CN109251962B - Micron tube sensor and preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of miRNAs detection and analysis, in particular to a micron tube sensor and a preparation method and application thereof. The invention provides a micro-tube sensor and a probe set for detecting miRNA of single base mutation, and also provides a method for detecting single base mutation, wherein the method is based on that target molecules catalyze self-assembly of hairpin probes to form a double-chain structure, BHQ3 hybridized on a micro-tube is replaced by toehold, so that optical waveguide at the end of the micro-tube is recovered from quenching, and a method for distinguishing similar miRNA under the action of a competitive probe is provided. As the reaction cycle progresses, a high signal is generated by a low concentration of the target molecule, while a very low signal is generated by the inhibited single base mutated non-target molecule, thereby achieving the target of detection in a serum environment.

Description

Micron tube sensor and preparation method and application thereof

Technical Field

The invention relates to the technical field of miRNAs detection and analysis, in particular to a micron tube sensor and a preparation method and application thereof.

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.

Single base mutations refer to variations in which one nucleic acid in DNA or RNA is replaced with another nucleic acid, and also include insertions and deletions of a single base pair. The detection of single base mutation is of great significance to early diagnosis and prevention of many diseases. At present, many methods for detecting single base mutation are developed, such as nanopore sequencing, enzyme labeling, microarray chip technology, real-time fluorescence quantitative PCR, electrochemical methods, colorimetric analysis, and the like. These methods have certain limitations, such as the inability to distinguish miRNA with small sequence difference, complex operation, low repeatability, large consumption of raw materials, low detection sensitivity, high price, etc., which limits the potential applications in clinical diagnosis.

The fluorescence detection method attracts more and more eyes due to the advantages of high sensitivity, good repeatability, safety, no toxicity and the like. However, some difficulties still exist in fluorescence detection, such as severe leakage of background signal, photobleaching, overlapping with background fluorescence spectrum, etc. Therefore, it is still a very significant challenge to develop a new fluorescent sensor system capable of detecting single base mutations rapidly, with high selectivity and high sensitivity.

Disclosure of Invention

In view of this, the technical problem to be solved by the present invention is to provide a microtube sensor, and a preparation method and an application thereof, wherein the sensor has good specificity and high sensitivity.

The micron tube sensor provided by the invention is a polydiacetylene micron tube for modifying a quenching group;

the quencher group is modified on the polydiacetylene nanotube by two complementary ssDNAs.

The quenching group is BHQ 3.

The sequences of the complementary ssDNA are shown in SEQ ID NO 1 and SEQ ID NO 2, respectively.

The quenching group is connected to the 3' end of SEQ ID NO. 2.

The polydiacetylene microtubes are sequentially connected with-CH₂-CH₂-NH-complementary ssDNA-BHQ 3.

The preparation method of the amino-diacetylene monomer for preparing the polydiacetylene microtubes comprises the following steps:

dissolving 10, 12-pentacosadiynoic acid 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 dichloromethane, and stirring at 25 ℃ for reacting for 6 hours; performing rotary evaporation and extraction on the product, adding 1.1 times of equivalent weight of ethylenediamine liquid, dissolving in a dichloromethane solvent, and performing magneton stirring reaction at 30 ℃ for 1 hour; and extracting and rotary evaporating the product to obtain the amino-diacetylene monomer.

The preparation method of the epoxy modified polydiacetylene nanotube comprises the following steps:

dissolving amino diacetylene and octadecylamine-substituted melamine in an absolute ethanol solution, then pouring the solution into ultrapure water (150 times of ethanol volume) at 75 ℃, carrying out ultrasonic treatment for 60min, then placing in a dark place, naturally cooling to room temperature, and reacting at 4 ℃ overnight (8-12 h) to obtain a composite vesicle;

mixing the composite vesicle solution with a lead nitrate solution (v: v ═ 1000:1), and reacting at room temperature for 2-3 weeks to obtain a polydiacetylene nanotube;

irradiating the polydiacetylene microtube by 254nm ultraviolet for 10 minutes, and then heating at 80 ℃ for 10 minutes to obtain a red-phase polydiacetylene microtube;

and (3) reacting ethylene glycol diglycidyl ether (volume fraction is 10%) with the red-phase polydiacetylene microtubes at 30 ℃ for overnight to obtain the epoxy-modified microtubes.

The preparation method of the micron tube sensor comprises the following steps:

reacting the polydiacetylene microtubes with ethylene glycol diglycidyl ether to obtain epoxy-modified polydiacetylene microtubes;

reacting the epoxy-modified polydiacetylene microtube with ssDNA1 modified with amino at the 5' -end to obtain a ssDNA1 modified microtube;

the microtube modified with ssDNA1 was reacted with ssDNA2 modified with BHQ3 at the 3' end to obtain a microtube sensor.

The conditions for the reaction of the epoxy-modified polydiacetylene microtubes with the 5' -end-modified amino-group ssDNA1 are as follows: the reaction is carried out for 4h at 30 ℃.

The buffer solution for the reaction of the ssDNA1 modified micron tube and the ssDNA2 of the 3' -end modified BHQ3 is Tris buffer solution; the reaction conditions were: 5min at 90 ℃; room temperature (18-30 ℃) for 2 hours.

The invention also provides a probe set comprising hairpin probe H1 and hairpin probe H2;

the hairpin probe H1 is, in order from the 5 'end to the 3' end: h1-1 sequence, H1-2 sequence, H1-3 sequence and H1-4 sequence;

the H1-1 sequence is complementary to a target sequence;

the H1-2 sequence is not consistent with or complementary to a target sequence and is a loop structure of a probe;

the H1-3 sequence is complementary with H1-1 to form a stem structure of the probe;

the H1-4 sequence is identical to the ssDNA2 sequence;

the hairpin probe H2 is, in order from the 5 'end to the 3' end: h2-1 sequence, H2-2 sequence, H2-3 sequence and H2-4 sequence;

the H2-1 sequence is a cohesive end sequence;

the H2-2 sequence is complementary to the H1-2 sequence;

the sequence of H2-3 is consistent with H1-3 and is a ring structure of the probe;

the H2-4 sequence is complementary to H2-2, forming the stem structure of the probe.

In the invention, the length of the H1-2 sequence is 12-14 bp.

The length of the H1-3 sequence is smaller than that of the H1-1 sequence, and the difference is 8 bp.

The H2-1 sequence was 7bp in length and was not complementary to any of the other sequences in hairpin probe H2.

The H2-3 sequence is as long as the H1-3 sequence.

ssDNA1 and ssDNA2 match each other. ssDNA1 is immobilized by epoxy on the microtube under the action of the terminal amino group for hybridization with ssDNA2, while BHQ3 at the end of ssDNA2 quenches the microtube fluorescence, which weakens the optical waveguide signal. In the detection process, the sequence of the target molecule catalyzes the self-assembly of the hairpin probe to form a double-chain structure, and the BHQ3 hybridized on the microtube is replaced by the toehold (H1-4 sequence), so that the optical waveguide at the end of the microtube is recovered from quenching. And can realize the differentiation of approximate miRNA under the action of competitive probe.

The probe set provided by the invention further comprises at least one pair of competition probes: hairpin probe H1 'and hairpin probe H2';

the hairpin probe H1 ' is, in order from the 5 ' end to the 3 ' end: h1 ' -1 sequence, H1 ' -2 sequence and H1 ' -3 sequence;

the H1' -1 sequence is complementary to a non-target sequence;

the H1' -2 column is not identical or complementary to a non-target sequence and is a loop structure of a probe;

the H1 '-3 sequence is complementary with H1' -1 to form a stem structure of the probe;

the hairpin probe H2 ' is, in order from the 5 ' end to the 3 ' end: h2 '-1 sequence, H2' -2 sequence, H2 '-3 sequence and H2' -4 sequence;

the H2' -1 sequence is a cohesive end sequence;

the H2 '-2 sequence is complementary to the H1' -2 sequence;

the sequence of H2 '-3 is consistent with H1' -3 and is a ring structure of the probe;

the H2 '-4 sequence is complementary to H2' -2, forming the stem structure of the probe.

In the present invention, the length of the H1' -1 sequence is 7bp, and it is not complementary to any other sequence in the hairpin probe H2.

When a sequence similar to the target sequence exists, a competitive probe is added to the reaction system. The hairpin probe H1 can replace ssDNA2 on the micron tube and hybridize with ssDNA1 to recover the micron tube waveguide; the hairpin probe H1' cannot replace ssDNA2 on the microtube due to the deletion of a part of base sequence, so that the waveguide signal cannot be recovered, the target signal is greatly different from the non-target signal, and the efficient distinction between single base mutations is realized.

The invention also provides a miRNA detection method, wherein the microtube sensor, the probe set and a sample to be detected are incubated together, and the miRNA content is obtained according to the optical waveguide fluorescence intensity.

In the invention, the detection substrate of the detection method is a hydrophobic tablet.

The buffer solution of the detection method is Tris buffer solution.

When two probes are used for detection, the molar ratio of the two probes in the probe set is 1: 4.

when competitive probes are added for detection, the molar ratio of the probes in the probe set is 1:4:0.125: 0.5.

The co-incubation conditions are room temperature (18-30 ℃) and 2 hours.

The sample to be detected is serum.

And after incubation, carrying out fluorescence detection after ultrapure water cleaning.

The invention also provides a detection kit of the let-7a, which comprises a micron tube sensor and a specific probe set;

a polydiacetylene microtube modified BHQ 3;

the BHQ3 was modified on the polydiacetylene microtube by two complementary ssDNA;

the sequences of the complementary ssDNA are respectively shown as SEQ ID NO 1 and SEQ ID NO 2;

the specific probe set comprises two probes of nucleotide sequences shown in SEQ ID NO. 3-4.

The kit of the invention also comprises at least one pair of the following competition probes;

let-7c competitor probe: h1 c: the nucleotide sequence is shown as SEQ ID NO. 5;

h2 c: the nucleotide sequence is shown as SEQ ID NO. 6;

let-7e competitor probe: h1 e: the nucleotide sequence is shown as SEQ ID NO. 7;

h2 e: the nucleotide sequence is shown as SEQ ID NO. 8;

let-7f competitor probe: h1 f: the nucleotide sequence is shown as SEQ ID NO. 9;

h2 f: the nucleotide sequence is shown as SEQ ID NO. 10.

The invention also provides a method for detecting the let-7a, which comprises the steps of mixing the micron tube sensor, SEQ ID NO: 3-4, incubating the probe group of the nucleotide sequence shown in the specification and a sample to be detected together, and obtaining the miRNA content according to the optical waveguide fluorescence intensity.

SEQ ID NO: 3-4, wherein the molar ratio of the quantitative probes in the probe group of the nucleotide sequences is 1: 4.

the sample to be detected is serum.

The co-incubation conditions are room temperature (18-30 ℃) and 2 hours.

The fluorescence intensity of the optical waveguide is the fluorescence intensity excited by 532nm light.

In the let-7a detection method, the co-incubation components further comprise at least one pair of the following competition probes;

let-7c competitor probe: h1 c: the nucleotide sequence is shown as SEQ ID NO. 5;

h2 c: the nucleotide sequence is shown as SEQ ID NO. 6;

let-7e competitor probe: h1 e: the nucleotide sequence is shown as SEQ ID NO. 7;

h2 e: the nucleotide sequence is shown as SEQ ID NO. 8;

let-7f competitor probe: h1 f: the nucleotide sequence is shown as SEQ ID NO. 9;

h2 f: the nucleotide sequence is shown as SEQ ID NO. 10.

The reaction system for adding the competitive probe comprises:

mu.L of Tris was metered in.

The invention provides a micro-tube sensor and a probe set for detecting miRNA of single base mutation, and also provides a method for detecting single base mutation, wherein the method is based on that target molecules catalyze self-assembly of hairpin probes to form a double-chain structure, BHQ3 hybridized on a micro-tube is replaced by toehold, so that optical waveguide at the end of the micro-tube is recovered from quenching, and a method for distinguishing similar miRNA under the action of a competitive probe is provided. As the reaction cycle progresses, a high signal is generated by a low concentration of the target molecule, while a very low signal is generated by the inhibited single base mutated non-target molecule, thereby achieving the target of detection in a serum environment. Experiments show that the detection method has high sensitivity and selectivity on let-7 families, the detection process is simple, convenient, sensitive and rapid, and the detection result is accurate.

Drawings

FIG. 1 shows the reaction mechanism of the detection method of the present invention;

FIG. 2 shows the process of example 1 in which ssDNA1 modifies a microtube; modifying the microtube with epoxy, and then accessing the modified microtube into Raman spectrum of ssDNA1 microtube; FIG. 2a is the process of amino-nanotube modification of single-stranded DNA; FIG. 2b is a Raman spectrum, wherein i represents the Raman spectrum of the epoxy-grafted microtube, ii represents the Raman spectrum after the ssDNA1 is grafted, and the more prominent peaks are the characteristic peaks of the DNA;

FIG. 3 shows the change in optical waveguide fluorescence after modification of a microtube with BHQ3-ssDNA2 of example 2; FIG. 3a shows the fluorescence quenching process of the ends of microtubes after hybridization of microtubes-ssDNA 1 and BHQ3-ssDNA 2; FIG. 3b is a graph showing the fluorescence spectrum change at the end of the microtube after hybridization between the microtube-ssDNA 1 and BHQ3-ssDNA2 with different concentrations; with the continuous increase of the concentration of BHQ3-ssDNA2, the fluorescence intensity of the end of the micrometer tube is continuously reduced;

FIG. 4 shows the fluorescence change of the end light waveguide after dripping let-7a, H1a, H2a probes on the micron tube of example 3; FIG. 4a shows that in the BHQ 3-micro tube system, after excitation by 532nm light, the fluorescence of the end of the micro tube is weak, and when detected let-7a is added, the fluorescence brightness of the end of the micro tube is enhanced; FIG. 4b shows that the fluorescence of the end of the microtube gradually recovers by gradually adding a solution of let-7a at an enhanced concentration;

FIG. 5 is a schematic diagram showing the drop-wise addition of different concentrations of let-7a onto a hydrophobic plate, the change in fluorescence of an end optical waveguide, and the detection limit of a 4 μm tube according to example; adding let-7a solutions with different concentrations into a BHQ 3-micron tube system, wherein the concentration is from 10pM to 20fM, and the fluorescence of the end of the micron tube is gradually recovered; and the fluorescence change of the end of the microtube and the concentration of let-7a are found to be in a proportional relation in the concentration range from 20fM to 100 fM;

FIG. 6 shows the fluorescence kinetics curves of the end light waveguides of the solution of example 5, in which 20 μ Llet-7a, (H1c, H2c) and let-7c, (H1e, H2e) and let-7e, (H1f, H2f) and let-7f, and all solutions containing H1a and H2a are respectively dripped on five micro-tubes; FIG. 6a is a graph showing the fluorescence kinetics of let-7a, c, e, f without the addition of competitor probe; FIG. 6b is a fluorescence kinetics curve of let-7a, c, e, f after adding competitive probes H1c/e/f and H2c/e/f, which shows that the competitive probes greatly improve the capability of the system to distinguish single base mutation;

FIG. 7 shows fluorescence changes of the tip optical waveguide in example 6, in which four microtubes were respectively dropped with a solution containing serum of a pancreatic cancer patient, serum of a lung cancer patient, serum of an ovarian cancer patient, a serum solution of a stomach cancer patient, and 4 solutions of H1 and H2 containing a, c, e, and f.

Detailed Description

The invention provides a micron tube sensor and a preparation method and application thereof, and a person skilled in the art can appropriately improve process parameters by referring to the content. 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 applications of this 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 in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention relates to sequences as shown in table 1:

TABLE 1 nucleic acid sequences

The invention is further illustrated by the following examples:

example 1

The preparation method of the amino-diacetylene monomer for preparing the polydiacetylene microtubes comprises the following steps: 10, 12-twenty five carbon double alkyne acid molecule and 1.2 times of equivalent of 1- (3-dimethylamino propyl) -3-ethyl carbodiimide hydrochloride (EDC) and 1.2 times of equivalent of N-hydroxy succinimide (NHS) are dissolved in 30mL of refined dichloromethane, and the mixture is stirred magnetically for reaction for 6 hours at 25 ℃. The obtained product is subject to rotary evaporation and extraction, then ethylenediamine liquid with 1.1 times of equivalent weight is added and dissolved in dichloromethane solvent, and magneton stirring reaction is carried out for 1 hour at the temperature of 30 ℃. And finally, extracting and rotary evaporating to obtain the amino-diacetylene monomer.

0.0056g of aminodiyne was dissolved in 2mL of absolute ethanol with 0.00075g of octadecylamine-substituted melamine. And pouring the solution into 300mL of ultrapure water at 75 ℃, carrying out ultrasonic treatment for 60min, placing in a dark place, naturally cooling to room temperature, and placing in a refrigerator at 4 ℃ overnight to obtain the composite vesicle. And respectively adding 10ml of the composite 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.

Taking the polydiacetylene microtube out of the weighing bottle, placing the microtube under an ultraviolet tube with the wavelength of 254nm, and irradiating for 10 minutes to ensure that the diacetylene is polymerized into a blue phase. The blue-phase polydiacetylene microtube is heated at 80 ℃ for 10 minutes to change into a red-phase polydiacetylene microtube. Diluting 10 mu L of ethylene glycol diglycidyl ether to 100 mu L, adding the diluted solution into a red-phase microtube, and reacting at 30 ℃ overnight to obtain the epoxy-modified microtube. And uniformly mixing the epoxy-modified micron tube with 10 mu L of amino-modified ssDNA1, and reacting at 30 ℃ for 4h to obtain the ssDNA 1-modified micron tube. The micron tube raman spectrum modified with ssDNA1 is shown in figure 2. a is a schematic diagram of amino microtube modified epoxy; and b is a Raman spectrum, i represents the Raman spectrum of the micron tube with the surface connected with the epoxy, ii represents the Raman spectrum after the micron tube is connected with ssDNA1, and the more-come peak is the characteristic peak of the DNA.

Example 2

BHQ3-ssDNA2 was hybridization paired with microtube-ssDNA 1 by the base complementary pairing principle. The buffer used for hybridization was Tris buffer. A single ssDNA 1-micron tube was picked up by a needle tube and immersed in 100. mu.L of BHQ3 modified ssDNA2 solution in a water bath environment at 90 ℃ for 5 minutes. And then, the small test tube is moved out of the water bath pot and slowly cooled to the room temperature, and the cooling time is about 2 hours. After the reaction was completed, the microtube was picked out of the solution with a needle and gently rinsed several times with ultrapure water to remove the adsorbed BHQ3-ssDNA 2. By this step, the microtube tip optical waveguide fluorescence is quenched. The wavelength is used as an abscissa, and the change of the fluorescence brightness of the microtube is used as an ordinate to be plotted. The change of the fluorescence spectrum of the microtube optical waveguide is shown in FIG. 2. The b is a graph showing the change of fluorescence spectrum of the end of the microtube after the hybridization of the ssDNA 1-microtube and ssDNA2-BHQ3 with different concentrations. The fluorescence intensity at the ends of the microtubes decreased with increasing concentrations of ssDNA-BHQ 3.

Example 3

The microtube prepared in example 2 was placed on a hydrophobic plate, and 10. mu.L of a solution containing H1a (300nM) probe, H2a (1200nM) probe, let-7a, in Tris buffer, was added dropwise. After being placed for 2h at room temperature and cleaned by ultrapure water, the change of fluorescence of the optical waveguide is detected, as shown in FIG. 3. and a picture a shows that in a BHQ 3-micro tube system, after excitation by 532nm light, the fluorescence of the end of the micro tube is weak, and when the detected let-7a is added, the fluorescence brightness of the end of the micro tube is enhanced. And b, gradually adding a let-7a solution with an enhanced concentration, and gradually recovering the fluorescence of the end of the micro tube.

Example 4

The microtube was placed on a hydrophobic plate and 10. mu.L of a solution containing H1a (300nM) probe, H2a (1200nM) probe, let-7a in Tris buffer was added dropwise. let-7a concentration was increased from 20fM to 10pM and changes in fluorescence intensity of the light guide at the end of the microtube were observed. Let-7a concentration is plotted on the abscissa and the change in fluorescence intensity of the optical waveguide of the microtube is plotted on the ordinate, as shown in FIG. 4. Graph a shows that in BHQ 3-micron tube system, let-7a solution with different concentrations is added, the concentration is from 20fM to 10pM, and the fluorescence of the end of the micron tube is gradually recovered. The fluorescence intensity of the end of the microtube gradually increases with the increasing concentration of the detected let-7 a. b is a plot of changes in microtube tip fluorescence proportional to the concentration of let-7a over a concentration range from 20fM to 100 fM. The lowest detection limit was 2 fM.

Example 5

mu.L of a solution containing all H1a (300nM), H2a (1200nM) probe, H1c (37.5nM) and H2c (150nM), H1e (37.5nM) and H2e (150nM), H1f (37.5nM) and H2f (150nM) probe, and 0.1pM of let-7a, let-7c, let-7e, and let-7f was added dropwise to each of the 4 microtubes. And (3) drawing by taking the DNA concentration as an abscissa and the change of the fluorescence brightness of the optical waveguide of the microtube as an ordinate, and observing the differentiation of the single-base differential miRNA by the microtube detector, wherein the drawing is shown in figure 5. As can be seen from the analysis of FIG. a, the fluorescence of the microtubes is restored to different degrees after the microtubes react with the let-7c, let-7e and let-7f before the competitive probes H1c/e/f and H2c/e/f are added. When competitive probes were added, let-7c, let-7e, and let-7f were found to recover little of the microtube fluorescence, while let-7a recovered the microtube fluorescence. The method has better distinguishing capability for let-7a, let-7c, let-7e and let-7 f.

Example 6

After 20. mu.L of a probe comprising H1a (300nM) and H2a (1200nM), H1c (37.5nM) and H2c (150nM), H1e (37.5nM) and H2e (150nM), H1f (37.5nM) and H2f (150nM) and 10. mu.L of serum from a pancreatic cancer patient, 10. mu.L of serum from a lung cancer patient, 10. mu.L of serum from an ovarian cancer patient, 10. mu.L of serum from a gastric cancer patient were dropped onto each of 4 microtubes, respectively, they were left at room temperature for 2 hours, and after washing with ultrapure water, the change in fluorescence of the optical waveguide was examined, as shown in FIG. 6.

The serum of pancreatic cancer patients is not obviously signaled, while the serum of other cancer patients is highly expressed, which indicates that let-7a is reduced in the serum of pancreatic cancer patients, and the microtubes can distinguish the types of cancer patients. Different kinds of miRNA can affect the health conditions of different parts of a human body, and the detection of specific miRNA becomes more important for quickly and accurately positioning the focus. The cancer detection in the present hospital has the defects of difficult detection, complex operation, long time consumption and the like, and the micron tube detector has the advantages of convenience, rapidness, obvious distinguishing effect and the like for detecting different cancers. Therefore, the microtube detector based on the competitive amplification principle can be applied to serum detection of cancer patients, and the method is rapid, convenient, economical and feasible and has application value in future cancer detection.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

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Claims (3)

1. A detection product for miRNA, comprising a microtube sensor and a probe set;

the microtube sensor is a polydiacetylene microtube for modifying a quenching group;

the quenching group is modified on the polydiacetylene nanotube through two complementary ssDNAs; the quenching group is BHQ 3; the sequences of the complementary ssDNA are respectively shown as SEQ ID NO 1 and SEQ ID NO 2;

the preparation method of the micron tube sensor comprises the following steps:

reacting the polydiacetylene microtubes with ethylene glycol diglycidyl ether to obtain epoxy-modified polydiacetylene microtubes;

reacting the epoxy-modified polydiacetylene microtube with ssDNA1 modified with amino at the 5' -end to obtain a ssDNA1 modified microtube;

reacting the microtube modified by ssDNA1 with ssDNA2 modified by a quenching group at the 3' end to obtain a microtube sensor;

the probe set comprises hairpin probe H1 and hairpin probe H2;

the hairpin probe H1 is, in order from the 5 'end to the 3' end: h1-1 sequence, H1-2 sequence, H1-3 sequence and H1-4 sequence;

the H1-1 sequence is complementary to a target sequence;

the H1-2 sequence is not consistent with or complementary to a target sequence and is a loop structure of a probe;

the H1-3 sequence is complementary with H1-1 to form a stem structure of the probe;

the H1-4 sequence is identical to the ssDNA2 sequence;

the hairpin probe H2 is, in order from the 5 'end to the 3' end: h2-1 sequence, H2-2 sequence, H2-3 sequence and H2-4 sequence;

the H2-1 sequence is a cohesive end sequence;

the H2-2 sequence is complementary to the H1-2 sequence;

the sequence of H2-3 is consistent with H1-3 and is a ring structure of the probe;

the H2-4 sequence is complementary with H2-2 to form a stem structure of the probe;

the probe set also comprises at least one pair of competitive probes: hairpin probe H1 'and hairpin probe H2';

the hairpin probe H1 ' is, in order from the 5 ' end to the 3 ' end: h1 ' -1 sequence, H1 ' -2 sequence and H1 ' -3 sequence;

the H1' -1 sequence is complementary to a non-target sequence;

the H1' -2 column is not identical or complementary to a non-target sequence and is a loop structure of a probe;

the H1 '-3 sequence is complementary with H1' -1 to form a stem structure of the probe;

the hairpin probe H2 ' is, in order from the 5 ' end to the 3 ' end: h2 '-1 sequence, H2' -2 sequence, H2 '-3 sequence and H2' -4 sequence;

the H2' -1 sequence is a cohesive end sequence;

the H2 '-2 sequence is complementary to the H1' -2 sequence;

the sequence of H2 '-3 is consistent with H1' -3 and is a ring structure of the probe;

the H2 '-4 sequence is complementary with H2' -2 to form a stem structure of the probe;

the non-target sequence is an approximate miRNA of the miRNA to be detected.

2. A method for detecting miRNA of non-diagnosis purpose, which comprises incubating the micro-tube sensor of claim 1, probe set and sample to be detected together, and obtaining miRNA content according to optical waveguide fluorescence intensity.

3. A let-7a detection kit, comprising a microtube sensor and a specific probe set;

a polydiacetylene microtube modified BHQ 3;

the BHQ3 was modified on the polydiacetylene microtube by two complementary ssDNA;

the sequences of the complementary ssDNA are respectively shown as SEQ ID NO 1 and SEQ ID NO 2;

the specific probe group comprises two probes of nucleotide sequences shown in SEQ ID NO. 3-4;

at least one pair of the following competitive probes is also included;

let-7c competitor probe: h1 c: the nucleotide sequence is shown as SEQ ID NO. 5;

h2 c: the nucleotide sequence is shown as SEQ ID NO. 6;

let-7e competitor probe: h1 e: the nucleotide sequence is shown as SEQ ID NO. 7;

h2 e: the nucleotide sequence is shown as SEQ ID NO. 8;

let-7f competitor probe: h1 f: the nucleotide sequence is shown as SEQ ID NO. 9;

h2 f: the nucleotide sequence is shown as SEQ ID NO. 10.

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