CN112608983A - Paper-based detection method for exosome - Google Patents
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Abstract
The invention provides a paper-based detection method of exosomes, which comprises the following steps: (1) an identification and molecular conversion system, (2) an amplification reaction system, and (3) an LFB detection analysis system. The invention converts exosome into single-stranded DNA target, amplifies the single-stranded DNA by nucleic acid amplification technology, and further utilizes PtAu-NiO2And detecting the amplified single-stranded DNA by using lateral flow chromatography test paper with nano enzyme as a signal probe. The invention uses the nucleic acid lateral flow chromatography test paper as a terminal detection platform, solves the problem that exosomes cannot be simultaneously detected qualitatively and quantitatively, and realizes simple and convenient low-cost detection of exosomes.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a paper-based detection method of exosomes.
Background
The nanometer-scale particle size of exosomes and the ultra-trace-level concentration in body fluid bring huge challenges to the identification and analysis of exosomes. The existing analysis method not only depends on large and expensive instruments, but also needs complex pretreatment process and professional operators, and in addition, the traditional exosome detection method cannot simultaneously determine the nature and quantity of the exosome. Therefore, the development of a rapid, simple, low-cost and highly sensitive method for detecting exosomes is the key to the rapid development and popularization of clinical disease diagnosis and treatment methods using exosomes as biomarkers.
Lateral flow test paper (LFB) is a sensitive, cheap, simple and convenient visual rapid detection method. The early LFB is based on an immune recognition technology, and then a researcher combines functional nucleic acid with LFB, so that the nucleic acid lateral flow chromatography biosensor is invented, and the application range of the LFB is greatly expanded. At present, the application of nucleic acid LFB includes various kinds of nucleic acid, and non-nucleic acid substances, such as microorganism, protein, small molecule, metal ion, etc. Compared with antibodies, the aptamer has the advantages of stable chemical property, easy preparation and modification, easy signal amplification combined with a nucleic acid amplification technology and the like, but no report of using nucleic acid LFB for exosome detection exists at present.
Disclosure of Invention
The invention aims to provide a paper-based detection method of exosomes.
It is another object of the present invention to provide a method for detecting exosomes based on biosensor technology.
In order to achieve the purpose of the invention, the inventor designs a magnetic aptamer three-nucleic acid nanoprobe (MB-ATDP) to perform recognition and molecular conversion of an exosome, designs a template according to a converted single-stranded DNA target to perform an amplification reaction, and uses PtAu-NiO as a reaction product2The functional nucleic acid lateral flow chromatography test paper with the nano enzyme as the signal probe is used for analysis, and a novel exosome visual biosensor based on the functional nucleic acid lateral flow chromatography test paper is constructed.
In a first aspect, the invention provides a paper-based detection method of exosomes, comprising the following steps: (1) an identification and molecular conversion system, (2) an amplification reaction system, and (3) an LFB detection analysis system.
The detection system is used for carrying out lateral flow chromatography analysis on a product obtained after a sample to be detected sequentially reacts through the recognition and molecular conversion system and the amplification reaction system;
wherein, the LFB detection analysis system comprises: functional nucleic acid detection test paper, PtAu-NiO2Nano enzyme signal probe, LFB system buffer solution and TMB color developing solution.
Wherein the PtAu-NiO2The nano enzyme signal probe is PtAu-NiO with the surface modified with a single-chain nucleic acid sequence A2A nanoparticle; the detection line of the functional nucleic acid detection test paper is modified with a single-stranded nucleic acid sequence B, and the quality control line is modified with a single-stranded nucleic acid sequence C; the single-stranded nucleic acid sequence A is partially or fully complementary paired with the single-stranded nucleic acid sequence C.
The A, B, C is used only to distinguish between different sequences and not for sorting.
The complementation includes complementation or reverse complementation defined by the prior art or the common general knowledge and/or complementation or reverse complementation according to the complementation principle defined by the prior art or the common general knowledge.
The test paper comprises test paper prepared by the prior art or common knowledge; the preparation method comprises the preparation method described in the prior art or common general knowledge.
Specifically, the LFB detection and analysis process of the functional nucleic acid comprises the following steps: adding 40-60 muL system buffer solution and 1-2 muL amplified sample solution into a sample pool, and dripping 0.5-2 muL PtAu-NiO at the superposition position of a sample pad and a combination pad of the functional nucleic acid detection test paper2After the nano enzyme signal probe is detected, inserting a detection test paper sample pad into a sample pool, waiting for 2-5min, changing the detection line of the test paper from colorless to black, observing by naked eyes, taking out the test paper, dropwise adding 1-3 mu L TMB color development liquid on the test paper, changing the detection line of the test paper into blue after the color development is carried out for 3-8min, observing by naked eyes again, and extracting the gray pixel area of the detection line by using Image J software. In still another embodiment, the LFB detection and analysis process of the functional nucleic acid includes: adding 50 muL system buffer solution into a sample pool, and dissolving the sample after 1 muL amplificationDropping and coating 1 mu L PtAu-NiO at the superposition position of the sample pad and the combination pad of the functional nucleic acid detection test paper2After the nanoenzyme signal probe is detected, inserting a detection test paper sample pad into a sample pool, changing the detection line of the test paper from colorless to black after waiting for 3min, observing by naked eyes, taking out the test paper, dropwise adding 2 mu L TMB color development liquid on the line, changing the detection line of the test paper into blue after 5min of color development, observing by naked eyes again, and extracting the gray pixel area of the detection line by using Image J software.
Specifically, the detection method further comprises at least one of the following 1) to 4):
1) the PtAu-NiO2The nano enzyme signal probe comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 2 is modified in PtAu-NiO through interaction2Obtaining a probe after the surface of the nano-particles;
2) the functional nucleic acid detection test paper comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 3 at a detection line through interaction modification, and the nucleotide sequence shown in the sequence table SEQ ID NO: 4, modifying the nucleotide sequence shown in the figure at the position of the quality control line through interaction to obtain detection test paper;
3) the PtAu-NiO2The nano enzyme signal probe comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 2 is substituted and/or deleted and/or added by one or more nucleotides and has a nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO: 2 has the same function and is modified in PtAu-NiO through interaction2Obtaining a probe after the surface of the nano-particles;
4) the functional nucleic acid detection test paper comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 3 is substituted and/or deleted and/or added by one or more nucleotides and has the nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO: 3, modifying the nucleotide sequence with the same function at a detection line, and carrying out the following steps of: 4 is substituted and/or deleted and/or added by one or more nucleotides and has the nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO: 4, modifying the nucleotide sequence with the same function at the position of the quality control line through interaction to obtain the detection test paper.
The invention also provides the application of the detection method in the aspect of detecting the exosome, wherein the detection can be qualitative detection or quantitative detection.
In a second aspect, the present invention provides a method for qualitatively detecting exosomes by using the aforementioned detection method, comprising the following steps:
s1: adding a sample to be detected into the recognition and molecular conversion system, and carrying out target conversion reaction;
s2: adding the product obtained in the step S1 into the amplification reaction system to carry out amplification reaction;
s3: and (3) detecting and analyzing the amplification product by using the LFB detection system.
S3 is specifically as follows: adding 40-60 muL system buffer solution and 1-2 muL amplified sample solution into a sample pool, and dripping 0.5-2 muL PtAu-NiO at the superposition position of a sample pad and a combination pad of the functional nucleic acid detection test paper2After the nano enzyme signal probe is detected, inserting a detection test paper sample pad into a sample pool, waiting for 2-5min, changing the detection line of the test paper from colorless to black, observing by naked eyes, taking out the test paper, dropwise adding 1-3 mu L TMB color development liquid on the test paper, changing the detection line of the test paper into blue after the color development is carried out for 3-8min, observing by naked eyes again, and extracting the gray pixel area of the detection line by using Image J software.
Still specifically, 50 muL system buffer solution and 1 muL sample solution after amplification are added into the sample pool, and 1 muL PtAu-NiO is dripped and coated at the superposition position of the sample pad and the combination pad of the functional nucleic acid detection test paper2After the nanoenzyme signal probe is detected, inserting a detection test paper sample pad into a sample pool, changing the detection line of the test paper from colorless to black after waiting for 3min, observing by naked eyes, taking out the test paper, dropwise adding 2 mu L TMB color development liquid on the line, changing the detection line of the test paper into blue after 5min of color development, observing by naked eyes again, and extracting the gray pixel area of the detection line by using Image J software.
In a third aspect, the present invention provides a method for quantitative detection of exosomes by using the aforementioned detection method, comprising the following steps:
and (3) SI: making a standard curve:
diluting the exosome solution by 2 times in a gradient manner, constructing recognition and molecular conversion systems with different exosome concentrations, and carrying out amplification and LFB detection and analysis by the same steps as the qualitative detection;
taking the concentration of exosomes as a vertical coordinate, extracting the area of a T-line gray pixel by Image J software as a horizontal coordinate, and drawing a standard curve;
and (3) SII: and detecting the sample to be detected according to the qualitative detection method, substituting the area value of the extracted T-line gray pixel into the standard curve, and calculating to obtain the content of exosomes in the sample to be detected so as to realize quantitative detection of exosomes.
The invention provides a method for detecting exosomes based on a biosensor technology, which comprises the steps of firstly designing a magnetic aptamer trinucleotide nanoprobe with triple functions of target identification, molecular conversion and separation and purification, wherein the exosomes are combined with aptamers on the ring part of the probe, and the nucleic acid undergoes conformational change, so that a trinucleotide structure on the stem part of the probe is disintegrated, a single-stranded DNA target is released, and then the trinucleotide structure is separated through an external magnetic field; carrying out amplification reaction on the supernatant obtained after magnetic separation to obtain a large number of single-stranded DNA targets; after amplification, the single-stranded DNA target in the solution is connected with PtAu-NiO on the sample pad2Partial hybridization of a single-stranded nucleic acid sequence A on the nano enzyme signal probe occurs, the complex then flows laterally, when the complex passes through a detection line (T line), the other part of a single-stranded DNA target is hybridized with a single-stranded nucleic acid sequence B on the T line to form a sandwich structure, the sandwich structure is developed on the T line, and redundant PtAu-NiO is generated2The nano enzyme signal probe continuously flows, is finally captured by the quality control line (C line), and develops color on the C line, if the color development is too light, TMB color development liquid can be dripped, and due to PtAu-NiO2Has peroxidase-like activity, and can catalyze substrate to generate blue substance and enhance signal.
Preferably, the aptamer sequence is a CD63 protein specific sequence
A part of the single-stranded DNA target and PtAu-NiO2The single-stranded nucleic acid sequence A on the surface of the nano enzyme signal probe is partially or completely complementary, one part of the single-stranded nucleic acid sequence A is partially or completely complementary with the single-stranded nucleic acid sequence B at the detection line of the functional nucleic acid detection test paper, and the single-stranded nucleic acid sequence C at the quality control line is partially or completely complementary PtAu-NiO2Single-stranded nucleic acid sequence A on the surface of the nano enzyme signal probe.
Preferably, the aforementioned method, single-stranded DNA target, PtAu-NiO2The base sequences of the single-stranded nucleic acid on the surface of the nano enzyme signal probe, the detection line of the functional nucleic acid detection test paper and the single-stranded nucleic acid at the quality control line are as follows:
single-stranded DNA target: 5'-GAGAGAGAGAGAGAGAGGGAAAAGGAAAGG-3'
PtAu-NiO2Single-stranded nucleic acid on the surface of the nano enzyme signal probe: 5'-TTTTTTCCTTTCCTTTTC-3'
Single-stranded nucleic acid at detection line: 5'-CTCTCTCTCTCTCTCCCCCCCC-3'
Single-stranded nucleic acid at the control line: 5'-CCCCCCCGAAAAGGAAAGG-3'
The invention also provides a detection kit matched with the method, and the kit at least comprises the following components: magnetic aptamer trinucleotide nano-probe, template, detection test paper and PtAu-NiO2Nano enzyme signal probe, TMB color development liquid and the like.
The detection and analysis principle of the kit of the invention is as follows: firstly, combining an exosome with an aptamer on a magnetic aptamer trinucleotide probe ring to disintegrate a trinucleotide structure of a probe stem part, releasing a single-stranded DNA target, and separating through an external magnetic field; carrying out nucleic acid amplification on the supernatant obtained after magnetic separation to obtain a large number of single-stranded DNA targets; single-stranded DNA target in solution after amplification utilizes PtAu-NiO with single-stranded nucleic acid modified on surface2The detection test paper with the nanoenzyme as the signal probe carries out lateral flow chromatography due to PtAu-NiO2The aggregation of the nano enzyme signal probe finally develops color on a T line and a C line of the test paper; if the color development is too light, the TMB color development liquid can be added dropwise, because PtAu-NiO2Has peroxidase-like activity, and can catalyze substrate to generate blue substance and enhance signal.
The specific detection method comprises the following steps:
1) recognition and molecular conversion: adding an exosome solution into a magnetic aptamer trinucleotide probe solution, incubating under a dark condition, and then carrying out magnetic separation to obtain a supernatant solution containing a free single-stranded DNA target;
2) and (3) amplification reaction: preparing single-stranded DNA target, template, enzyme, dNTP and reaction obtained by conversionBuffer and ddH2O, performing nucleic acid amplification reaction;
3) LFB detection assay: adding 40-60 mu L LFB system buffer solution and 1-2 mu L amplified sample solution into a sample pool, and dripping 0.5-2 mu L PtAu-NiO at the superposition position of a detection test paper sample pad and a combination pad2After the nano enzyme signal probe is detected, inserting the detection test paper sample pad into a sample pool, waiting for 2-5min, and judging whether the sample to be detected contains exosomes or not and the concentration of the exosomes according to whether a black strip appears on a T line or not; and if the color development is not obvious, dripping 1-3 mu L of TMB color development liquid on the line, developing for 3-8min, and judging whether the sample to be detected contains exosomes and the concentration of the exosomes according to the condition whether a blue strip appears on the T line or not. The change of the color of the T line can be directly observed by naked eyes, or the gray pixel area of the T line is extracted by using Image J software.
Wherein, the formula of the LFB system buffer solution in the step 3) is as follows: 4 XSSC buffer (containing 10nM Tris-HCl, 0.002% Trtion X-100, 2% BSA, 0.05% Tween-20) at pH 7.4.
Preferably, a series of exosome standard solutions are prepared at concentrations and assayed according to the method described above. And 3) extracting the gray pixel area of the T line by using Image J software to judge the color development condition, and drawing a standard color development curve according to the color change of the T line, wherein the exosome concentration is y, and the pixel area is x: y = 6.8502e0.0006x，R2= 0.966, so that quantitative detection of exosomes can be achieved.
The visual detection sensitivity of the method to exosomes is 4.5pM, and the visual detection limit is reduced to 0.6pM after the color development of the TMB is catalyzed by the nano enzyme.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the invention is used for simultaneously, qualitatively and quantitatively detecting the exosome simply and conveniently at low cost by establishing the paper-based detection method of the exosome. Designing a magnetic aptamer trinucleotide nano probe to recognize exosomes and carry out molecular conversion according to specific proteins on the surface of exosomes, designing a template to carry out nucleic acid amplification reaction according to converted single-stranded DNA targets, and using PtAu-NiO2The lateral flow chromatography test paper with nano enzyme as a signal probe is used for analysis, a novel exosome detection method based on the functional nucleic acid lateral flow chromatography test paper is constructed, and a novel method capable of qualitatively and quantitatively detecting exosomes simultaneously is provided. The method has the advantages of simple and convenient detection operation of the exosome, lower requirement and no dependence on a large instrument, successfully solves the problem that the exosome cannot be simultaneously detected qualitatively and quantitatively, and is expected to be used as a low-cost primary screening means for rapid field screening of diseases in primary medical institutions or poor regions.
The method utilizes a magnetic aptamer trinucleotide nanoprobe to convert exosomes which are difficult to measure into single-stranded nucleic acid targets which are easy to detect.
And (II) after the exosomes are subjected to target conversion and amplification, the number of ultra-trace exosome molecules is amplified by 1000 times.
And (III) the method based on the flow measurement chromatography test paper can not only utilize the color change generated by aggregation induction of the nano enzyme signal probe for visual qualitative analysis, but also utilize the peroxidase-like activity of the nano enzyme to enhance the visual signal, and can realize quantitative determination by means of image analysis software.
And (IV) the method can realize simple and convenient detection of exosome with low cost.
Drawings
FIG. 1 shows PtAu-NiO in example 1 of the present invention2And (3) detecting the single-stranded DNA target by the LFB with the nanoenzyme as the signal probe.
FIG. 2 shows PtAu-NiO in example 1 of the present invention2Nano enzyme is used as a signal probe, and TMB color development liquid is dripped to detect the single-chain DNA target through the LFB amplified by the enzyme.
FIG. 3 is a result of lateral flow assay test paper for detecting exosomes diluted by 2-fold concentration gradient, without adding TMB color-developing solution.
FIG. 4 is a result of lateral flow assay test paper for detecting exosomes diluted 2-fold in concentration gradient, with TMB color-developing solution added.
FIG. 5 is a T-line grayscale pixel area standard curve fit for lateral flow dipstick assay detection of exosomes diluted 2-fold in concentration gradient.
FIG. 6 shows the results of lateral flow test strips for detection of exosomes extracted from equal volumes of HepG2 and L02 cell supernatants.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
In the invention, the LFB system buffer solution formula is as follows: 4 XSSC buffer (containing 10nM Tris-HCl, 0.002% Trtion X-100, 2% BSA, 0.05% Tween-20) at pH 7.4.
Example 1 establishment of paper-based detection method of exosomes
1. Experimental Material
20 XSSC, Tris-HCl, sodium phosphate dodecahydrate, Bovine Serum Albumin (BSA), Tween-20, sucrose, TMB color developing solution and the like.
The sequence was designed as follows (SEQ ID NOS: 1-4):
2. detection and verification of LFB on single-stranded DNA target
Diluting the single-chain DNA target by 2-time concentration gradient, adding 50 mu L system buffer solution and 1 mu L single-chain DNA target solution into a sample pool, and dripping 1 mu L PtAu-NiO at the superposition position of a detection test paper sample pad and a combination pad2And after the nano enzyme signal probe is detected, inserting the detection test paper sample pad into the sample pool, waiting for 3min, observing the result by naked eyes, and judging the test paper to be positive when the C line and the T line are black. The color of the T-line becomes lighter with decreasing concentration of single-stranded DNA target, and the visible detection limit of LFB for single-stranded DNA target is 78.13nM (fig. 1).
3. Detection and verification of LFB on single-stranded DNA target after enzymatic amplification
Diluting the single-chain DNA target by 2-time concentration gradient, adding 50 mu L system buffer solution and 1 mu L single-chain DNA target solution into a sample pool, and dripping and coating the solution at the superposition position of a detection test paper sample pad and a combination pad1µL PtAu-NiO2And after the nano enzyme signal probe is detected, inserting the detection test paper sample pad into a sample pool, waiting for 3min, dropwise adding 2 mu L TMB color development liquid on the line, developing for 5min, observing the result by naked eyes, and judging the detection test paper sample pad to be positive if a blue line appears on the T line, and finally judging the detection test paper sample pad to be negative if the detection test paper sample pad is colorless. The color of the T line becomes lighter with the decrease of the concentration of the single-stranded DNA target, and after the color development of the TMB catalyzed by the nano-enzyme, the visual detection limit of the LFB to the single-stranded DNA target is 9.77nM (FIG. 2).
4. Detection and verification of exosomes by LFB
According to the optimization system, 2-fold concentration gradient dilution is carried out on the exosome solution, and the MB-ATDP solution and the exosome solution are mixed according to the volume ratio of 1: 1 mixing (20. mu.L each), incubating for 2h, magnetic separating, leaving a supernatant; taking 1 mu L of supernatant for amplification; adding 50 mu L system buffer solution and 1 mu L amplification product into a sample pool, and dripping 1 mu L PtAu-NiO at the superposition position of a detection test paper sample pad and a combination pad2And after the nano enzyme signal probe is detected, inserting the detection test paper sample pad into the sample pool, waiting for 3min, observing the result by naked eyes, and judging the test paper to be positive when the C line and the T line are black. The color of the T-line became lighter with decreasing exosome concentration, and the limit of visible detection of exosomes by LFB was 4.5pM (fig. 3).
5. Detection and verification of LFB on exosome after enzymatic amplification
According to the optimization system, 2-fold concentration gradient dilution is carried out on the exosome solution, and the MB-ATDP solution and the exosome solution are mixed according to the volume ratio of 1: 1 mixing (20. mu.L each), incubating for 2h, magnetic separating, leaving a supernatant; taking 1 mu L of supernatant to carry out EXPAR amplification; adding 50 mu L system buffer solution and 1 mu L EXPAR amplification product into a sample pool, and dripping 1 mu L PtAu-NiO at the superposition position of a detection test paper sample pad and a combination pad2And after the nano enzyme signal probe is detected, inserting the detection test paper sample pad into a sample pool, waiting for 3min, dropwise adding 2 mu L TMB color development liquid on the line, developing for 5min, observing the result by naked eyes, and judging the detection test paper sample pad to be positive if a blue line appears on the T line, and finally judging the detection test paper sample pad to be negative if the detection test paper sample pad is colorless. The color of the T line becomes lighter along with the reduction of the concentration of the exosome, and after the color development of the TMB catalyzed by the nano enzyme, the visual detection limit of the LFB to the exosome is 0.6pM (figure 4).
6. Quantitative detection of LFB on exosomes
Preparing a series of exosome standard solutions with concentration according to the optimization system, carrying out identification and molecular conversion, carrying out amplification on obtained supernate, detecting an amplification product by using LFB, taking the exosome concentration as a vertical coordinate, extracting the area of a T-line gray pixel by using Image J software as a horizontal coordinate, and drawing a standard curve (figure 5). y = 6.8502e0.0006x，R2 = 0.966。
Example 2 examination of the applicability of the detection method
Exosomes extracted from equal volumes of HepG2 and L02 cell supernatants were detected according to the detection method constructed in example 1. LFB was able to effectively distinguish tumor exosomes from non-tumor exosomes (fig. 6).
Example 3 verification of amplification Effect of detection method
After exosome conversion and amplification, substituting the gray pixel area of each T line in the LFB detection result into a formula obtained by carrying out standard curve fitting on the gray pixel area of the T line obtained by LFB detection after diluting a single-stranded DNA target by 2 times of gradient to obtain: y = 14.829e0.0005xObtaining the concentration of the single-stranded DNA target after exosome conversion and amplification. Exosome concentrations were converted to substance quantity concentrations and compared to single stranded DNA targets. The molecular amplification of exosomes by this method was 1000-fold (table 1).
TABLE 1 molecular amplification of exosomes after target conversion and amplification
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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Claims (9)
1. A paper-based detection method of exosomes is characterized by comprising the following steps: (1) a recognition and molecular conversion system, (2) an amplification reaction system, and (3) a lateral flow chromatography test paper LFB detection analysis system;
the paper-based detection method is used for carrying out lateral flow chromatographic analysis on a product obtained after a sample to be detected sequentially reacts through the identification and molecular conversion system and the amplification reaction system;
wherein, the LFB detection analysis system comprises: functional nucleic acid detection test paper, PtAu-NiO2Nano enzyme signal probe, LFB system buffer solution and TMB color developing solution.
2. The detection method according to claim 1,
the PtAu-NiO2The nano enzyme signal probe is PtAu-NiO with the surface modified with a single-chain nucleic acid sequence A2A nanoparticle;
the detection line of the functional nucleic acid detection test paper is modified with a single-stranded nucleic acid sequence B, and the quality control line is modified with a single-stranded nucleic acid sequence C;
the single-stranded nucleic acid sequence A is partially or fully complementary paired with the single-stranded nucleic acid sequence C.
3. The detection method according to claim 1 or 2, wherein the LFB detection assay system comprises the steps of: adding a buffer solution and an amplified sample solution into a sample pool, and dripping PtAu-NiO at the superposition position of a sample pad and a combination pad of the functional nucleic acid detection test paper2After the nano enzyme signal probe is detected, inserting a detection test paper sample pad into a sample pool, changing the color of a detection line of the test paper from colorless to black after waiting for a plurality of minutes, observing by naked eyes, taking out the test paper, dropwise adding TMB color development liquid on the line, changing the detection line of the test paper into blue after color development, observing by naked eyes again, and extracting the gray pixel area of the detection line by software.
4. The method for detecting according to claim 3, wherein the LFB detection assay process for functional nucleic acid comprises: adding 40-60 muL system buffer solution and 1-2 muL amplified sample solution into a sample pool, and dripping 0.5-2 muL PtAu-NiO at the superposition position of a sample pad and a combination pad of the functional nucleic acid detection test paper2After the nano enzyme signal probe is detected, inserting a detection test paper sample pad into a sample pool, waiting for 2-5min, changing the detection line of the test paper from colorless to black, observing by naked eyes, taking out the test paper, dropwise adding 1-3 mu L TMB color development liquid on the test paper, changing the detection line of the test paper into blue after the color development is carried out for 3-8min, observing by naked eyes again, and extracting the gray pixel area of the detection line by using Image J software.
5. The detection method according to claim 1 or 2, further comprising at least one of the following 1) or 2):
1) the PtAu-NiO2The nano enzyme signal probe comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 2 is modified in PtAu-NiO through interaction2Obtaining a probe after the surface of the nano-particles;
2) the functional nucleic acid detection test paper comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 3 at a detection line through interaction modification, and the nucleotide sequence shown in the sequence table SEQ ID NO: 4, modifying the nucleotide sequence shown in the figure at the position of the quality control line through interaction to obtain the detection test paper.
6. The detection method according to claim 1 or 2, further comprising at least one of the following 1) or 2):
1) the PtAu-NiO2The nano enzyme signal probe comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 2 is substituted and/or deleted and/or added by one or more nucleotides and has a nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO: 2 has the same function and is modified in PtAu-NiO through interaction2Obtaining a probe after the surface of the nano-particles;
2) the functional nucleic acid detection test paper comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 3 is substituted and/or deleted and/or added by one or more nucleotides and has the nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO: 3, modifying the nucleotide sequence with the same function at a detection line, and carrying out the following steps of: 4 is substituted and/or deleted and/or added by one or more nucleotides and has the nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO: 4, modifying the nucleotide sequence with the same function at the position of the quality control line through interaction to obtain the detection test paper.
7. The detection method of claim 3, further comprising at least one of the following 1) or 2):
1) the PtAu-NiO2The nano enzyme signal probe comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 2 by modifying the nucleotide sequence shown in the figureDecorated with PtAu-NiO2Obtaining a probe after the surface of the nano-particles;
2) the functional nucleic acid detection test paper comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 3 at a detection line through interaction modification, and the nucleotide sequence shown in the sequence table SEQ ID NO: 4, modifying the nucleotide sequence shown in the figure at the position of the quality control line through interaction to obtain the detection test paper.
8. The detection method of claim 3, further comprising at least one of the following 1) or 2):
1) the PtAu-NiO2The nano enzyme signal probe comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 2 is substituted and/or deleted and/or added by one or more nucleotides and has a nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO: 2 has the same function and is modified in PtAu-NiO through interaction2Obtaining a probe after the surface of the nano-particles;
2) the functional nucleic acid detection test paper comprises: and (3) mixing the amino acid sequence shown in SEQ ID NO: 3 is substituted and/or deleted and/or added by one or more nucleotides and has the nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO: 3, modifying the nucleotide sequence with the same function at a detection line, and carrying out the following steps of: 4 is substituted and/or deleted and/or added by one or more nucleotides and has the nucleotide sequence which is similar to the nucleotide sequence shown in SEQ ID NO: 4, modifying the nucleotide sequence with the same function at the position of the quality control line through interaction to obtain the detection test paper.
9. Use of the detection method according to any one of claims 1 to 8 for the detection of exosomes.
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