A method for specifically detecting shrimp allergens using a label-free fluorescent sensor and a label-free fluorescent sensor

Abstract

The present invention discloses a method for specifically detecting shrimp allergens by a label-free fluorescence sensor, which comprises the following steps: forming an aptamer-complementary strand complex by binding a biotin-modified tropomyosin aptamer and a complementary strand, then adding streptavidin-modified magnetic nanoparticles for incubation and washing, obtaining an aptamer-complementary strand-magnetic nanoparticle complex, mixing it with a sample to be detected, then performing magnetic separation, sucking the supernatant, and adding melamine to the supernatant; after the incubation ends, adding MOPS and ascorbic acid for oscillation, adding CuSO4 to the oscillated mixture and incubating it in the dark; testing the fluorescence intensity of the mixture to obtain the content of shrimp allergens in the sample. The present invention also discloses a label-free fluorescence sensor. The present invention uses DNA as a template to synthesize copper nanoclusters with aggregation-induced emission characteristics as sensing signals, and uses aptamers as recognition elements, and has high sensitivity and specificity for the detection of shrimp allergens.

Description

Technical Field

[0001] The invention belongs to the technical field of food safety and rapid detection, and in particular relates to a method for specifically detecting shrimp allergens using a label-free fluorescent sensor and a label-free fluorescent sensor. Background Art

[0002] Food allergy refers to the abnormal reaction of the body's immune system to certain food proteins. When it occurs, it can affect multiple systems throughout the body. In severe cases, it can lead to anaphylactic shock and even endanger life. Tropomyosin (TM) is the main allergic protein in shrimp. It can cause a variety of allergic symptoms and seriously affect the quality of life of people with allergies. Therefore, it is extremely important to carry out allergen detection.

[0003] Allergen detection methods can be divided into immunology-based, molecular biology-based, mass spectrometry-based, and sensor-based methods according to different detection principles. Fluorescence sensing technology stands out in the field of food safety detection with its advantages of convenient detection, fast analysis speed, and high sensitivity. Commonly used recognition elements in fluorescent sensing systems include antibodies and aptamers. Aptamers are single-stranded oligonucleotides that can bind to target molecules with high specificity. Compared with antibodies, they have the advantages of easy synthesis, high stability, and good biocompatibility. They have been widely used in protein research, drug analysis, and other fields. After the aptamer specifically recognizes the target, it does not have the ability to convert the recognition information into a detectable signal. It is usually necessary to introduce a detectable signal into the aptamer, such as a fluorescent group. According to the fluorescent signal formed between the fluorescent group and the aptamer, it can be divided into labeled and label-free fluorescent sensors.

[0004] Patent CN114317543A discloses a fluorescent biosensor method for detecting α-lactalbumin by aptamers, which has the advantages of strong specificity and rapid detection, but requires the FAM fluorescent group to be labeled on the aptamer. Such labeling work not only increases the detection cost but also leads to a decrease in the affinity between the aptamer and the detection target. Compared with labeled sensors, label-free fluorescent sensors omit the cumbersome labeling process, are simple to prepare and have low detection costs. Label-free fluorescent signals are usually organic fluorescent dyes, inorganic quantum dots, metal nanoclusters, etc. Patent CN107389919B discloses a method for preparing a label-free fluorescent aptamer sensor and its application in shrimp allergenic proteins. The sensing system uses OliGreen fluorescent dye to achieve label-free detection of shrimp allergenic proteins. However, the photobleaching phenomenon of organic fluorescent dyes is relatively serious, and fluorescence quenching is very likely to occur at high concentrations or in an aggregated state, thereby limiting its development. Patent application CN111426667A discloses a fluorescence method for detecting β-lactoglobulin based on quantum dots-nucleic acid aptamers-graphene oxide, using CdTe quantum dots as fluorescent agents. Quantum dots have the advantages of strong stability and high photobleaching threshold, but quantum dots have strong cytotoxic effects and poor biocompatibility. Summary of the invention

[0005] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the object of the present invention is to provide a method for specifically detecting shrimp allergens using a label-free fluorescent sensor, which has high sensitivity and specificity for detecting shrimp allergens.

[0006] Another object of the present invention is to provide a label-free fluorescent sensor based on the method of specifically detecting shrimp allergens using the labeled fluorescent sensor.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A method for specifically detecting shrimp allergens using a label-free fluorescent sensor comprises the following steps:

[0009] (1) Preparation of aptamer-complementary chain-nanomagnetic bead complex:

[0010] The biotin-modified tropomyosin aptamer and the complementary chain are combined to form an aptamer-complementary chain complex, and then streptavidin-modified nanomagnetic beads are added for incubation and washing to obtain an aptamer-complementary chain-nanomagnetic bead complex;

[0011] (2) mixing the aptamer-complementary chain-nanomagnetic bead complex obtained in step (1) with the sample to be detected, and then performing magnetic separation, aspirating the supernatant, and adding melamine to the supernatant for incubation to obtain a reaction solution;

[0012] After the incubation, 3-(N-morpholino)propanesulfonic acid and ascorbic acid were added and shaken, and then CuSO4 was added and shaken again to obtain a mixed solution. After the mixed solution was incubated in the dark, CuNCs were formed in the mixed solution.

[0013] (3) Testing the fluorescence intensity of the mixed solution obtained in step (2), and obtaining the content of shrimp allergens in the sample to be tested based on quantitative analysis of the fluorescence intensity.

[0014] Preferably, the sequence of the tropomyosin aptamer is 5'-biotin-TACTAACGGTACAAGCTACCAGGCCGCCAACGTTGACCTAGAAGC ACTGCCAGACCCGAACGTTGACCTAGAAGC-3'; and the complementary strand is 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCTTCTAGGTCAA CGTT-3'.

[0015] Preferably, the step (1) of combining the tropomyosin aptamer and the complementary strand to form an aptamer-complementary strand complex comprises:

[0016] The tropomyosin aptamer and the complementary chain were diluted with Tris-HCl buffer respectively and then mixed, first heated at 94-96°C for 5-10 minutes, and then reacted at 3-5°C for 10-30 minutes to form an aptamer-complementary chain complex.

[0017] Preferably, the step (1) of adding streptavidin-modified nanomagnetic beads for incubation and washing to obtain an aptamer-complementary chain-nanomagnetic bead complex is specifically as follows: adding streptavidin-modified nanomagnetic beads pre-washed in phosphate buffer, then incubating at 3-5° C. for 1-2 h, washing the nanomagnetic beads with Tris-HCl buffer, and fixing the volume with Tris-HCl buffer to obtain a mixed solution of 100-500 nM aptamer-complementary chain-nanomagnetic bead complex.

[0018] Preferably, the magnetic separation in step (2) and the supernatant are drawn are specifically:

[0019] After incubation at 36-38° C. in a shaker for 1-2 hours, magnetic separation was performed, and the supernatant was aspirated and fixed to a volume to obtain a complementary chain (cDNA) concentration of 50-250 nM in the supernatant.

[0020] Preferably, the step (2) of adding melamine for incubation is specifically as follows:

[0021] Melamine is added to the supernatant and reacted at room temperature for 5 to 10 minutes to obtain a reaction solution; the concentration of melamine in the reaction solution is 10 to 50 nM.

[0022] Preferably, in step (2), 3-(N-morpholinyl)propanesulfonic acid and ascorbic acid are added for shaking, and then CuSO4 is added, specifically:

[0023] 10-50 nM 3-(N-morpholino)propanesulfonic acid (MOPS) and 0.1-0.5 M anhydrous antacid are added to the reaction solution of step (2), the mixture is shaken, and then 2-5 mM CuSO4 is added.

[0024] Preferably, the light-proof incubation in step (2) is specifically: reacting in the dark for 3 to 5 minutes.

[0025] Preferably, the fluorescence intensity of the mixed solution obtained in the test step (2) is specifically:

[0026] The fluorescence emission spectrum of the mixed solution obtained in step (2) at 650 nm was tested under an excitation wavelength of 340 nm.

[0027] A label-free fluorescent sensor, a method for specifically detecting shrimp allergens based on the label-free fluorescent sensor, comprising the aptamer-complementary chain-nanomagnetic bead complex;

[0028] When the label-free fluorescent sensor is used for detecting shrimp allergens, the method for specifically detecting shrimp allergens with the label-free fluorescent sensor is used.

[0029] The principle of the present invention is:

[0030] The aptamer in the sensor of the present invention is modified with biotin, and the complementary chain is divided into two parts, one part is a plurality of thymines, which serve as a synthesis template of CuNCs; the other part is the complementary sequence of the aptamer. According to the principle of base complementary pairing, the aptamer-complementary chain complex is first formed. Since the streptavidin is modified on the nanomagnetic beads, it can specifically bind to the biotin modified on the aptamer. Under the action of streptavidin-biotin, the aptamer-complementary chain-nanomagnetic bead complex is finally formed. When an allergen is present, since the affinity between the allergen and the aptamer is greater than the affinity between the aptamer and the complementary chain, the complementary chain is separated during the process of the allergen and the complementary chain competing for the aptamer. Through magnetic separation, the complementary chain remains in the supernatant. Since the polythymine in the complementary chain is the synthesis template of CuNCs, and melamine can combine with thymine through hydrogen bonds, the single-stranded polythymine forms a double chain. The rigid structure of the double-stranded polythymine-melamine complex improves the stability of the template, making the CuNCs formed after the addition of copper sulfate and ascorbic acid more aggregated, and the generated fluorescence signal changes more sensitively. If there is no allergen, there is no complementary chain in the supernatant, and no fluorescence signal will be generated after the addition of copper sulfate and ascorbic acid. Finally, the detection of allergens is achieved by constructing a quantitative relationship between the change in fluorescence signal and the concentration of allergens.

[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0032] (1) The method of the label-free fluorescent sensor for specific detection of shrimp allergens of the present invention uses DNA as a template to synthesize copper nanoclusters (CuNCs) with aggregation-induced emission properties as a sensing signal, and uses aptamers as recognition elements to construct a label-free fluorescent sensor to achieve specific detection of shrimp allergens. The detection of shrimp allergens has high sensitivity and specificity.

[0033] (2) The method of the present invention for specifically detecting shrimp allergens using a label-free fluorescent sensor enhances the rigidity of the DNA template by adding melamine and further improves the sensitivity of shrimp allergen detection by utilizing the aggregation-induced luminescence properties of metal nanoclusters.

[0034] (3) The label-free fluorescent sensor of the present invention specifically detects shrimp allergens, and reduces background fluorescence by adding nanomagnetic beads to quickly separate the complex.

[0035] (4) The method of the present invention for specifically detecting shrimp allergens using a label-free fluorescent sensor omits the tedious labeling process and avoids the problem of decreased affinity of the nucleic acid aptamer for the target substance caused by the labeling work. It has the advantages of simple operation, low detection cost and short detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1This is a schematic diagram of a method for specifically detecting shrimp allergens using a label-free fluorescent sensor according to an embodiment of the present invention.

[0037] Figure 2A The fluorescence excitation and emission spectra of CuNCs synthesized using the complementary chain (5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCTTCTAGGTCA ACGTT-3') as a template in an embodiment of the present invention; Figure 2B This is the UV absorption spectrum of CuNCs synthesized using the complementary chain (5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCTTCTAGGTCA ACGTT-3') as template.

[0038] Figure 3 This is a transmission electron micrograph of CuNCs synthesized using a complementary chain (5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCTTCTAGGTCA ACGTT-3') as a template according to an embodiment of the present invention.

[0039] Figure 4A The fluorescence emission spectra of the fluorescence sensor of the embodiment of the present invention in response to different concentrations of TM (the TM concentrations from bottom to top are 0, 0.05, 0.5, 1, 1.5, 2, 2.5, 3.5, and 5 μg / mL); Figure 4B This is the linear relationship between the fluorescence intensity at 650 nm and different TM concentrations.

[0040] Figure 5 The fluorescence response graphs of different proteins (TM, bovine serum albumin (BSA), soybean β-conglobulin, streptavidin, β-lactoglobulin, thrombin protein) under the fluorescence sensor of the embodiment of the present invention are shown.

[0041] Figure 6 Fluorescence intensity diagram of the label-free fluorescent sensor constructed using polythymine of different lengths as templates to specifically detect shrimp allergens.

[0042] Figure 7 4 is a comparison diagram of the effect of melamine on the fluorescence intensity of the fluorescence sensor according to the embodiment of the present invention. DETAILED DESCRIPTION

[0043] The present invention will be further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited thereto.

[0044] The method for specifically detecting shrimp allergens using a label-free fluorescent sensor according to an embodiment of the present invention comprises the following steps:

[0045] (1) Preparation of aptamer-complementary chain-nanomagnetic bead complex:

[0046] The biotin-modified tropomyosin aptamer and the complementary chain are combined to form an aptamer-complementary chain complex, and then streptavidin-modified nanomagnetic beads are added for incubation and washing to obtain an aptamer-complementary chain-nanomagnetic bead complex;

[0047] (2) adding different concentrations of TM to the aptamer-complementary chain-nanomagnetic bead complex obtained in step (1) to react, then performing magnetic separation, absorbing the supernatant, and adding melamine to the supernatant for incubation to obtain a reaction solution;

[0048] After the incubation, 3-(N-morpholino)propanesulfonic acid and ascorbic acid were added and shaken, and then CuSO4 was added and shaken again to obtain a mixed solution. After the mixed solution was incubated in the dark, CuNCs were formed in the mixed solution; the fluorescence intensity F of the mixed solution was detected. The detection model was constructed by linearly correlating different concentrations of TM with F;

[0049] (3) Detection of samples to be tested:

[0050] The sample to be tested is added to the aptamer-complementary chain-nanomagnetic bead complex obtained in step (1) to react, and then magnetic separation is performed, the supernatant is aspirated, and melamine is added to the supernatant for incubation to obtain a reaction solution;

[0051] After the incubation, 3-(N-morpholino)propanesulfonic acid and ascorbic acid are added and shaken, and then CuSO4 is added and shaken again to obtain a mixed solution. After the mixed solution is incubated in the dark, CuNCs are formed in the mixed solution. The fluorescence intensity of the obtained mixed solution is tested, and the content of shrimp allergens in the tested sample is obtained by quantitative analysis based on the fluorescence intensity and the detection model obtained in step (2).

[0052] In other embodiments of the present invention, a method for specifically detecting shrimp allergens using a label-free fluorescent sensor comprises the following steps:

[0053] (1) 10-50 μL of 1 μM aptamer and 10-50 μL of 1 μM complementary chain were mixed, heated at 95°C for 5-10 min, and then reacted at 4°C for 10-30 min to form an aptamer-complementary chain complex. Then, 10-50 μL of 1 mg / mL nanomagnetic beads pre-washed in phosphate buffer (0.01-0.1 mol / L, pH 7.5) were added, and the mixture was incubated at 4°C for 1-2 h. Then, the nanomagnetic beads were washed with Tris-HCl buffer (0.01-0.1 mol / L, pH 7.5) and the volume was fixed to 100 μL to obtain an aptamer-complementary chain-nanomagnetic bead complex.

[0054] (2) Add 100 μL of TM of different concentrations to the above-mentioned aptamer-complementary chain-nanomagnetic bead complex, incubate at 37°C in a shaker for 1-2 hours, then perform magnetic separation and aspirate the supernatant to make the volume 200 μL. Add 2 μL of 1-5 μM melamine to the constant volume solution, react at room temperature for 5-10 minutes, then add 10-50 nM MOPS and 0.1-0.5 M antacid and shake, then add 2-5 mM CuSO4 and shake again. The mixture is reacted in the dark for 3-5 minutes for fluorescence intensity measurement. Use a fluorescence spectrophotometer at room temperature to measure the fluorescence emission spectrum of the solution at 650 nm at an excitation wavelength of 340 nm, and use different concentrations of TM to linearly correlate with F to construct a detection model;

[0055] (3) Detection of samples to be tested:

[0056] The sample to be tested is added to the aptamer-complementary chain-nanomagnetic bead complex obtained in step (1) to react, and then magnetic separation is performed, the supernatant is aspirated, and melamine is added to the supernatant for incubation to obtain a reaction solution;

[0057] After the incubation, 3-(N-morpholino)propanesulfonic acid and ascorbic acid are added and shaken, and then CuSO4 is added and shaken again to obtain a mixed solution. After the mixed solution is incubated in the dark, CuNCs are formed in the mixed solution. The fluorescence intensity of the obtained mixed solution is tested, and the content of shrimp allergens in the tested sample is obtained by quantitative analysis based on the fluorescence intensity and the detection model obtained in step (2).

[0058] Figure 1The schematic diagram of the method for specifically detecting shrimp allergens by a label-free fluorescent sensor according to an embodiment of the present invention shows that: in this embodiment, biotin is modified on the aptamer in the sensor, and the complementary chain is divided into two parts, one part is 40 thymines, which serves as a synthesis template for CuNCs; the other part is the complementary sequence of the aptamer. According to the principle of base complementary pairing, the aptamer-complementary chain complex is first formed. Since streptavidin is modified on the nanomagnetic beads, it can specifically bind to the biotin modified on the aptamer. Under the action of streptavidin-biotin, the aptamer-complementary chain-nanomagnetic bead complex is finally formed. When allergens are present, since the affinity between the allergen and the aptamer is greater than the affinity between the aptamer and the complementary chain, the complementary chain is separated during the competition between the allergen and the complementary chain for the aptamer. Through magnetic separation, the complementary chain remains in the supernatant. Since the polythymine in the complementary chain is the synthesis template of CuNCs, and melamine can combine with thymine through hydrogen bonds to form a double-stranded polythymine, the rigid structure of the double-stranded polythymine-melamine complex improves the stability of the template, making the CuNCs formed after the addition of copper sulfate and ascorbic acid more aggregated, and the generated fluorescence signal changes more sensitively. If there is no allergen, there is no complementary chain in the supernatant, and no fluorescence signal will be generated after the addition of copper sulfate and ascorbic acid. Finally, the detection of allergens is achieved by constructing a quantitative relationship between the change in fluorescence signal and the concentration of allergens.

[0059] In order to better illustrate the effect of the method of the present invention for specifically detecting shrimp allergens using a label-free fluorescent sensor, the following experiments and tests were performed in this example:

[0060] Synthesis of CuNCs using DNA as template:

[0061] Take 5 μL of 1 μM complementary chain, add 283 μL of 10 mM MOPS, 4 μL of 0.1 M ascorbic acid, shake for 60 seconds, then add 8 μL of 2 mM CuSO4, continue shaking for 30 seconds, and then react for 5 minutes in the dark to obtain CuNCs. After the reaction is completed, the fluorescence excitation spectrum and emission spectrum of the solution are measured at room temperature using a fluorescence spectrophotometer, and the ultraviolet absorption spectrum of the solution is measured using a UV-visible spectrophotometer. Figure 2A It can be seen that the excitation wavelength of CuNCs is at 340nm and the emission wavelength is at 650nm. Figure 2B It can be seen that there is a characteristic absorption peak of CuNCs at 340nm. Figure 3 It can be seen that the morphology of CuNCs can be observed by transmission electron microscopy, thus proving the synthesis of CuNCs.

[0062] Establishment of a detection model for label-free fluorescent sensor to specifically detect shrimp allergens:

[0063] The streptavidin-modified nanomagnetic beads were pre-washed 3 times in phosphate buffer (20mM PBS, 100mM NaCl, 5mMMgCl2) and placed at 4°C for use. 20μL of 1μM biotin-modified aptamer was mixed with 20μL of 1μM complementary chain, first heated at 95°C for 10min, and then reacted at 4°C for 10min. Then 30μL of 1mg / mL pre-washed nanomagnetic beads were added to the mixture and incubated at 4°C for 60min. The reaction solution was then washed 4 times with 300μL Tris-HCl buffer (50mM Tris-HCl, 150mMNaCl, 2mM MgCl2) and fixed to 100μL to form an aptamer-complementary chain-nanomagnetic bead complex.

[0064] Take 100 μL of TM of different concentrations and mix it with the solution, place it in a shaker at 37°C and incubate it for 2 hours, then use magnetic separation to aspirate the supernatant and make it up to 200 μL. Add 2 μL of 1 μM melamine to the constant volume and react at room temperature for 5 minutes, then add 86 μL of 10mM MOPS and 4 μL of 0.1M ascorbic acid, shake for 60 seconds, then add 8 μL of 2mM CuSO4, continue shaking for 30 seconds, and finally let the mixed solution react for 5 minutes in the dark. Use a fluorescence spectrophotometer to measure the fluorescence emission spectrum of the solution at room temperature (excitation wavelength is 340 nm), and establish a standard curve for the specific detection of shrimp allergens by a label-free fluorescent sensor: F is the fluorescence intensity of the system at 650 nm; in the concentration range of 0.05-5 μg / mL TM, the value of F shows a good linear relationship with the TM concentration (such as Figure 4B As shown), the standard regression equation is Y=1068X+133 (where Y is the change in fluorescence intensity, X is the concentration of TM), R 2 =0.9952, and the minimum detection limit (S / N=3) is 25.25ng / mL. Compared with other current methods for detecting shrimp allergens, the method of the present invention, which uses DNA as a template to construct a label-free fluorescent sensor for specific detection of shrimp allergens, has high sensitivity. In addition, the method omits the cumbersome labeling process, does not require complicated preparations and means, is simple to operate, and has more application prospects.

[0065] Evaluation of the specificity of the label-free fluorescent sensor of the present invention:

[0066] Six different proteins, including TM, bovine serum albumin (BSA), soybean β-conglobulin, streptavidin, β-lactoglobulin, and thrombin protein, were selected to evaluate the selectivity of the constructed fluorescent sensing system. The selectivity evaluation experiment was carried out under the same sample treatment conditions as in Example 1, and the concentration of all proteins was 5 μg / mL. The results are shown in Figure 5As shown, BSA, soybean β-conglobulin, streptavidin, β-lactoglobulin, and thrombin protein had no effective response to the sensing system, while TM caused the fluorescence of the sensing system to turn on, proving that the sensing system has excellent selectivity and can be used for the specific detection of shrimp allergens.

[0067] The fluorescence sensing method was verified by spike recovery to be applicable to the detection of shrimp allergens in complex food systems:

[0068] 0.5 μg / mL, 1 μg / mL, and 5 μg / mL TM were added to mayonnaise diluted 100 times with Tris-HCl buffer to obtain spiked samples for detection. The content of shrimp allergens in mayonnaise samples was detected according to the method in Example 1. As shown in Table 1, the recovery rate of TM added to mayonnaise was between 94.0% and 102.0%, and the relative standard deviation was 3.1% to 5.2%. The results show that this method is suitable for detecting shrimp allergens in complex food matrices and has high accuracy and reliability.

[0069] Table 1: Detection of shrimp allergens in complex food systems using the fluorescent sensing strategy developed by the present invention (n=3)

[0070]

[0071] Comparison of fluorescence intensity of label-free fluorescent sensors specifically detecting shrimp allergens using polythymine of different lengths as templates:

[0072] The complementary sequence was replaced with the DNA in Table 2, and the corresponding CuNCs were prepared by adding 5 μg / mL TM in step 2 to detect the fluorescence value. The fluorescence results of CuNCs synthesized with polythymine of different lengths as templates were analyzed, as shown in FIG. Figure 6 As shown in the figure, the detection fluorescence value of CuNCs synthesized with DNA4 as template is stronger than that of DNA1, DNA2, and DNA3, and is not much different from that of DNA5. Therefore, from the perspective of fluorescence sensitivity and detection cost, the detection effect of DNA4 as template is the best.

[0073] Table 2: Complementary sequences formed by polythymine of different lengths

[0074]

[0075] To investigate the effect of melamine on the fluorescence intensity of the label-free fluorescent sensor for specific detection of shrimp allergens:

[0076] The control was made by not adding melamine and keeping other conditions unchanged, and the corresponding CuNCs were prepared to detect the fluorescence value. Referring to the method of specific detection of shrimp allergens by label-free fluorescence sensor in Example 2, the effect of melamine on CuNCs was analyzed. Figure 7 The results showed that the addition of melamine at different concentrations of TM could induce the aggregation of CuNCs and significantly enhance the fluorescence intensity, thereby improving the detection sensitivity and achieving better detection results.

[0077] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for specifically detecting shrimp allergens using a label-free fluorescent sensor, characterized in that: The following steps are involved: (1) Preparation of aptamer-complementary chain-nanomagnetic bead complex: The biotin-modified tropomyosin aptamer and the complementary chain are combined to form an aptamer-complementary chain complex, and then streptavidin-modified nanomagnetic beads are added for incubation and washing to obtain an aptamer-complementary chain-nanomagnetic bead complex; The sequence of the tropomyosin aptamer is 5'-biotin-TACTAACGGTACAAGCTACCAGGCCGCCAACGTTGACCTAGAAGCACTGCCAGACCCGAACGTTGACCTAGAAGC-3'; the complementary strand is 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCTTCTAGGTCAACGTT-3'; The step of combining the tropomyosin aptamer and the complementary chain to form an aptamer-complementary chain complex is specifically as follows: The tropomyosin aptamer and complementary chain were diluted with Tris-HCl buffer and mixed, first heated at 94-96°C for 5-10 min, and then reacted at 3-5°C for 10-30 min to form an aptamer-complementary chain complex; The step of adding streptavidin-modified nanomagnetic beads, incubating and washing to obtain an aptamer-complementary chain-nanomagnetic bead complex is specifically as follows: adding streptavidin-modified nanomagnetic beads pre-washed in phosphate buffer, then incubating at 3-5° C. for 1-2 h, washing the nanomagnetic beads with Tris-HCl buffer, and then constant-volume-adjusting with Tris-HCl buffer to obtain a 100-500 nM mixed solution of the aptamer-complementary chain-nanomagnetic bead complex; (2) mixing the aptamer-complementary chain-nanomagnetic bead complex obtained in step (1) with the sample to be detected, and then performing magnetic separation, aspirating the supernatant, and adding melamine to the supernatant for incubation to obtain a reaction solution; After the incubation, 3-(N-morpholino)propanesulfonic acid and ascorbic acid were added and shaken, and then CuSO4 was added and shaken again to obtain a mixed solution. After the mixed solution was incubated in the dark, CuNCs were formed in the mixed solution. (3) Testing the fluorescence intensity of the mixed solution obtained in step (2), and quantitatively analyzing the fluorescence intensity to obtain the content of shrimp allergens in the sample to be tested.

2. The method for specific detection of shrimp allergens by a label-free fluorescent sensor according to claim 1, characterized in that: Perform magnetic separation as described in step (2) and absorb the supernatant, specifically: After incubation at 36-38°C in a shaker for 1-2 h, magnetic separation was performed, the supernatant was aspirated, and the concentration of the complementary chain in the supernatant obtained after constant volume was 50-250 nM.

3. The method for specific detection of shrimp allergens by a label-free fluorescent sensor according to claim 2, characterized in that: The step (2) of adding melamine for incubation is specifically as follows: Melamine is added to the supernatant and reacted at room temperature for 5 to 10 minutes to obtain a reaction solution; the concentration of melamine in the reaction solution is 10 to 50 nM.

4. The method for specific detection of shrimp allergens by a label-free fluorescent sensor according to claim 3, characterized in that: In step (2), 3-(N-morpholinyl)propanesulfonic acid and ascorbic acid are added and shaken, and then CuSO4 is added, specifically: 10-50 nM 3-(N-morpholino)propanesulfonic acid and 0.1-0.5 M anhydrous hematoxylin are added to the reaction solution in step (2), the mixture is shaken, and then 2-5 mM CuSO4 is added.

5. The method for specific detection of shrimp allergens by a label-free fluorescent sensor according to claim 1, characterized in that: The light-proof incubation in step (2) is specifically: reacting in the dark for 3 to 5 minutes.

6. The method for specific detection of shrimp allergens by a label-free fluorescent sensor according to claim 1, characterized in that: The fluorescence intensity of the mixed solution obtained in the test step (2) is specifically: The fluorescence emission spectrum of the mixed solution obtained in step (2) at 650 nm was tested under an excitation wavelength of 340 nm.

7. A label-free fluorescent sensor, characterized in that: A method for specifically detecting shrimp allergens based on the label-free fluorescent sensor according to any one of claims 1 to 6, comprising the aptamer-complementary chain-nanomagnetic bead complex; When the label-free fluorescent sensor is used for detecting shrimp allergens, the method for specifically detecting shrimp allergens with a label-free fluorescent sensor according to any one of claims 1 to 6 is used.

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