state, when target molecules exist, the DNA3 is specifically combined with the target molecules, the neck ring structure of the DNA3 is opened, a gold nanoparticle-H1 compound and a gold nanoparticle-H2 compound are added, the DNA3 after the neck ring structure is opened can induce the neck ring structures of H1 and H2 to be opened, so that the gold nanoparticle-H1 and the gold nanoparticle-H2 are subjected to hybrid chain reaction, the gold nanoparticles are close to each other, the color is changed, the identification and the detection of the target molecules can be realized through a colorimetric method, and the specific schematic diagram is shown in FIG. 2.
One of the keys of the invention is the design of a probe with the detection capability on miRNA and/or a target molecule with an aptamer, namely how to design H1 and H2, so that the miRNA and/or the target molecule can stably exist on one hand, and on the other hand, the neck ring structure can be smoothly opened under the induction of a target object recognition complex, and further, a hybridization chain reaction can occur. If the number of hybridized bases in H1 and H2 is too large, the neck ring structures of H1 and H2 are too stable to be easily opened under the induction of a target recognition complex; if the number of bases hybridized between H1 and H2 is too small, the neck ring structures of H1 and H2 may be unstable, and the neck ring structures may be easily opened even in the absence of the target substance, resulting in false positive detection. In the invention, a plurality of groups of H1 and H2 with different structures and different hybridization base numbers are designed in the test process, and through comparison of actual detection effects, H1 and H2 shown in SEQ ID NO.1 and SEQ ID NO.2 are adopted, so that the detection effect on miRNA and target molecules is the most excellent.
The invention has the beneficial effects that:
the invention skillfully associates gold nanoparticles with DNA hybridization chain reaction for the first time, detects miRNA and target molecules with nucleic acid aptamers through the color change caused by the approach of the mutual distance of the gold nanoparticles, has simple detection method and high sensitivity, and can reach the minimum concentration of 1.0 multiplied by 10 for miRNA detection-11M; the lowest concentration of ATP detection can reach 1.0 multiplied by 10-8M。
Drawings
FIG. 1: the invention detects the schematic diagram of miRNA;
FIG. 2: the invention detects the schematic diagram of ATP;
FIG. 3: transmission electron micrograph of gold nanoparticles of example 1;
FIG. 4: detecting a working curve (A) and a detection solution photo (B) of miR-203 with different concentrations;
FIG. 5: the selectivity of the invention for detecting miR-203;
FIG. 6: and detecting working curves of miR-21 with different concentrations.
FIG. 7A: UV absorption spectra and photographs of ATP at different concentrations were obtained in example 7;
FIG. 7B: working curves for different concentrations of ATP were tested.
Detailed Description
The present invention will be further described with reference to examples, but the following description is only for the purpose of explaining the present invention and does not limit the contents thereof.
Example 1: preparing a miRNA detection probe:
the probe includes: the gold nanoparticle-H1 compound, the gold nanoparticle-H2 compound and the carboxyl magnetic microsphere-DNA 1/2 compound; wherein: the sequences of H1 and H2 are as follows:
H1：5’-SH-GCG ATT CCT AGG TTG AGC CCA GGG TTT TTT CACAGT CCC TGG GCT CAA CCT AGG-3’；(SEQ ID NO.1)
H2：5’-CCC TGG GCT CAA CCT AGG AAT CGC TTT TTT CCT AGG TTGAGC CCA GGG ACT GTG-SH-3’；(SEQ ID NO.2)。
the DNA1 contains a sequence complementary and/or partially complementary to the miRNA to be detected; DNA1 is partially complementary to DNA 2.
The preparation method of the gold nanoparticle-H1 compound and the gold nanoparticle-H2 compound comprises the following steps:
(1) preparation of gold nanoparticles with diameter of 5 nm: to a 20mL mixture of chloroauric acid (0.25mM) and trisodium citrate (0.25mM) was added 0.6mL of 0.1M ice sodium borohydride, and the mixture was stirred until the solution became pink, and reacted for 2-5 h. And storing the gold nanoparticle solution at 4 ℃ for later use. The transmission electron microscope picture of the gold nanoparticles is shown in figure 3, which shows better dispersibility and particle size uniformity.
(2) Preparing a gold nanoparticle-H1 complex and a gold nanoparticle-H2 complex: h1 and H2 were incubated with gold nanoparticles at a ratio of 1:2 in 0.5 XTBE buffer (89mM Tris, 89mM boric acid, 2mM EDTA, pH 8.0), respectively, and reacted at room temperature for 6 hours, to which NaCl was added in small amounts several times during the reaction to a concentration of 50 mM. The separation was carried out by electrophoresis on a 3% agarose gel (electrophoresis gel buffer: 0.5 XTBE, 70V,1.5 h). The strips were dialyzed against dialysis membrane (molecular weight cut-off 14000) and the gold nanoparticle-DNA complexes were stored at 4 ℃.
The preparation method of the carboxyl magnetic microsphere-DNA 1/2 compound comprises the following steps:
mu.L of carboxyl modified magnetic beads (1 μ M) were added with 100 μ L of 0.01M N-hydroxysuccinimide sodium salt (NHS) and 100 μ L of 0.01M 1M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and reacted at room temperature for 1h, the mixture was magnetically separated and washed three times with deionized water, the activated magnetic beads were resuspended in 200 μ L of deionized water, and 200 μ L of 1.0X 10 beads were added-5mol/L DNA1, then, at room temperature for 4h reaction, finally, the complexes were magnetic separation, 0.01M pH 7.4 PBS buffer solution washing 3 times and then heavy suspension in 200 u Lml PBS buffer solution, get carboxyl magnetic microsphere-DNA 1 complexes. 200. mu.L of 1.0X 10 was added to the above carboxyl magnetic microsphere-DNA 1 complex-4mol/L DNA2, then reacting for 2h at room temperature, and magnetism is added to the obtained carboxylThe microsphere-DNA 1/2 complex was magnetically separated, washed 3 times with 0.01M PBS buffer at pH 7.4 and resuspended in 100. mu.L PBS buffer.
Example 2: application of probe in miR-203 detection
1. Test materials:
the nucleotide sequence of miR-203 is as follows:
5’-GUG AAA UGU UUA GGA CCA CUA G-3’。
the composition and preparation method of the probe are the same as example 1, wherein:
the sequences of H1 and H2 are as follows:
H1：5’-SH-GCG ATT CCT AGG TTG AGC CCA GGG TTT TTT CACAGT CCC TGG GCT CAA CCT AGG-3’；(SEQ ID NO.1)
H2：5’-CCC TGG GCT CAA CCT AGG AAT CGC TTT TTT CCT AGG TTGAGC CCA GGG ACT GTG-SH-3’；(SEQ ID NO.2)；
the sequences of DNA1 and DNA2 are as follows:
DNA1：5’-GGG C TAG TGG TCC TAA ACA TTT CAC-NH2-3’；(SEQ ID NO.3)
DNA2：5’-GGA CCA CTA G CCC TGG GCT CAA CCT AGG AAT CGC-3’；(SEQ ID NO.4)。
2. the detection method comprises the following steps:
the specific method comprises the following steps:
(1) preparing a miR-203 working solution: preparing a miR-203 working solution: sterilizing a PBS buffer solution (pH 7.4) containing 1 per mill (volume ratio) of DEPC water, and preparing miR-203 solutions with different concentrations by using the PBS buffer solution.
(2) Drawing a working curve: 100 mu L of carboxyl magnetic microsphere-DNA 1/2 compound and 100 mu L of miR-203 solution (0, 1.0X 10) with different concentrations-11M，5.0×10-11M，1.0×10-10M，3.0×10-10M) are mixed evenly, after reaction for 2H at 25 ℃, magnetic separation is carried out, supernatant is absorbed, then 100 mu L of gold nanoparticle-H1 compound and 100 mu L of gold nanoparticle-H2 compound are added into the supernatant, the color change of the solution is recorded after incubation for 4H, and ultraviolet absorption spectrum is measured. Taking the concentration of miR-203 as the abscissa and ultraviolet absorption peaks at 620nm and 520nmThe value ratio plots a working curve for the ordinate. As can be seen from FIG. 4, with the increase of the concentration of miR-203, the ultraviolet absorption peak ratios at 620nm and 520nm are correspondingly changed. The working curve equation of miR-203 detected by the method is that Y is 0.022X +0.437(Y represents the ultraviolet absorption peak ratio at 620nm and 520nm, X represents the concentration of miR-203, and the unit is 10-10M, R ═ 0.99), the concentration range of miR-203 was 1.0 × 10-11M-3.0×10-10And M. With the increase of the concentration of miR-203, the color of the system is gradually changed from light wine red to purple to light blue (figure 4), which shows that the increase of the concentration of miR-203 improves the aggregation degree of gold nanoparticles, and the color of the system is different.
(3) Detection of miR-203: and (3) extracting RNA in the sample to be detected, measuring the ultraviolet absorption spectrum of the incubated solution by adopting the method in the step (1), and substituting the ultraviolet absorption spectrum into the working curve to obtain the concentration of miR-203 in the sample to be detected.
The minimum concentration of miR-203 detected by adopting the probe colorimetric method of gold nanoparticle-DNA hybridization chain reaction is 1.0 multiplied by 10-11M。
Example 3: selective investigation of miR-203 detection method
Preparation of miR-203, miR-21 (nucleic acid sequence: 5'-UAG CUU AUC AGA CUG AUG UUG A-3') and miR-16 (nucleic acid sequence: 5'-UAG CAG CAC GUA AAU AUU GGC G-3') solutions: sterilizing PBS buffer solution (pH 7.4) containing 1 ‰ (volume ratio) DEPC water, and preparing into 1.0 × 10-10M miR-203、2.0×10-9M miR-21 and 2.0X 10-9M miR-16 solution.
100 μ L of carboxyl magnetic microsphere-DNA 1/2 complex and 100 μ L of Lmur-203 solution (1.0X 10)-10M) or miR-21 solution (1.0X 10)-9M) or miR-16 solution (1.0X 10)-9M), uniformly mixing, carrying out magnetic separation after reacting for 2H at 25 ℃, sucking supernatant, then adding 100 mu L of gold nanoparticle-H1 compound and 100 mu L of gold nanoparticle-H2 compound into the supernatant, incubating for 4H, and recording the ultraviolet absorption peak ratio at 620nm and 520 nm. As shown in FIG. 5, only the ultraviolet spectrum of the miR-203 solution on the gold nanoparticles is obviously changed, and the color of the solution is also changed.Although the concentration of the miR-21 solution and the miR-16 solution is 1 order of magnitude higher than that of the miR-203 solution, the ultraviolet absorption peak ratio is still not obviously changed, so that the method has good selectivity on the miR-203.
Example 4: intracellular miR-203 concentration detection
Extraction of RNA from cells: at 37 deg.C, 5% CO2In a humid environment of (1), MCF-7 cells were cultured in a DMEM medium containing 10% fetal bovine serum and 1% double antibody, and 1.0X 10 cells were counted on a hemocytometer4MCF-7 cells are washed twice with PBS (10mM, pH 7.4), MCF-7 cells are placed in a circulation mode under the environment of 37 ℃ and liquid nitrogen for 3 times within 3min, 300 mu L of cell lysis solution is added, then 40 mu L of chloroform and 200 mu L of PBS (10mM, pH 7.4) are added to the cells and stirred vigorously for 15s, then the cells are placed at room temperature for 3min, the mixed solution is centrifuged at 12000 for 15min, the supernatant is collected, then 100 mu L of isopropanol is added to the supernatant, the mixture is placed at room temperature for 10min after being mixed uniformly, the mixture is centrifuged at 13000 for 10min to precipitate RNA, and finally the obtained RNA is redispersed in DEPC-treated PBS buffer solution (pH 7.4).
Detecting miR-203 in cells by a colorimetric probe method: uniformly mixing 100 mu L of carboxyl magnetic microsphere-DNA 1/2 compound with 100 mu L of LRNA solution extracted from cells, carrying out magnetic separation after reacting for 2H at 25 ℃, sucking supernatant, then adding 100 mu L of gold nanoparticle-H1 compound and 100 mu L of gold nanoparticle-H2 compound into the supernatant, recording the color change of the solution after incubating for 4H, and detecting miR-203 by a colorimetric method. And measuring an ultraviolet absorption spectrum, calculating the peak ratio of 620nm to 520nm, and calculating according to a working curve chart drawn in the figure 4 to obtain that the concentration of miR-203 in the cell is 460 copies/cell.
Example 5: application of probe based on gold nanoparticle-DNA hybrid chain reaction in miR-21 detection
1. Test materials:
the nucleotide sequence of miR-21 is as follows:
5’-GUG AAA UGU UUA GGA CCA CUA G-3’。
the composition and preparation method of the probe are the same as example 1, wherein:
the sequences of H1 and H2 are as follows:
H1：5’-SH-GCG ATT CCT AGG TTG AGC CCA GGG TTT TTT CACAGT CCC TGG GCT CAA CCT AGG-3’；(SEQ ID NO.1)
H2：5’-CCC TGG GCT CAA CCT AGG AAT CGC TTT TTT CCT AGG TTGAGC CCA GGG ACT GTG-SH-3’；(SEQ ID NO.2)；
the sequences of DNA1 'and DNA 2' are as follows:
DNA1’：5’-GGG T CAA CAT CAG TCT GAT AAG CTA-NH2-3’；(SEQ ID NO.5)
DNA2’：5’-CTG ATG TTG A CCC TGG GCT CAA CCT AGG AAT CGC-3’；(SEQ ID NO.6)。
2. the detection method comprises the following steps:
the specific method comprises the following steps:
(1) preparing miR-21 working solution: preparing miR-21 working solution: sterilizing a PBS buffer solution (pH 7.4) containing 1 per mill (volume ratio) of DEPC water, and preparing miR-21 solutions with different concentrations by using the PBS buffer solution.
(2) Drawing a working curve: 100 mu L of carboxyl magnetic microsphere-DNA 1/2 compound and 100 mu L of miR-21 solution (0, 1.0X 10) with different concentrations-11M，5.0×10-11M，1.0×10-10M，3.0×10-10M) are mixed evenly, after reaction for 2H at 25 ℃, magnetic separation is carried out, supernatant is absorbed, then 100 mu L of gold nanoparticle-H1 compound and 100 mu L of gold nanoparticle-H2 compound are added into the supernatant, the color change of the solution is recorded after incubation for 4H, and ultraviolet absorption spectrum is measured. And drawing a working curve graph by taking the concentration of miR-21 as an abscissa and taking the ultraviolet absorption peak ratio at 620nm and 520nm as an ordinate. As can be seen from FIG. 6, with the increase of the concentration of miR-21, the ultraviolet absorption peak ratios at 620nm and 520nm are correspondingly changed. The working curve equation for detecting miR-21 by the method is that Y is 0.025X +0.359(Y represents the ultraviolet absorption peak value ratio at 620nm and 520nm, X represents the concentration of miR-21, and the unit is 10-10M, R ═ 0.99), the concentration range of miR-21 was 1.0 × 10-11M-3.0×10-10And M. With the increase of the concentration of miR-21, the system color is observed to be gradually changed from light wine red to purple and then to lightBlue, which shows that the concentration of miR-21 is increased, the aggregation degree of gold nanoparticles is improved, and the colors of the systems are different.
(3) Detection of miR-21: and (3) extracting RNA in the sample to be detected, measuring the ultraviolet absorption spectrum of the incubated solution by adopting the method in the step (1), and substituting the ultraviolet absorption spectrum into the working curve to obtain the concentration of miR-21 in the sample to be detected.
Example 6: detection of intracellular miR-21 concentration
Extraction of RNA from cells: at 37 deg.C, 5% CO2In a humid environment of (1), MCF-7 cells were cultured in a DMEM medium containing 10% fetal bovine serum and 1% double antibody, and 1.0X 10 cells were counted on a hemocytometer4MCF-7 cells are washed twice with PBS (10mM, pH 7.4), MCF-7 cells are placed in a circulation mode under the environment of 37 ℃ and liquid nitrogen for 3 times within 3min, 300 mu L of cell lysis solution is added, then 40 mu L of chloroform and 200 mu L of PBS (10mM, pH 7.4) are added to the cells and stirred vigorously for 15s, then the cells are placed at room temperature for 3min, the mixed solution is centrifuged at 12000 for 15min, the supernatant is collected, then 100 mu L of isopropanol is added to the supernatant, the mixture is placed at room temperature for 10min after being mixed uniformly, the mixture is centrifuged at 13000 for 10min to precipitate RNA, and finally the obtained RNA is redispersed in DEPC-treated PBS buffer solution (pH 7.4).
Detecting miR-21 in cells by a colorimetric probe method: uniformly mixing 100 mu L of carboxyl magnetic microsphere-DNA 1/2 compound with 100 mu L of LRNA solution extracted from cells, carrying out magnetic separation after reacting for 2H at 25 ℃, sucking supernatant, then adding 100 mu L of gold nanoparticle-H1 compound and 100 mu L of gold nanoparticle-H2 compound into the supernatant, recording the color change of the solution after incubating for 4H, and detecting miR-21 by a colorimetric method. And measuring an ultraviolet absorption spectrum, calculating the peak ratio of 620nm to 520nm, and calculating according to a working curve chart drawn in figure 6 to obtain the concentration of miR-21 in the cell of 7600 copies/cell.
Example 7: application of probe of the invention in detecting ATP
(1) mu.L of DNA3 was mixed with 100. mu.L of ATP solution (0, 1.0X 10)-8M，5.0×10-8M，1.0×10-7M，5.0×10-7M) are mixed evenly, after reaction for 1H at 25 ℃, 100 mu L of gold nanoparticle-H1 compound and 100 mu L of gold nanoparticle-H2 compound are added, and the color change of the solution is recorded after incubation for 4H. As can be seen from FIG. 7A, the color of the system gradually changed from light wine red to purple and then to light blue with the increase of ATP concentration (FIG. 7A), which shows that the increase of ATP concentration increases the aggregation degree of gold nanoparticles and the color of the system is different. And drawing a working curve by taking the ATP concentration as an abscissa and the ultraviolet absorption peak ratio at 620nm and 520nm as an ordinate. As can be seen from FIG. 7B, the UV absorption peak ratios at 620nm and 520nm change with increasing ATP concentration. The work curve equation of the method for detecting ATP is that Y is 1.62X +0.17(Y represents the ultraviolet absorption peak value ratio at 620nm and 520nm, X represents the ATP concentration, and the unit is 10-6M, R ═ 0.99), the ATP concentration range was 1.0 × 10-8M-5.0×10-7M。
(2) Detection of ATP: extracting ATP in the sample to be detected, measuring the ultraviolet absorption spectrum of the incubated solution by adopting the method in the step (1), and substituting the ultraviolet absorption spectrum into the working curve to obtain the concentration of ATP in the sample to be detected.
The sequence of DNA3 is as follows:
5’-CCC AGG T CCC TGG GCT CAA CCT AGG AAT CGC GGG ACC TGG GGG AGT ATT GCG GAG GAA GGT-3’。
example 8: detection of ATP concentration in cell disruption solution
At 37 deg.C, 5% CO2In a humid environment of (1%), MCF-7 cells were cultured in a DMEM medium containing 10% fetal bovine serum and 1% double antibody. MCF-7 cells were washed twice with PBS (10mM, pH 7.4) and sonicated in an ice bath to give cell lysates. Mixing 100 μ L DNA3 and 100 μ L cell disruption solution uniformly, reacting at 25 deg.C for 1H, adding 100 μ L gold nanoparticle-H1 complex and 100 μ L gold nanoparticle-H2 complex, incubating for 4H, measuring ultraviolet absorption spectrum, calculating peak ratio at 620nm and 520nm, and calculating according to the working curve chart drawn in FIG. 7B to obtain cell disruption solution with ATP concentration of 3.2 × 10-6M。
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> Linyi university
<120> method for detecting miRNA and/or aptamer-containing target molecule, and detection probe
<130> 2017
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 54
<212> DNA
<213> Artificial sequence
<400> 1
gcgattccta ggttgagccc agggtttttt cacagtccct gggctcaacc tagg 54
<210> 2
<211> 54
<212> DNA
<213> Artificial sequence
<400> 2
ccctgggctc aacctaggaa tcgctttttt cctaggttga gcccagggac tgtg 54
<210> 3
<211> 25
<212> DNA
<213> Artificial sequence
<400> 3
gggctagtgg tcctaaacat ttcac 25
<210> 4
<211> 34
<212> DNA
<213> Artificial sequence
<400> 4
ggaccactag ccctgggctc aacctaggaa tcgc 34
<210> 5
<211> 25
<212> DNA
<213> Artificial sequence
<400> 5
gggtcaacat cagtctgata agcta 25
<210> 6
<211> 34
<212> DNA
<213> Artificial sequence
<400> 6
ctgatgttga ccctgggctc aacctaggaa tcgc 34
<210> 7
<211> 61
<212> DNA
<213> Artificial sequence
<400> 7
cccaggtccc tgggctcaac ctaggaatcg cgggacctgg gggagtattg cggaggaagg 60
t 61

Claims (7)

1. A method for detecting miRNA or other target molecules with aptamers by using a probe not used for disease diagnosis,
the probe includes: the gold nanoparticle-H1 complex, the gold nanoparticle-H2 complex and the target recognition complex;
wherein the gold nanoparticle-H1 complex and the gold nanoparticle-H2 complex are formed by combining the gold nanoparticles with the neck ring DNAs H1 and H2 respectively;
the target recognition complex is a carboxyl magnetic microsphere-DNA 1/2 complex for recognizing miRNA or DNA3 for recognizing target molecules; wherein, DNA1 and DNA2 are partially complementary;
the sequence of the neck ring DNA H1 is shown as SEQ ID NO.1, and the sequence of H2 is shown as SEQ ID NO. 2;
the preparation method of the carboxyl magnetic microsphere-DNA 1/2 compound comprises the following steps: activating the magnetic beads modified by carboxyl, adding DNA1 into the activated magnetic beads, reacting for 3-5h, and carrying out magnetic separation on the obtained compound to obtain a carboxyl magnetic microsphere-DNA 1 compound; then adding DNA2, reacting for 1-3h, and performing magnetic separation to obtain carboxyl magnetic microsphere-DNA 1/2 compound;
the activating treatment comprises the following steps: adding N-hydroxysuccinimide sodium salt and 1M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride into the carboxyl modified magnetic beads, reacting at room temperature, performing magnetic separation on the mixture, and washing with deionized water;
the preparation method of the gold nanoparticle-H1 compound and the gold nanoparticle-H2 compound comprises the following steps: respectively incubating the neck ring DNA H1 and H2 and the gold nanoparticles in a buffer solution, reacting for 5-7H at room temperature, and adding a small amount of NaCl for multiple times in the reaction process to make the concentration of NaCl reach 50 mM; separating by agarose gel electrophoresis, and dialyzing the separated strip in a dialysis membrane to obtain the final product;
the buffer is 0.5 xTBE buffer, the composition of the 0.5 xTBE buffer is 89mM Tris, 89mM boric acid and 2mM EDTA, and the pH is 8.0;
the DNA3 is a cervical loop structure comprising a nucleic acid aptamer sequence capable of specifically binding to a target molecule to be detected;
the detection steps are as follows:
(1) respectively mixing the target identification compound with target solutions to be detected with a series of concentration gradients, reacting, adding the gold nanoparticle-H1 compound and the gold nanoparticle-H2 compound into the reacted solution, incubating for 3-5H, measuring the ultraviolet absorption spectrum of the incubated solution, and constructing a working curve between the concentration of the target to be detected and the ultraviolet absorption peak;
(2) and (3) extracting a target object in the sample to be detected, measuring the ultraviolet absorption spectrum of the incubated solution by adopting the method in the step (1), and substituting the ultraviolet absorption spectrum into the working curve to obtain the concentration of the target object in the sample to be detected.
2. The detection method according to claim 1, wherein the miRNA is miR-203 or miR-21; the target molecule is ATP, thrombin, lysozyme or epidermal growth factor receptor.
3. The detection method of claim 2, wherein when the miRNA is miR-203, the probe comprises a DNA1 sequence shown in SEQ ID No.3 and a DNA2 sequence shown in SEQ ID No. 4.
4. The detection method of claim 2, wherein when the miRNA is miR-21, the probe comprises a DNA1 sequence shown in SEQ ID No.5 and a DNA2 sequence shown in SEQ ID No. 6.
5. The assay of claim 2 wherein, when the target molecule is ATP, the sequence of DNA3 is shown in SEQ ID No. 7.
6. The detection method according to claim 1, wherein the gold nanoparticles are prepared by a method comprising: adding ice sodium borohydride into a mixed solution of chloroauric acid and trisodium citrate, stirring until the solution becomes pink, and reacting for 2-5h to obtain the sodium hydrogen bromide.
7. The method of claim 6, wherein the mixed solution of chloroauric acid and trisodium citrate has a chloroauric acid concentration of 0.25mM, trisodium citrate concentration of 0.25mM, and sodium borohydride ice concentration of 0.1M.
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