CN106947811B - Method, probe set and kit for detecting miRNAs-21 - Google Patents
Method, probe set and kit for detecting miRNAs-21 Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology, and particularly discloses a method, a probe set and a kit for detecting miRNAs-21. In the method for detecting miRNAs-21, the single-stranded DNA of the modified gold nanorod and the single-stranded DNA of the modified polydiacetylene nanotube are hybridized and paired to form the polydiacetylene nanotube modified by the gold nanorod, so that the end light waveguide of the polydiacetylene nanotube is subjected to fluorescence quenching, and after the polydiacetylene nanotube is mixed with the H1 probe, the H2 probe and a sample to be detected, the miRNA-21 can catalyze the self-assembly of the hairpin probe to form a double-stranded structure, and single-stranded substitution is carried out through the toehold, so that the end light waveguide fluorescence of the polydiacetylene nanotube is recovered, and the content of the miRNAs-21 in the sample to be detected can be detected according to the fluorescence change intensity. Experiments show that the detection method of the invention has high sensitivity and selectivity on miRNA-21, the detection process is simple, convenient, sensitive and rapid, and the detection result is accurate.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a method, a probe set and a kit for detecting miRNAs-21.
Background
MicroRNAs (miRNAs) are non-coding single-stranded small molecular RNAs consisting of 18-24 basic groups, play a role in negative regulation and control of gene expression at the level after transcription, and participate in important biological processes such as cell growth, development, apoptosis and the like. Recent medical studies have shown that the expression level of miRNA, either increased or decreased, is in some way inseparable linked to major human diseases such as cancer. This allows mirnas to serve as tumor markers to monitor the development of cancer early.
The encoding gene of miRNA-21 is located at 17q23.2, namely a vacuole membrane protein gene (VMP 1) encoding region and a tenth intron of VMP1 gene. VMP1 is also known as transmembrane protein 49 (TMEM-49). miRNA-21 is highly expressed in various cancers, such as: pancreatic cancer, gastric cancer, liver cancer, breast cancer, and the like. Therefore, miRNA-21 is a recognized oncogenic small RNA, and the occurrence of cancer can be effectively monitored by quantitatively detecting miRNA-21.
Many methods for detecting mirnas have been developed, such as Northern blotting techniques, microarray chips (microarray), real-time reverse transcription polymerase chain reaction, electrochemical methods, fluorescence analysis, electrochemiluminescence, colorimetric analysis, and the like. However, the above methods have limitations such as low detection sensitivity, lack of sufficient analyte, poor specificity, high time and labor consumption, and often require expensive and cumbersome tools and skilled professionals, which limits their potential applications in clinical diagnosis.
Fluorescence detection methods are gaining more and more attention at present due to their high sensitivity and simple operation. However, current fluorescence sensors have some special limitations, including small probe size, low stability, photobleaching, overlap with background fluorescence spectra, low abundance and high sequence similarity, among others. Therefore, the development of a method for rapidly detecting miRNA-21 with high selectivity and high sensitivity is of great significance.
Disclosure of Invention
In view of the above, the present invention provides a method, a probe set and a kit for detecting miRNAs-21, which are directed to the deficiencies of the prior art. The method for detecting the miRNAs-21 can quickly detect the content of the miRNAs-21 in a sample to be detected with high selectivity and high sensitivity.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a method for detecting miRNAs-21 comprises the steps of mixing a gold nanorod modified polydiacetylene microtube, an H1 probe, an H2 probe and a sample to be detected, and detecting the change of the fluorescence brightness of an end light waveguide of the gold nanorod modified polydiacetylene microtube;
the gold nanorods and the polydiacetylene nanotubes are respectively modified by single-stranded DNA, and the single-stranded DNA of the modified gold nanorods is complementary with the single-stranded DNA base of the modified polydiacetylene nanotubes; the H1 probe and the H2 probe are hairpin probes, can self-assemble to form a double-chain structure in the presence of miRNAs-21, and then generate a chain displacement reaction with the single-chain DNA of the modified gold nanorod through the toehold.
Preferably, the sequence of the single-stranded DNA of the modified gold nanorod is shown as SEQ ID NO: 1 is shown in the specification; the sequence of the single-stranded DNA of the modified polydiacetylene nanotube is shown as SEQ ID NO: 2, respectively.
Preferably, the sequence of the H1 probe is as shown in SEQ ID NO: 3 is shown in the specification; the sequence of the H2 probe is shown as SEQ ID NO: 4, respectively.
Preferably, the gold nanorod modified polydiacetylene microtubes are fixed on a hydrophobic sheet.
Preferably, the sample to be tested is serum.
The invention also provides a probe set for detecting the miRNAs-21, which comprises an H1 probe and an H2 probe, wherein the H1 probe and the H2 probe are hairpin probes, and can be self-assembled to form a double-stranded structure in the presence of the miRNAs-21, and then generate a strand displacement reaction with the single-stranded DNA of the modified gold nanorod through the toehold.
Preferably, the sequence of the H1 probe is as shown in SEQ ID NO: 3 is shown in the specification; the sequence of the H2 probe is shown as SEQ ID NO: 4, respectively.
The invention also provides a kit for detecting miRNAs-21, which is characterized by comprising the probe set.
Preferably, the kit further comprises gold nanorod modified polydiacetylene nanotubes, wherein the gold nanorods and the polydiacetylene nanotubes are modified by single-stranded DNAs respectively, and the single-stranded DNAs of the modified gold nanorods are complementary to the single-stranded DNAs of the modified polydiacetylene nanotubes.
Preferably, the gold nanorod modified polydiacetylene microtubes are fixed on a hydrophobic sheet.
Preferably, the sequence of the single-stranded DNA of the modified gold nanorod is shown as SEQ ID NO: 1 is shown in the specification; the sequence of the single-stranded DNA of the modified polydiacetylene nanotube is shown as SEQ ID NO: 2, respectively.
The invention also provides an application of the probe set and the kit in preparation of cancer diagnosis products.
According to the technical scheme, the invention provides a method, a probe set and a kit for detecting miRNAs-21. The method for detecting the miRNAs-21 comprises the steps of mixing a gold nanorod modified polydiacetylene microtube, an H1 probe, an H2 probe and a sample to be detected, and detecting the change of the fluorescence brightness of an end light waveguide of the gold nanorod modified polydiacetylene microtube; the gold nanorods and the polydiacetylene nanotubes are respectively modified by single-stranded DNA, and the single-stranded DNA of the modified gold nanorods is complementary with the single-stranded DNA base of the modified polydiacetylene nanotubes; the H1 probe and the H2 probe are hairpin probes, can self-assemble to form a double-chain structure in the presence of miRNAs-21, and then generate a chain displacement reaction with the single-chain DNA of the modified gold nanorod through the toehold. In the method for detecting miRNAs-21, single-stranded DNA of a modified gold nanorod and single-stranded DNA of a modified polydiacetylene nanotube are hybridized and paired to form a gold nanorod modified polydiacetylene nanotube, the end of the polydiacetylene nanotube is subjected to optical waveguide fluorescence quenching, the gold nanorod modified polydiacetylene nanotube is mixed with an H1 probe, an H2 probe and a sample to be detected, a target molecule miRNA-21 in the sample to be detected can catalyze a hairpin probe to self-assemble to form a double-chain structure, single-stranded substitution is carried out on the single-stranded DNA of the modified gold nanorod through toehold, the gold nanorod is separated from the polydiacetylene nanotube, so that the end of the polydiacetylene nanotube is subjected to optical waveguide fluorescence recovery from quenching, and the content of the miRNAs-21 in the sample to be detected can be detected according to the fluorescence change intensity. Experiments show that the detection method of the invention has high sensitivity and selectivity on miRNA-21, the detection process is simple, convenient, sensitive and rapid, and the detection result is accurate.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a schematic diagram showing a specific reaction principle of optical waveguide fluorescence recovery of a polydiacetylene microtube tip;
FIG. 2 shows a schematic diagram of the reaction process of the single-stranded DNA modified microtube of example 1 (a) and the infrared spectrum (b) and Raman spectrum (c) of the modified microtube; wherein in the figure (b), i represents the infrared spectrogram of the polydiacetylene micro-tube, and ii represents the infrared spectrogram of the double-bond modified polydiacetylene micro-tube obtained after the reaction of the polydiacetylene micro-tube and allyl glycidyl ether, and 1130cm is-1Stretching vibration of C-O-C bond, 923cm-1Stretching vibration representing a C ═ CH bond; in the figure (c), i represents a Raman spectrum of the double-bond modified polydiacetylene micro-tube, ii represents a Raman spectrum of the single-strand DNA modified polydiacetylene micro-tube, and the added peaks are characteristic peaks of DNA;
FIG. 3 shows the reaction process of example 3 hybridization of single-stranded DNA modified gold nanorods with single-stranded DNA modified polydiacetylene nanotubes (a), and the XPS spectra (b) and optical waveguide fluorescence change (c) of the resulting gold nanorod modified polydiacetylene nanotubes; wherein in the graph (b), i represents an XPS spectrogram of the single-stranded DNA modified polydiacetylene micro-tube, ii represents an XPS spectrogram obtained by hybridizing a single-stranded DNA modified gold nanorod with the single-stranded DNA modified polydiacetylene micro-tube, and gold elements are arranged on the surface of the micro-tube after hybridization, which indicates that the hybridization is successful; the graph (c) shows that the fluorescence spectrum change graph of the end of the micrometer tube is continuously reduced along with the continuous increase of the concentration of the gold nanorods modified by the single-stranded DNA after the gold nanorods modified by the single-stranded DNA are hybridized with the polydiacetylene micrometer tube modified by the single-stranded DNA;
FIG. 4 is a schematic diagram of the concentration and enrichment effect of the gold nanorod-modified polydiacetylene nanotube on a hydrophobic plate in example 4, a schematic diagram of miRNA-21 detection by the gold nanorod-modified polydiacetylene nanotube (b), and optical waveguide fluorescence change of the gold nanorod-modified polydiacetylene nanotube after reaction of miRNA-21 with different concentrations (c); wherein in the diagram (a), the reaction is carried out on the hydrophobic sheet, and the reactants are concentrated on the surface of the micron tube to react according to the concentration and enrichment effect; in the figure (b), in a gold nanorod-microtube system, after excitation by 532nm light, the fluorescence of the end of the microtube is weak, and after the detected miRNA-21 is added, the fluorescence brightness of the end of the microtube is enhanced; adding miRNA-21 solutions with different concentrations into a gold nanorod-micron tube system, wherein the concentration is from 10fM to 50pM, the fluorescence of the end of the micron tube is gradually recovered, and the change of the fluorescence of the end of the micron tube is in proportional relation with the concentration of miRNA-21 in the concentration range from 10fM to 100 fM;
FIG. 5 shows a graph (a) of fluorescence change of miRNA-21 optical waveguides and a graph (b) of detection limit for detecting different concentrations of miRNA-21 optical waveguides in the gold nanorod-modified polydiacetylene microtube of example 5; adding miRNA-21 solutions with different concentrations into the gold nanorod-micron tube body system, wherein the concentration is from 10fM to 50pM, the fluorescence of the end of the micron tube is gradually recovered, and the fluorescence change of the end of the micron tube is in a proportional relation with the concentration of miRNA-21 in the concentration range from 10fM to 100 fM;
FIG. 6 is a graph showing the optical waveguide fluorescence changes of the gold nanorod-modified polydiacetylene microtubes of example 6 for detecting different concentrations of miRNA-21, single base mismatch miRNA-21, three base mismatch miRNA-21, miRNA-144, and miRNA-199 a;
FIG. 7 is a graph showing the change of optical waveguide fluorescence of the gold nanorod-modified polydiacetylene microtube in example 7 in the detection of serum of healthy people, serum of pancreatic cancer patients, serum of breast cancer patients and serum of gastric cancer patients.
Detailed Description
The invention discloses a method, a probe set and a kit for detecting miRNAs-21. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
In the method for detecting miRNAs-21, the single-stranded DNA of the modified gold nanorod and the single-stranded DNA of the modified polydiacetylene nanotube are matched through hybridization to form the polydiacetylene nanotube modified by the gold nanorod, and the end of the polydiacetylene nanotube is subjected to optical waveguide fluorescence quenching; after the polydiacetylene microtube modified by the gold nanorods, the H1 probe, the H2 probe and a sample to be detected are mixed, target molecule miRNA-21 in the sample to be detected can catalyze the hairpin probe to self-assemble to form a double-chain structure, single-chain substitution is carried out on single-chain DNA of the modified gold nanorods through the toehold, and the gold nanorods are separated from the polydiacetylene microtube, so that the fluorescence of the optical waveguide at the end of the polydiacetylene microtube is recovered from quenching. The content of the miRNAs-21 in the sample to be detected can be detected according to the fluorescence change intensity.
The specific reaction principle of the optical waveguide fluorescence recovery of the polydiacetylene microtube end is shown in figure 1. miRNA-21 opens a hairpin structure of the H1 probe through a red toehold end, and is hybridized with an H1 probe to form H1-miR-21. The H2 probe replaces miRNA-21 in H1-miR-21 through a blue toehold end, and H1 and H2 are hybridized to form H1-H2. In the process, the miRNA-21 can be used as a catalyst, so that the reaction is circularly generated, and meanwhile, the miRNA-21 is not consumed, thereby improving the sensitivity of the micro-tube sensor for detecting Target and reducing the reaction limit.
The polydiacetylene micron tube disclosed by the invention adopts a polydiacetylene compound material, can integrate detection and display into a whole, and is an ideal sensor material. The invention prepares the one-dimensional polydiacetylene nanotube by an assembly method, constructs a novel biosensor with high sensitivity and high selectivity, and is used for detecting miRNA in a complex biological environment.
The method for preparing the amino-diacetylene monomer of the polydiacetylene nanotube comprises the following steps: dissolving diacetylene molecules, 1.2 times of equivalent of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 1.2 times of equivalent of N-hydroxysuccinimide (NHS) in 30mL of refined dichloromethane, carrying out magneton stirring reaction for 6 hours at 25 ℃, carrying out rotary evaporation and extraction on the obtained product, adding 1.1 times of equivalent of ethylenediamine liquid, dissolving the mixture in a dichloromethane solvent, carrying out magneton stirring reaction for 1 hour at 30 ℃, and finally carrying out extraction and rotary evaporation to obtain the amino-diyne monomer.
The preparation method of the polydiacetylene microtube comprises the following steps: dissolving 0.0056g of aminodiyne and 0.00075g of octadecylamine-substituted melamine in 2mL of absolute ethanol solution, pouring the solution into 300mL of ultrapure water at 75 ℃, carrying out ultrasonic treatment for 60min, naturally cooling to room temperature in a dark place, and putting the dark place in a refrigerator at 4 ℃ overnight to obtain the composite vesicle. And respectively adding 10ml of vesicle solution and 10 mu L of lead nitrate solution into a weighing bottle, placing the weighing bottle in a ventilated place at room temperature in an open manner, and separating out white filamentous substances after two to three weeks to obtain the polydiacetylene micron tube.
In some embodiments, the method for preparing the single-stranded DNA modified polydiacetylene nanotube of the invention specifically comprises: taking the polydiacetylene microtube out of the weighing bottle, and placing the microtube under an ultraviolet tube with the wavelength of 254nm for irradiating for 10 minutes to ensure that the diacetylene is polymerized into a blue phase. The diacetylene in the blue phase was heated at 80 ℃ for 10 minutes to change it to the red phase. And (3) diluting 20 mu L of allyl glycidyl ether to 10mL, adding the diluted allyl glycidyl ether into the microtube, and reacting at 30 ℃ overnight to obtain the double-bond modified microtube. The single-stranded microtubes were mixed with 200. mu.L of thiol-modified single-stranded DNA and 2959 photoinitiator (n (2959): n (DNA): 1:100) and reacted under 365nm light for 6 hours to give single-stranded DNA-modified microtubes.
Further, in some embodiments, the method for preparing the single-stranded DNA modified gold nanorods specifically comprises: preparing the gold nanorods by a seed growth method, and incubating the gold nanorods with the sulfydryl modified single-stranded DNA to obtain the single-stranded DNA modified gold nanorods.
In some embodiments, the sequence of the single-stranded DNA of the modified gold nanorods is as set forth in SEQ ID NO: 1 is shown in the specification; the sequence of the single-stranded DNA of the modified polydiacetylene nanotube is shown as SEQ ID NO: 2, respectively.
In some embodiments, the sequence of the H1 probe is as set forth in SEQ ID NO: 3 is shown in the specification; the sequence of the H2 probe is shown as SEQ ID NO: 4, respectively.
In some embodiments, the gold nanorod-modified polydiacetylene microtubes are immobilized on a hydrophobic sheet. The hydrophobic sheet has a concentration and enrichment effect, so that liquid drops formed by mixing the H1 probe, the H2 probe and a sample to be detected can be gathered on the surface of the gold nanorod-modified polydiacetylene microtube, the reaction sensitivity is improved, and the reaction limit is reduced.
In light of the present invention, it will be understood by those skilled in the art that the test sample of the present invention includes, but is not limited to, serum.
The invention also provides a probe set for detecting the miRNAs-21, which comprises an H1 probe and an H2 probe, wherein the H1 probe and the H2 probe are hairpin probes, and can be self-assembled to form a double-stranded structure in the presence of the miRNAs-21, and then generate a strand displacement reaction with the single-stranded DNA of the modified gold nanorod through the toehold.
In some embodiments, the sequence of the H1 probe is as set forth in SEQ ID NO: 3 is shown in the specification; the sequence of the H2 probe is shown as SEQ ID NO: 4, respectively.
The invention also provides a kit for detecting miRNAs-21, which comprises the probe set.
In some embodiments, the kit further comprises gold nanorod-modified polydiacetylene nanotubes, wherein the gold nanorod and the polydiacetylene nanotubes are respectively modified by single-stranded DNA, and the single-stranded DNA of the modified gold nanorod is complementary to the single-stranded DNA of the modified polydiacetylene nanotubes in base.
In some embodiments, the sequence of the single-stranded DNA of the modified gold nanorods is as set forth in SEQ ID NO: 1 is shown in the specification; the sequence of the single-stranded DNA of the modified polydiacetylene nanotube is shown as SEQ ID NO: 2, respectively.
In a certain embodiment, miRNA-21, single-base mismatching miRNA-21, three-base mismatching miRNA-21, miRNA-144 and miRNA-199a are respectively dripped onto different single polydiacetylene microtubes, and then an H1 probe and an H2 probe are added, so that the result shows that the fluorescence change of the end light waveguide of the polydiacetylene microtube is strong only when the miRNA-21 is added, and the detection of miRNA-21 by the polydiacetylene microtubes has good selectivity.
In one embodiment, the H1 probe, the H2 probe, the serum of a healthy person, the serum of a pancreatic cancer patient, the serum of a breast cancer patient or the serum of a gastric cancer patient are dripped on a single micron tube, and the result shows that the miRNA-21 signal expression of the serum of a patient suffering from cancer is obviously higher than that of the serum of the healthy person.
Therefore, the invention also provides the application of the probe set and the kit in preparing cancer diagnosis products.
Compared with the prior art, the invention has at least one of the following advantages and effects: the miRNA-21 is detected with high sensitivity and selectivity, the detection process is simple, convenient, sensitive and rapid, the detection result is accurate, and the detection means is simple.
In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.
Example 1: preparation of Single-stranded DNA-modified microtubes
And (3) diluting 20 mu L of allyl glycidyl ether to 10mL, adding the diluted allyl glycidyl ether into a polydiacetylene micron tube, and reacting at 30 ℃ overnight to obtain the double-bond modified polydiacetylene micron tube. And uniformly mixing a single polydiacetylene micro-tube with 200 mu L of 5' -end sulfhydryl modified single-stranded DNA (ssDNA1 sequence is shown as SEQ ID NO: 1) and 2959 photoinitiator (n (2959): n (ssDNA1) ═ 1:100), and reacting for 6 hours under 365nm illumination to obtain the single-stranded DNA modified polydiacetylene micro-tube. Wherein the micrometer tube infrared spectrum of the single-stranded DNA modified polydiacetylene micrometer tube is shown in figure 2b, and the Raman spectrum of the single-stranded DNA modified polydiacetylene micrometer tube is shown in figure 2 c.
Example 2: single-stranded DNA modified gold nanorod
Preparing a gold nanorod by a seed growth method, and incubating the gold nanorod with 5' -end sulfhydryl modified single-stranded DNA (ssDNA2 sequence is shown as SEQ ID NO: 2) after purification (the molar ratio of the gold nanorod to the DNA is 1:25600) to obtain the single-stranded DNA modified gold nanorod. The specific operation is as follows: to 7.5mL of a 0.1M solution of cetyltrimethylammonium bromide (CTAB) was first added 250. mu.L of 0.01M chloroauric acid and stirred well. Then 600. mu.L of a 0.01M solution of sodium borohydride (sodium borohydride was left at-20 ℃ C. for 10 minutes in advance) was added, followed by stirring for 2 minutes under mechanical stirring (400 rpm). After 2 minutes, a light brown solution, namely the gold seed solution, is formed. The gold seed solution was allowed to stand at room temperature for 1 hour. Preparation of growth solution 9.5mL of 0.1M cetyltrimethylammonium bromide and 400. mu.L of 0.01M chloroauric acid were mixed under mechanical stirring (stirring speed of 250rpm), stirred uniformly, 60. mu.L of 0.01M silver nitrate was added, and then 64. mu.L of 0.1M ascorbic acid was added to the above solution, at which time it was observed that the color of the solution changed from bright yellow to colorless transparent, indicating that the weak reducing agent ascorbic acid was presentMixing Au3+Reduction to Au+. And finally, adding 20 mu L of gold seeds into the solution, stirring for 1 minute, stopping stirring, and standing for one night to obtain the gold nanorods. The whole experimental process requires protection from light and the temperature is 25 ℃. . The prepared gold nanorods were centrifuged at 13000rcf for 30 minutes, the supernatant was removed, and the pellet was dissolved in 10mM PBS (pH 8.0, containing 0.3% (w/v) sodium dodecyl sulfate). This process was repeated three times to obtain 10mL of purified gold nanorods suspended in the above buffer solution. 10mL of an ethanol solution of polyvinylpyrrolidone (10% w/v) was then added and stirred at 40 ℃ for 18 hours. After completion of the reaction, the reaction mixture was centrifuged, and the supernatant was removed and redispersed in PBS containing 0.3% sodium lauryl sulfate. This process was repeated three times. Then, the ultraviolet absorption spectrum is tested, and the concentration of the gold nanorods is calculated according to the Lambert beer law A-Kbc. After the specific concentration of the gold nanorods is obtained, sulfhydryl modified DNA is added. The added DNA was previously treated with tris (2-carboxyethyl) phosphine for 2 hours, so that the oxidized thiol group was reduced. Then, according to the mole ratio of the DNA to the gold nanorods of 25600: 1 DNA was added, sonicated for 5 seconds and allowed to stand at room temperature for 16 hours. After the reaction is finished, salt is added for curing to weaken the electrostatic interaction between the gold nanorods and the DNA, so that more DNA reacts with the gold nanorods. The salt solution was a mixed solution of 10mM PB (pH 8.0), 300mM sodium chloride, 4mM magnesium chloride, and 0.3% sodium lauryl sulfate. Every half hour to give a final salt concentration of 150mM sodium chloride, 2mM magnesium chloride. . After 16 hours at room temperature, centrifuge 3 times at 9300rcf for 30 minutes each time. This process was repeated 3 times. And finally suspending the prepared single-stranded DNA modified gold nanorods in a buffer solution, and storing the gold nanorods in a refrigerator at 4 ℃.
Example 3: gold nanorod modified polydiacetylene microtube
And hybridizing the gold nanorods modified by the single-stranded DNA with the polydiacetylene microtubes modified by the single-stranded DNA to obtain the polydiacetylene microtubes modified by the gold nanorods. The specific operation is as follows: a single polydiacetylene nanotube is picked out by a needle tube and put into a small test tube, and the small test tube is immersed into 200 mu L of single-stranded DNA modified gold nanorod solution and is placed in a water bath environment at 90 ℃ for 5 minutes. And then the small test tube is moved out of the water bath pot to be slowly cooled to the room temperature, and the cooling time is about 1-2 h. After the reaction is finished, the polydiacetylene microtubes are picked out from the gold nanorod solution by using a needle tube, and the microtubes are slightly washed by ultrapure water for a plurality of times to remove the unadsorbed gold nanorods.
The detection of the optical waveguide fluorescence change is carried out on the polydiacetylene microtube modified by the obtained gold nanorods, and the result is shown in figure 3 c. The XPS spectrum results are shown in FIG. 3 b. The result shows that gold elements exist on the surface of the microtube after hybridization, which indicates that the hybridization is successful, and the gold nanorod-modified polydiacetylene microtube is obtained.
Example 4: detection of miRNA
The gold nanorod-modified polydiacetylene microtube obtained in example 3 was placed on a hydrophobic plate, 10. mu.L of a solution containing H1(300nM, sequence shown in SEQ ID NO: 3), H2(1000nM, sequence shown in SEQ ID NO: 4), and miRNA-21 (sequence shown in SEQ ID NO: 5) was added dropwise, the plate was left at room temperature for 2 hours, and after being washed with ultrapure water, the change in fluorescence of the optical waveguide was detected, and the result is shown in FIG. 4. The results show that the reaction is carried out on the hydrophobic plate, and the reactants are concentrated on the surface of the micron tube to react according to the concentration and enrichment effect. In the gold nanorod-microtube system, after excitation by 532nm light, the fluorescence of the end of the microtube is weak, and after the detected miRNA-21 is added, the fluorescence brightness of the end of the microtube is enhanced. The fluorescence of the ends of the microtubes gradually recovers with the gradual increase of the concentration of the miRNA-21 added. Example 5: sensitivity detection
The gold nanorod-modified polydiacetylene microtubes obtained in example 3 were placed on a hydrophobic plate, and 10. mu.L of a solution containing H1(300nM) probe, H2(1000nM) probe, and miRNA-21 was added dropwise. Wherein the concentration of miRNA-21 is increased from 10fM to 50pM, and the result of observing the change of fluorescence brightness of the end optical waveguide of the gold nanorod-modified polydiacetylene microtube is shown in figure 5 a. The miRNA-21 concentration is taken as the abscissa, the fluorescence brightness of the gold nanorod-modified polydiacetylene microtube optical waveguide is changed into the ordinate, the detection limit is calculated, and the result is shown in figure 5 b.
The results show that, in the gold nanorod-micron tube system, miRNA-21 solutions with different concentrations are added, the fluorescence of the end of the micron tube is gradually recovered from 10fM to 50pM, and the fluorescence change of the end of the micron tube is in proportion to the concentration of miRNA-21 in the concentration range from 10fM to 100 fM.
Example 6: specificity detection
10 mu of LmiRNA-21, single-base mismatching miRNA-21 (shown as SEQ ID NO: 6), three-base mismatching miRNA-21 (shown as SEQ ID NO: 7), miRNA-144 (shown as SEQ ID NO: 8) and miRNA-199a (shown as SEQ ID NO: 9) are respectively dripped on the single polydiacetylene nanotube modified by the gold nanorods obtained in different examples 3, then an H1(300nM) probe and an H2(1000nM) probe are respectively added, the mixture is placed at room temperature for 2H, after being washed by ultrapure water, the fluorescence change of the optical waveguide is detected, the specificity of the polydiacetylene nanotube modified by the gold nanorods to the miRNA-21 is observed, and the result is shown in figure 6.
The result shows that the fluorescence of the gold nanorod-modified polydiacetylene microtubes cannot be recovered after the gold nanorod-modified polydiacetylene microtubes react with single-base mismatched miRNA-21, three-base mismatched miRNA-21, miRNA-144 and miRNA-199a respectively at four concentrations. When miRNA-21 was added, fluorescence recovery of gold nanorod-modified polydiacetylene nanotubes was found. The detection method of the invention has better detection specificity to miRNA-21.
Example 7: serum detection
Serum containing healthy people, serum containing pancreatic cancer patients, serum containing breast cancer patients and serum containing gastric cancer patients are respectively dripped on a single polydiacetylene nanotube modified by the gold nanorods obtained in different examples 3, then an H1(300nM) probe and an H2(1000nM) probe are respectively added, the mixture is placed for 2H at room temperature, and after being washed by ultrapure water, the change of optical waveguide fluorescence is detected, and the result is shown in figure 7.
The results show that the signal expression of the serum of patients with three cancers is obviously higher than that of the serum of healthy people.
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<110> university of science and technology in China
<120> method, probe set and kit for detecting miRNAs-21
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Claims (4)
1. A kit for detecting miRNA-21 is characterized by comprising a probe set for detecting miRNA-21 and a gold nanorod modified polydiacetylene nanotube;
the gold nanorods and the polydiacetylene nanotubes are respectively modified by single-stranded DNA, and the single-stranded DNA of the modified gold nanorods is complementary with the single-stranded DNA base of the modified polydiacetylene nanotubes;
the probe set for detecting miRNA-21 comprises an H1 probe and an H2 probe, the H1 probe and the H2 probe are hairpin probes, the hairpin probes can be self-assembled to form a double-stranded structure in the presence of miRNA-21, and then the double-stranded structure and the single-stranded DNA of the modified gold nanorod undergo a strand displacement reaction through toehold.
2. The kit according to claim 1, wherein the gold nanorod-modified polydiacetylene microtubes are immobilized on a hydrophobic sheet; the sequence of the single-stranded DNA of the modified gold nanorod is shown as SEQ ID NO: 1 is shown in the specification; the sequence of the single-stranded DNA of the modified polydiacetylene nanotube is shown as SEQ ID NO: 2, respectively.
3. The kit of claim 1, wherein the sequence of the H1 probe is as set forth in SEQ ID NO: 3 is shown in the specification; the sequence of the H2 probe is shown as SEQ ID NO: 4, respectively.
4. Use of the kit of any one of claims 1 to 3 for the preparation of a product for cancer diagnosis.
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| CN109251964B (en) * | 2018-04-25 | 2021-09-14 | 深圳市乾康医药科技有限公司 | Circulating microRNAs detection kit, method for specifically detecting circulating microRNAs and application |
| CN109251962B (en) * | 2018-09-17 | 2020-12-25 | 中国科学技术大学 | Micron tube sensor and preparation method and application thereof |
| CN110346334B (en) * | 2018-12-27 | 2022-02-11 | 华东理工大学 | Nano-gold dual-probe system based on DNA coding circulation program |
| CN110564657A (en) * | 2019-08-27 | 2019-12-13 | 北京理工大学 | Microbial sensor for early detection of breast cancer and detection system thereof |
| CN112961906B (en) * | 2021-02-05 | 2024-01-23 | 南京邮电大学 | Gold nanorod detection probe, preparation method, detection method and application thereof |
| CN113151403B (en) * | 2021-04-06 | 2023-06-16 | 中国科学技术大学 | DNA and methylation level detection kit, preparation method and application thereof |
| CN113999910B (en) * | 2021-11-24 | 2022-08-16 | 河北翰林生物科技有限公司 | Visual detection reagent, kit and detection method for gastric cancer marker miRNA-21 |
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| CN104450909A (en) * | 2014-12-09 | 2015-03-25 | 临沂大学 | Detection method for detecting micro ribonucleic acid and corresponding kit |
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| CN104450909A (en) * | 2014-12-09 | 2015-03-25 | 临沂大学 | Detection method for detecting micro ribonucleic acid and corresponding kit |
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