CN113341153A - Sandwich type sensor for detecting crustacean tropomyosin and application thereof - Google Patents
Sandwich type sensor for detecting crustacean tropomyosin and application thereof Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention relates to a sandwich-type sensor for detecting crustacean tropomyosin and application thereof. The sandwich type sensor comprises a molecular imprinting microsphere and a nucleic acid aptamer probe, wherein the nucleic acid aptamer probe has a nucleotide sequence shown in SEQ ID NO. 1. The sandwich type sensor not only has the advantages of detection of the traditional fluorescent probe, but also can specifically identify the allergen, and the sandwich type sensor combines the high-sensitive luminous performance of the carbon quantum dots with the high-specificity antigen identification performance of the aptamer, so that the crustacean tropomyosin allergen can be efficiently detected in vitro.
Description
Technical Field
The invention relates to the technical field of biological analysis, in particular to a sandwich type sensor for detecting crustacean tropomyosin and application thereof.
Background
The detection method of the crustacean tropomyosin mainly comprises the technologies such as ELISA, PCR, RT-PCR and the like, and the technologies have the problems of long detection time, low sensitivity and accuracy (easy occurrence of false positive) and the like. Therefore, the detection mode of tropomyosin is in need of improvement.
The aptamer is a single-chain nucleotide with specific recognition capability on a target substance, has no toxicity, good chemical stability, easy modification, flexible and changeable sequence design, no immunogenicity and capability of combining with almost any target molecule with high affinity and specificity, is expected to become a substitute of an antibody, and has huge application potential and diverse application range in the field of bioanalysis.
The molecularly imprinted polymer is used as an identification element and can identify the template molecule with high specificity and selectivity. But compared with the imprinting of small molecules, the imprinting template has the advantages of large volume, complex structure, easy change and high imprinting difficulty. Thus, imprinting large portions of protein macromolecules still presents a number of challenges.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Accordingly, the present invention is directed to a sandwich-type sensor for detecting crustacean tropomyosin and uses thereof. The sensor can quickly, sensitively and specifically detect the crustacean tropomyosin.
To this end, in a first aspect of the present invention, the present invention provides a sandwich sensor for detecting crustacean tropomyosin, comprising molecularly imprinted microspheres and an aptamer probe having a nucleotide sequence set forth in SEQ ID NO. 1.
The sandwich type sensor has the advantages of detection of the traditional fluorescent probe and can specifically recognize the allergen, and the sandwich type sensor combines the high-sensitivity luminescence property of the carbon quantum dot with the high-specificity antigen recognition property of the aptamer, so that the crustacean tropomyosin allergen can be efficiently detected in vitro.
Optionally, the preparation of the molecularly imprinted microsphere comprises:
mixing Fe3O4Dissolving the magnetic microspheres and the tropomyosin standard substance in a Tris buffer solution, carrying out oscillation incubation for 2h at room temperature, adding dopamine, continuing oscillation incubation for 3h at room temperature, cleaning, dissolving in PBS, adding a polypeptide solution, carrying out oscillation incubation for 24h at room temperature, washing the magnetic microspheres with an acetic acid solution of SDS, and washing with distilled water to obtain the molecularly imprinted microspheres.
Further, 100mg of Fe3O4The magnetic microspheres and 20mg tropomyosin standard were dissolved in 20mL 10mM Tris buffer pH 8.0.
Further, the amino acid sequence of the polypeptide is EKEKEKEPPPPC.
Further, 0.71mM of the polypeptide solution was added at a volume ratio of 10: 1.
Further, the dopamine is added in an amount of 40 mg.
Further, the magnetic microspheres collected by the magnet were washed with 3% (v/v) acetic acid solution containing 0.1% (w/v) SDS.
In a second aspect of the present invention, the present invention provides a method for detecting crustacean tropomyosin by using the above sandwich sensor, comprising:
dissolving 2mg of molecularly imprinted microspheres in PBS buffer solution, performing ultrasonic treatment for 30min to ensure that the molecularly imprinted microspheres are fully dispersed, adding a sample solution to be detected, placing the sample solution in a shaking table at 25 ℃ for incubation for 2h, then adding 200pmol of nucleic acid aptamer probe, performing shaking incubation at 25 ℃ for 1.5h, washing the sample solution for three times, performing magnetic separation and redissolution in 1mL of PBS solution, and measuring the fluorescence emission intensity at 480nm under the excitation of 370nm light by using an enzyme labeling instrument.
According to the detection method provided by the embodiment of the invention, the crustacean tropomyosin can be quickly, sensitively and specifically detected.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a schematic diagram of a molecularly imprinted-aptamer sandwich sensor;
FIG. 2 is Fe3O4@ PDANPs element analysis;
FIG. 3 is Fe3O4@ PDA NPs and imprinted Fe3O4Scanning Electron Microscopy (SEM) of @ TM-PDA NPs;
FIG. 4 is a blot Fe3O4The @ TM-PDA static adsorption curve;
FIG. 5 is a blot Fe3O4The specific adsorption of @ TM-PDA;
FIG. 6 shows the specificity of detection by the molecularly imprinted-aptamer sandwich sensor.
Detailed Description
The technical solution of the present invention is illustrated by specific examples below. It is to be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before or after the combination step or that other method steps may be inserted between the explicitly mentioned steps; it should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.
In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the invention have been shown, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
With reference to fig. 1, the sandwich type sensor is based on the principle that a separation and enrichment element (molecularly imprinted microspheres) and a signal element (fluorescent aptamer probes) are combined to jointly construct a molecularly imprinted-aptamer sandwich type biosensor. Separating tropomyosin in the enriched solution by using the molecularly imprinted microspheres, specifically identifying the tropomyosin by using the fluorescent aptamer probe, and indicating the change of the content of the tropomyosin according to the change of the fluorescent signal.
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.
Example 1
Preparation of tropomyosin:
(1) taking 50g of muscle of the penaeus vannamei boone, and removing the head, tail, shell and gut of the penaeus vannamei boone.
(2) The shrimp muscle was cut into a paste with a small knife and dissolved in Buffer A (50mmol/L KCl and 2mmol/L NaHCO)3) Fully homogenizing, and extracting at 4 deg.C for 20 min.
(3) Centrifuging the solution extracted in the step (2) at 4 ℃ and 10000r/min for 20min, taking the precipitate, suspending the precipitate in 10-time volume of Buffer A, centrifuging at 4 ℃ and 10000r/min for 20min, taking the precipitate, and repeating the steps for 5 times.
(4) And (4) fully washing the precipitate obtained in the step (3) by precooling acetone until the precipitate is colorless, filtering the precipitate by using six layers of gauze, taking the precipitate, drying the precipitate at room temperature, and removing impurities such as fat, fat-soluble pigment and the like to obtain the prawn acetone powder.
(5) Dissolving prawn acetone powder in Buffer B (0.02mol/L Tris-HCl, 1mol/L KCl and 0.1mmol/L DTT, pH 7.5), and extracting for 72 h.
(6) And (4) filtering the extract obtained in the step (5) by using six layers of gauze to obtain filtrate, and heating the filtrate for 20min in a water-proof manner.
(7) Centrifuging the filtrate heated in the step (6) at 4 ℃ and 10000r/min for 20min, taking supernatant, slowly adding ammonium sulfate according to the amount of 16.4g of ammonium sulfate added into every 100mL of supernatant to make the final concentration of ammonium sulfate in the filtrate be 30%, and standing at 4 ℃ for 1 h.
(8) Centrifuging the liquid after standing at 4 deg.C and 10000r/min for 20min, collecting precipitate, and re-dissolving with 1mol/L PBS to obtain tropomyosin liquid.
(9) Gradient eluting tropomyosin liquid with DEAE Sepharose F.F. anion exchange column and 0.5mol/L NaCl solution, collecting the eluted product to obtain tropomyosin for use.
Example 2
1. Screening of aptamers: screening of aptamers by capillary electrophoresis-exponential enrichment of ligand evolution (CE-SELEX)
Construction of random nucleotide (DNA) libraries and primers: random DNA library, upstream primer and downstream primer are all synthesized by Biotechnology engineering (Shanghai) GmbH (China); a random single-stranded nucleotide (ssDNA) library of 75nt in length was as follows:
5′-TACTAACGGT ACAAGCTA-N40-AACGTTGACCTAGAAGC-3′；
a random sequence of 40nt in the middle and fixed sequences (primer binding regions) of 8nt and 17nt in length at both ends.
An upstream primer: 5'-TACTAACGGTACAAGCTA-3', respectively;
a downstream primer: 5'-GCTTCTAGGTCAACGTT-3', respectively;
construction of a crustacean tropomyosin random ssDNA library: mu.L of the random ssDNA library (100. mu. mol/L) described above was mixed with 500. mu.L of borax-borate buffer (100mmol/L, pH 8.2), heat-denatured at 95 ℃ for 10min, immediately cooled in ice water for 10min to give a crustacean tropomyosin random ssDNA library, and the crustacean tropomyosin ssDNA library and tropomyosin were mixed in a final concentration ratio of 2: 1, mixing into a 200 mu L system, and incubating for 30min at 25 ℃ to obtain a mixed solution. Screening the mixed solution by a capillary electrophoresis method to obtain aptamers with nucleotide sequences of SEQ ID NO 1-n, carrying out PCR amplification on the aptamers by taking respective nucleotide sequences as primers, determining equilibrium dissociation constant, and selecting the aptamer with the smallest dissociation constant to obtain the aptamer with specificity combined with crustacean tropomyosin, namely the aptamer with the nucleotide sequence of SEQ ID NO 1-n
TABLE 1 aptamer sequences and dissociation constants
2. Preparation of aptamer probe:
preparing the carbon quantum dots: taking 6g of malic acid in a beaker with a cover, heating at 205 ℃ until the solid is completely melted, maintaining for 15min, cooling to room temperature, adding 50mL of 0.25M sodium hydroxide into the liquid, adjusting the pH of the solution to 6.0 with 0.05M NaOH, and dialyzing for 3d with a 1kDa dialysis membrane; and concentrating the carbon quantum dot solution at 40 ℃ by using a vacuum evaporator to obtain the carbon quantum dots.
Preparation of aptamer probe: mu.L of the carbon quantum dot after the spin evaporation is taken and put into a 1mL EP tube, 40 mu.L of 2 mu mol/L of the amino aptamer (SEQ ID NO:1) obtained above is added, and the mixture is incubated for 2 hours in a constant temperature shaking table at 25 ℃ to obtain the aptamer probe.
Example 3
Preparing the molecularly imprinted microspheres:
mixing 100mg of Fe3O4The magnetic microspheres and 20mg of Tropomyosin (TM) standard were dissolved in 20mL of 10mM Tris buffer (pH 8.0) and incubated with shaking at room temperature for 2 hours, then 40mg of dopamine (PDA) was added to the above mixed solution, incubation with shaking at room temperature was continued for 3 hours, after washing with distilled water sufficiently, the mixture was dissolved in PBS to a final concentration of 5mg/mL, and thereafter 0.71mM EKEKEKEPPPPC polypeptide solution was added in a volume ratio of 10:1 and incubated with shaking at room temperature for 24 hours. Washing the magnetic microspheres collected by the magnet with 3% (v/v) acetic acid solution containing 0.1% (w/v) SDS for 5 times, then thoroughly washing with distilled water for 8 times, and washing for multiple times to completely remove the template molecules (tropomyosin molecules) to obtain the molecularly imprinted microspheres (Fe)3O4@TM-PDA NPs)。
Example 4
1. Molecular imprinting microsphere element analysis
Elemental analysis of molecularly imprinted microspheres was performed with an Elementar Vario EL cube.
The results are shown in FIG. 2, Fe3O4The carbon content of the surface of the @ PDA NPs is about 8.35%. Thus, it was confirmed that Fe is present3O4The surface of the particles presents a PDA coating.
2、Fe3O4@ PDA NPs and molecularly imprinted microspheres (Fe)3O4@ TM-PDA NPs),
the results are shown in FIG. 3, Fe3O4@ PDA NPs specific imprinting Fe3O4The @ TM-PDA NPs microsphere is smoother, the shape of the microsphere is represented by a regular circle, and no cracks or ravines exist on the surface of the microsphere. As can be seen in FIG. 3b, the trace Fe3O4The surface of the @ TM-PDA NPs microspheres was shown to have cracks, which are indicated as binding sites for tropomyosin molecules.
3. Molecularly imprinted microspheres (Fe)3O4@ TM-PDA NPs) bind to tropomyosin statically
Suspending 2mg of the molecularly imprinted microspheres in 1.0mL of tropomyosin solutions (pH 7.5Tris buffer containing 0.01% SDS) with different concentrations, incubating the microspheres at room temperature with shaking for 2 hours, collecting the supernatant after magnetic separation, and measuring the tropomyosin content in the supernatant by using a Bradford kit; and calculating the bound tropomyosin content of the molecular imprinting microspheres by the difference between the total amount of the tropomyosin and the tropomyosin content of the supernatant.
The results are shown in FIG. 4, imprinting Fe3O4The amount of bound tropomyosin of @ TM-PDA NPs increases with the initial tropomyosin concentration, and the amount of protein adsorbed gradually reaches saturation as the protein concentration increases to a certain amount. Furthermore, imprinting Fe3O4@ TM-PDA NPs and Fe3O4The @ PDA NPs showed significant tropomyosin adsorption capacity compared to the previous sample. The static adsorption experimental data were further processed using Scatchard equation to evaluate the imprinting Fe3O4The ability of @ TM-PDA NPs to bind tropomyosin, and the analysis result shows that the imprinting Fe3O4The dissociation constant of @ TM-PDA NPs is 77.46. mu.g/mL, Fe3O4The dissociation constant of @ PDA NPs was 353.8. mu.g/mL, and the smaller the dissociation constant, the stronger the affinity was, and the imprinting Fe was observed3O4The affinity of @ TM-PDA NPs to TM is superior to that of Fe3O4@PDA NPs。
4. Molecularly imprinted microspheres (Fe)3O4@ TM-PDA NPs) selective recognition of allergen assays
For specificity testing, 2mg of the molecularly imprinted microspheres were suspended in 1.0mL of 2. mu.g/mL tropomyosin, bovine serum albumin, tropomyosin-depleted shrimp whole protein, chicken egg albumin, and shrimp arginine kinase solution (pH 7.5Tris buffer containing 0.01% SDS), incubated at room temperature for 2 hours with shaking, magnetically separated, and then the supernatant was extracted, collected, and the protein content in the supernatant was determined using the Bradford kit. The protein binding amount is the difference between the total mass of added protein and the mass of protein remaining in the solution after binding.
The results are shown in FIG. 5, in which the initial concentrations of 5 proteins were all 50. mu.g/mL. For Fe3O4For the @ PDA NPs, certain protein adsorption amount exists, but no obvious difference exists between different proteins, and the fact that the protein adsorption is non-specific adsorption is proved. For imprinting Fe3O4T test analysis and one-way variance analysis are carried out on the @ TM-PDA NPs group, and the analysis can know the imprinting Fe3O4@ TM-PDA NPs and Fe3O4@ PDA NPs have significant differences in the amount of tropomyosin bound (p < 0.05), and imprint Fe3O4The binding amount of the @ TM-PDA NPs tropomyosin is also significantly different from that of other proteins (p < 0.05), and the imprinting Fe3O4The highest ratio of the amount of bound tropomyosin of @ TM-PDA NPs compared to other proteins was up to 4.9 times. The above results show that the imprinting of Fe3O4The @ TM-PDA NPs recognize tropomyosin with high specificity.
5. Molecular imprinting-aptamer sandwich sensor specificity analysis
Taking molecular imprinting microspheres (Fe)3O4@ TM-PDA NPs)2mg was dissolved in PBS buffer, sonicated for 30min to ensure adequate dispersion of the molecularly imprinted microspheres, and a pre-diluted solution containing 2. mu.g of sample Protein (arginine kinase (AK) or Tropomyosin (TM), Bovine Serum Albumin (BSA) or chicken Ovalbumin (OVA) or whole Protein of shrimp (Protein without (TM)) was added and incubated in a shaker at 25 ℃ for 2 h. This is achieved byThen, 200pmol of aptamer fluorescent probe was added to the mixed system, followed by incubation with shaking at 25 ℃ for 1.5 hours. The fluorescence intensity at 480nm at an excitation wavelength of 370nm was measured by a microplate reader, and according to a protein concentration-fluorescence value standard curve y (22411 × x + 1556) (R20.9933) to determine the corresponding concentration, comparing the specificity of the detection method.
As shown in FIG. 6, the data obtained by adding the same amount of tropomyosin showed significant differences in fluorescence values compared with other proteins, indicating that other proteins did not significantly interfere with the detection of tropomyosin, demonstrating good method specificity. The assay data were analyzed using the software GraphPad Prism 7.00 for independent sample t-test and one-way anova with significant differences expressed as p <0.05, p <0.01, p <0.001, p < 0.0001.
Example 5
1. Detection of crustacean holoprotein tropomyosin:
extracting whole protein of crustacean (including Penaeus chinensis, Penaeus clarkii, Penaeus vannamei, Macrobrachium nipponensis and Penaeus prawni) and quantifying by BCA method, adding 1mL into the ultrasonically-mixed molecularly imprinted microspheres (Fe)3O4@ TM-PDA NPs), adding the aptamer probe, incubating for 1.5h, measuring the fluorescence intensity emitted at 480nm under the excitation of 370nm by using an enzyme-linked immunosorbent assay, and substituting the fluorescence value into a standard curve y ═ 22411 x +1556 (R)20.9933) the tropomyosin content in the actual sample was calculated.
The results are shown in table 2:
TABLE 2 detection of tropomyosin content in the Total protein
The results show that the detection result of the concentration of the tropomyosin and the proportion of the tropomyosin in the shrimp whole protein are similar to those described in other literatures, and certain differences exist probably because of inherent differences among different product batches and partial experimental errors, so that the detection method of the molecular imprinting-aptamer sandwich sensor is proved to have certain feasibility.
2. Detection of tropomyosin in shrimp-processed products.
Cutting processed shrimp products (shrimp dumpling, shrimp ball, shrimp slide, dried small shrimp and shrimp ball), placing into a beaker, and weighing. Subsequently, the leach solution (containing 50mM KCl and 2mM NaHCO) was added3The aqueous solution of (a) until no granular precipitate exists, leaching at 4 ℃ for 6h, centrifuging the leaching solution at 4 ℃ and 8000r/min for 20min, and collecting the supernatant.
Molecularly imprinted microspheres (Fe)3O4@ TM-PDA NPs), adding the supernatant of the processed shrimp product 1mL respectively, incubating for 2h, magnetically separating, adding 200pmol aptamer probe, incubating for 1.5h, washing with PBS solution three times, magnetically separating, re-dissolving in 1mL PBS solution, measuring the fluorescence emission intensity at 480nm under 370nm light excitation by an enzyme-linked immunosorbent assay, and using a standard curve y 22411 x +1556 (R)20.9933) the isolated tropomyosin content was calculated.
The results are shown in Table 3:
TABLE 3 detection of tropomyosin in shrimp processed products
The results of the detection of tropomyosin concentration indicate that the tropomyosin concentration is high in shrimp balls, shrimp slips and shrimp-flavored dumplings, particularly in shrimp balls, and this also relates to the processing raw materials and the processing manner of these shrimp-like processed products. Whereas a lower tropomyosin concentration was detected in the shrimp, possibly due to residual shrimp meat on the shrimp, the lower tropomyosin concentration also laterally demonstrates the detection accuracy of the molecularly imprinted-aptamer sandwich sensor herein.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above should not be understood to necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
SEQUENCE LISTING
<110> Zhejiang university of industry and commerce
<120> a sandwich-type sensor for detecting crustacean tropomyosin and applications thereof
<130> do not
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 40
<212> DNA
<213> Artificial sequence
<400> 1
gccaaaggag gcgagagata aaagattgcg aatccattcg 40
Claims (8)
1. A sandwich-type sensor for detecting crustacean tropomyosin, comprising molecularly imprinted microspheres and an aptamer probe having a nucleotide sequence set forth in SEQ ID NO. 1.
2. The sandwich sensor of claim 1 wherein the preparation of the molecularly imprinted microsphere comprises:
mixing Fe3O4Dissolving the magnetic microspheres and the tropomyosin standard substance in a Tris buffer solution, carrying out oscillation incubation for 2h at room temperature, adding dopamine, continuing oscillation incubation for 3h at room temperature, cleaning, dissolving in PBS, adding a polypeptide solution, carrying out oscillation incubation for 24h at room temperature, washing the magnetic microspheres with an acetic acid solution of SDS, and washing with distilled water to obtain the molecularly imprinted microspheres.
3. The sandwich sensor of claim 2 wherein 100mg Fe3O4The magnetic microspheres and 20mg tropomyosin standard were dissolved in 20mL 10mM Tris buffer pH 8.0.
4. The sandwich sensor of claim 2 wherein the amino acid sequence of the polypeptide is EKEKEKEPPPPC.
5. The sandwich sensor of claim 4 wherein 0.71mM of the polypeptide solution is added at a volume ratio of 10: 1.
6. The sandwich sensor of claim 2, wherein the dopamine is added in an amount of 40 mg.
7. The sandwich sensor of claim 2 wherein the collected magnetic microspheres are washed with 3% (v/v) acetic acid solution containing 0.1% (w/v) SDS.
8. A method for detecting crustacean tropomyosin using the sandwich sensor of any one of claims 1-7, comprising:
dissolving 2mg of molecularly imprinted microspheres in PBS buffer solution, performing ultrasonic treatment for 30min to ensure that the molecularly imprinted microspheres are fully dispersed, adding a sample solution to be detected, placing the sample solution in a shaking table at 25 ℃ for incubation for 2h, then adding 200pmol of nucleic acid aptamer probe, performing shaking incubation at 25 ℃ for 1.5h, washing the sample solution for three times, performing magnetic separation and redissolution in 1mL of PBS solution, and measuring the fluorescence emission intensity at 480nm under the excitation of 370nm light by using an enzyme labeling instrument.
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