Preparation of test strip for detecting tetrodotoxin based on magnetic fluorescence signal probe and Fab-tetrodotoxin-aptamer sandwich method

Abstract

The invention relates to a preparation method of a test strip for detecting tetrodotoxin based on a magnetic fluorescence signal probe and a Fab-tetrodotoxin-aptamer sandwich method. The preparation method comprises the following steps: preparing streptavidin magnetic beads with fluorescence signals through an amide reaction; the preparation method comprises the following steps: shearing an anti-tetrodotoxin monoclonal antibody stored in a laboratory by using papain to obtain a Fab fragment, biotinylating the Fab fragment, and then coupling the biotinylated Fab fragment with streptavidin magnetic beads with fluorescence signals through high affinity between biotin and streptavidin to obtain the magnetic fluorescence signal probe. The prepared signal probe has three functions: a matrix is separated and toxin is enriched through magnetic separation, the magnetic beads simultaneously generate a colorimetric signal and a fluorescence signal, and a biotin-streptavidin system amplifies a detection signal. In addition, the structure of the existing aptamer is optimized, and the obtained truncated aptamer is used for forming a detection line of the test strip. Tetrodotoxin is detected by using an antibody-toxin-aptamer sandwich method principle, so that non-specific binding can be effectively reduced, the anti-interference capability is improved, rapid qualitative and quantitative detection of a sample can be realized, and the kit has a good application prospect.

Description

TECHNICAL FIELD

[0001] The application relates to a preparation method of a test strip for detecting tetrodotoxin based on a magnetic fluorescent signal probe and a Fab-tetrodotoxin-aptamer sandwich method, in particular to a design of a magnetic fluorescent signal probe and a Fab-tetrodotoxin-aptamer sandwich method immunochromatography test strip. BACKGROUND

[0002] Tetrodotoxin (TTX) is a strong alkaloid marine toxin that can accumulate in puffer fish and other marine species. Tetrodotoxin is an amino full hydrogen quinazoline compound, which is one of the most toxic nerve toxins found in nature, and was once considered to be the most toxic non-protein toxin in nature. Tetrodotoxin has a local stimulating effect on the intestinal tract, and after absorption, it rapidly acts on nerve endings and nerve centers, can selectively and highly block the sodium ion channel on the nerve excitatory membrane, hinder nerve conduction, and thus cause nerve paralysis and death. Tetrodotoxin is very stable in chemical and thermal properties, and cannot be destroyed by general cooking methods such as salting or sunning. Only under alkaline conditions or at high temperature for more than 30 minutes can it be decomposed. The latent period of poisoning is very short, from 10-30 minutes to 3-6 hours, and if not rescued in time, the fastest death is within 10 minutes after poisoning, and the latest death is 4-6 hours. At present, there is no specific antidote for tetrodotoxin, and the main method to prevent poisoning is to limit the contact with contaminated food, so a sensitive, accurate and specific detection method is urgently needed for early detection of tetrodotoxin. The current conventional instrument methods for detecting tetrodotoxin in food, such as high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS / MS), have the advantages of high response sensitivity and accuracy, but have limitations such as expensive instruments, complex operation, high professional degree, long detection time, etc., and are not suitable for popularization. Therefore, it is the focus of research to construct a rapid, efficient, simple and sensitive method for detecting tetrodotoxin in samples on site. The immunochromatography test strip has the characteristics of low cost, simple operation, short detection time, high accuracy and the like, and is gradually favored by researchers and widely used in environmental monitoring, food detection, medical diagnosis and other fields.

[0003] Traditional immunochromatographic assay uses monoclonal antibody (mAb) as recognition element, which can accurately recognize target molecules and is the core of immunodetection. It plays an irreplaceable role in immunochromatographic assay and becomes the key raw material for early development of immunochromatographic test strip. However, with the increasing demand for immunochromatographic technology, the limitations of mAb are gradually exposed. The discovery and application of Fab fragment provide a new direction. From the structural characteristics, Fab fragment does not have Fc region, and its molecular weight is about 50 kDa, which is only one third of the complete monoclonal antibody. Compared with the complete mAb, Fab fragment has irreplaceable advantages in the application of immunochromatographic test strip: first, the smaller molecular volume significantly improves the chromatographic migration efficiency, which can quickly diffuse on the nitrocellulose membrane of the test strip, greatly shortening the detection reaction time and adapting to the core demand of instant detection; second, Fab fragment does not contain Fc segment, which can avoid non-specific binding mediated by Fc segment, reduce the false positive rate of test strip and improve the detection accuracy; third, the antigen binding region of Fab fragment is compact and easy to modify, which can efficiently couple with signal markers such as colloidal gold and fluorescent microspheres, enhance the signal response intensity of test strip and improve the detection sensitivity.

[0004] Due to its small size, small molecules are usually detected by competition method. However, the sensitivity of competitive immunoassay is insufficient, the batch-to-batch difference is large, the conditions are more difficult to optimize, and the preparation of toxin-signal molecule conjugate required by some immunoassay methods may also be challenging. Sandwich method is gradually favored due to its wider detection range and better specificity. There are many reports on antibody-based sandwich method immunochromatographic assay, which has shown great effect. As a biological recognition molecule, aptamer is almost suitable for any type of target, so it is considered as a substitute for antibody. Mixed antibody-aptamer sandwich system has become an attractive alternative, which combines the advantages of antibody and aptamer biological recognition molecules and the advantages of sandwich assay.

[0005] Based on the above background, a kind of test strip for detecting tetrodotoxin based on magnetic fluorescent signal probe and Fab-tetrodotoxin-aptamer sandwich method is designed. The streptavidin magnetic beads modified with fluorescent groups are coupled with biotinylated Fab antibody fragments. The signal probe prepared in this way has three functions: separating the matrix and enriching the toxin through magnetic separation, producing colorimetric and fluorescent signals at the same time, and amplifying the detection signal through the biotin-streptavidin system. In addition, the existing tetrodotoxin aptamer sequence is optimized in structure, and the antibody-toxin-aptamer sandwich method is designed to detect tetrodotoxin, which can effectively reduce non-specific binding and improve anti-interference ability. At present, there is no report on the design and synthesis of magnetic fluorescent signal probe and the use of structure-optimized aptamer to design Fab-toxin-aptamer sandwich method immunochromatographic test strip for the detection of tetrodotoxin. SUMMARY

[0006] The application relates to a test strip for detecting tetrodotoxin based on a magnetic fluorescent signal probe and a Fab-tetrodotoxin-aptamer sandwich method. Preparation of the magnetic fluorescent composite material: the magnetic fluorescent composite material is prepared through an amide reaction. Streptavidin magnetic beads are taken into a light-proof container, washed with 0.1-0.2 M PBS buffer solution for 2-3 times to remove the surface protective solution, separated on a magnetic stand and the supernatant is discarded, and then a certain volume of 0.1-0.2 M PBS buffer solution is added to resuspend the magnetic beads, so that the concentration is 0.5-1.5 mg / mL. Subsequently, esterified fluorescent dye is dissolved in anhydrous solvent to a concentration of 10-20 mM, and the solution is added to the pretreated magnetic bead solution at a molar ratio of 1:1-2, and then uniformly mixed by blowing and placed in a low-speed shaking bed for reaction at room temperature in the dark for 1-2 h. After the reaction is completed, 50-250 μL of an amino compound solution with a concentration of 0.05-0.2 M (final concentration of 10-50 mM) is added to the reaction system, and the amide reaction is terminated after incubation at room temperature in the dark for 15-30 min. The prepared mixture solution is placed on a magnetic stand, magnetically separated and the supernatant is discarded, and then the mixture is washed with washing buffer solution for 3-5 times, 0.1-0.2 M PBS buffer solution is added, and the mixture is stored at 2-8°C in the dark.

[0007] Preparation of the Fab antibody fragment: monoclonal antibody stored in the laboratory is placed in an enzyme digestion buffer (0.01-0.1 M PBS containing 0.2-2 mM EDTA, pH 7.0-7.5) and dialyzed overnight, the solution is replaced 2-3 times during the process, and the antibody concentration is adjusted to 0.5-2 mg / mL with the enzyme digestion buffer. Papain lyophilized powder is dissolved in the enzyme digestion buffer containing 1-5 mM cysteine (1-10 mg / mL), and activated at 25-37°C for 10-20 min. The activated papain solution is mixed with the monoclonal antibody solution at a ratio of 1:50-100, and reacted at 25-37°C for 6-8 h. Iodoacetamide is added and uniformly mixed to a final concentration of 10-20 mM, and then incubated at 25-37°C for 30-60 min. The solution is centrifuged at 4°C and 10000-20000 xg for 10-30 min, and the supernatant is taken. The Fab fragment is separated and purified through Protein A / G affinity chromatography, the penetration peak and the washing solution containing the Fab are combined, and the solution is concentrated to about 1 / 5-1 / 10 of the original volume through centrifugation at 2-8°C and 4,000-6000 xg by using a 30-50 kDa ultrafiltration tube, and the solvent is replaced with 0.01-0.1 M PBS (pH 7.0-7.5).

[0008] Preparation of the magnetic fluorescent signal probe: First, biotinylated Fab fragments are prepared. Biotinylation reagent is added dropwise to the Fab solution at a molar ratio of 1:5-20, and the reaction is carried out at room temperature in the dark for 30-60 min. Then, a termination solution with a final concentration of 50-100 mM is added to terminate the reaction for 10-20 min, and finally the sample is placed in 0.1-0.2 M dialysis solution for 8-12 h to remove free biotin and reaction byproducts, thereby obtaining biotinylated Fab. Then, an appropriate amount of magnetic fluorescent material solution (5-10 mg / mL) is taken, the supernatant is discarded after magnetic separation, and then a certain volume of deionized water is added. The biotinylated Fab antibody fragment (0.5-2.0 mg / mL) solution is mixed at a molar ratio of 1:1-2, incubated at room temperature for 60-90 min, and then again magnetic separation is performed to discard the supernatant. A resuspension solution (0.1-0.2 M PBS containing 1-2% BSA and 0.05-0.1% Tween-20) is added, and the magnetic fluorescent signal probe is obtained.

[0009] Design and synthesis of the aptamer: First, the secondary structure and three-dimensional structure information of the initial aptamer are obtained through the online platform mfold, and the three-dimensional structure model is obtained using Pymol. Then, tetrodotoxin and the aptamer are subjected to molecular docking using Autodock to obtain the binding site. Then, part of the redundant sequence not involved in binding is truncated, thereby obtaining the sequence of the truncated new aptamer. The truncated aptamer with 5' end modification of biotin is synthesized by Shanghai Generay Biotech Co., Ltd.

[0010] Preparation of the sandwich immunochromatographic test strip for tetrodotoxin detection: First, the sample pad is pretreated by soaking in buffer solution (0.5-1% BSA, 0.1-0.5% Tween-20, 0.5-0.8% NaCl in 0.01-0.05 M PBS, pH 7.0-7.5) for 20-30 min, and then vacuum-dried overnight at 20-40°C. Subsequently, streptavidin and biotinylated tetrodotoxin aptamers are mixed at a molar ratio of 1:5-10 and incubated at 2-8°C for 1-2 h. The resulting conjugate is sprayed onto a nitrocellulose membrane at a rate of 0.6-1.2 μL / cm to form the test line. Separately, 0.1-0.5 mg / mL anti-Fab mouse secondary antibody is sprayed onto a nitrocellulose membrane at a rate of 0.6-1.2 μL / cm to form the control line, with a distance of 3-8 mm between the test line and the control line. Then, the sample to be tested is mixed with a fluorescent magnetic signal probe in a centrifuge tube, incubated for 5-20 minutes, followed by magnetic separation to enrich the analyte. The supernatant is discarded, buffer solution is added again, and the mixture is thoroughly mixed before being dropped onto a sample pad. When the T line is not colored and the C line is yellow, the result is negative; when the T line is yellow and the C line gradually weakens until it disappears, the result is positive. After the color development is complete, the strip is irradiated with ultraviolet light to observe a gradient of fluorescence. After the test strip is completely dry, the T value is measured using a reader, and a standard curve is plotted to quantitatively detect the tetrodotoxin content in the sample.

[0011] The anhydrous solvent is one or more of dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile, and ethyl acetate.

[0012] The esterified fluorescent dye is one or more of the following series of dyes containing NHS ester groups: Fluorescein, Rhodamine B, Cyanine, FITC, Alexa Fluor, iFluor, and ATTO.

[0013] The amino-containing compound is one or more of glycine, ethanolamine, tris(hydroxymethyl)aminomethane, and lysine.

[0014] The biotinylation reagent is one or more of NHS-Biotin, Sulfo-NHS-Biotin, Sulfo-NHS-LC-Biotin, and Sulfo-NHS-SS-Biotin.

[0015] The termination solution is one or more of glycine, lysine, and Tris-HCl buffer.

[0016] The dialysate is one or more of PBS buffer or TBS buffer.

[0017] The initial aptamer sequence is: 5'-ATACCAGCTTATTCAATTTAATGCGGGGTGAGGCTCAATCAAGGAAA The truncated new aptamer sequence is: 5'-ATACCAGCTTATTCAATTTAATGCGGGGTGAGGCTCAATCAAGGAAAGATATAAGTAAGCAA-3' The present application relates to the immunochromatography test strip, and the magnetic fluorescent composite material is used as the Fab antibody fragment probe, and the Fab-toxin-aptamer sandwich method is used as the principle, compared with other immunochromatography test strips based on the colloidal gold and other marking carriers, and the antibody-toxin or aptamer-toxin is used as a single reaction, the prepared new immunochromatography test strip has the advantages of signal amplification, wide detection range, high sensitivity, fast response speed, good repeatability and high accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to specific embodiments of the present application and in conjunction with the drawings, wherein: Figure 1 It is a scanning electron microscope (SEM) image of the streptavidin magnetic beads used.

[0019] Figure 2 A is the chemical structure of tetrodotoxin.

[0020] Figure 2 B is the surface electrostatic potential analysis of tetrodotoxin.

[0021] Figure 2 C is the three-dimensional structure model of tetrodotoxin.

[0022] Figure 2 D is the secondary structure of the initial aptamer of tetrodotoxin.

[0023] Figure 2 E is the three-dimensional structure model of the initial aptamer of tetrodotoxin.

[0024] Figure 2 F is the molecular docking result of the initial aptamer of tetrodotoxin and tetrodotoxin.

[0025] Figure 2 G is the secondary structure of the truncated tetrodotoxin aptamer.

[0026] Figure 2 H is the secondary structure of the truncated tetrodotoxin aptamer.

[0027] Figure 2 I is the molecular docking result of truncated tetrodotoxin aptamer and tetrodotoxin.

[0028] Figure 3 is a schematic diagram of the sandwich method test strip for detecting TXX. DETAILED DESCRIPTION

[0029] The application will be described in detail below with specific examples: Examples

[0030] The specific steps are as follows: The magnetic fluorescent composite material was prepared by amide reaction. 10 μL of 10 mg / mL streptavidin magnetic beads was taken into a light-proof container, washed with 0.1 M PBS buffer for 3 times to remove the surface protective liquid, separated on a magnetic stand and the supernatant was discarded, then 90 μL of 0.1 M PBS buffer was added to resuspend the magnetic beads to a concentration of 1 mg / mL. Subsequently, NHS-Fluorescein was dissolved in anhydrous dimethyl sulfoxide to a concentration of 20 mM, and the solution was added to the pretreated magnetic bead solution at a molar ratio of 1:2, and then mixed by blowing and incubated in a low-speed shaking incubator at room temperature and in the dark for 1 h. After the reaction was completed, 100 μL of 0.1 M amino compound solution (final concentration of 10 mM) was added to the reaction system, and incubated at room temperature and in the dark for 30 min to terminate the amide reaction. The prepared mixture solution was placed on a magnetic stand, magnetically separated and the supernatant was discarded, washed with washing buffer for 3 times, 0.1 M PBS buffer was added, and stored at 4°C and in the dark.

[0031] The monoclonal antibody stored in the laboratory was dialyzed in enzyme digestion buffer (0.1 M PBS containing 2 mM EDTA, pH 7.4) overnight, and the solution was changed twice in the middle. The antibody concentration was adjusted to 1 mg / mL with enzyme digestion buffer. The papain freeze-dried powder was dissolved (2 mg / mL) with enzyme digestion buffer containing 2 mM cysteine, and activated at 37°C for 15 min. The activated papain solution was mixed with the monoclonal antibody solution at a ratio of 1:50, and reacted at 37°C for 6 h. Iodoacetamide was added and mixed to a final concentration of 20 mM, and incubated at 37°C for 30 min. Centrifugation was performed at 4°C and 10000 ×g for 30 min, and the supernatant was taken. The Fab fragment was separated and purified by Protein A / G affinity chromatography, the Fab-containing breakthrough peak and washing liquid were combined, and the volume was concentrated to 1 / 10 of the original volume by centrifugation at 4°C and 6000 ×g, and the solvent was replaced with 0.1 M PBS (pH 7.4).

[0032] First, biotinylated Fab fragments were prepared. Fab fragments were added dropwise to the Fab solution in a 1:10 molar ratio with NHS-biotin dissolved in anhydrous DSMO, and reacted at room temperature in the dark for 60 min. Then, a termination solution was added to a final concentration of 50 mM, and the reaction was terminated for 20 min. Finally, the free biotin and reaction byproducts were removed by placing the solution in 0.1 M dialysis fluid for 8 h, to obtain biotinylated Fab. Then, an appropriate amount of magnetic fluorescent material solution (10 mg / mL) was added, and the supernatant was removed by magnetic separation. Then, 100 μL of deionized water was added, and mixed with the biotinylated Fab antibody fragments (0.5 mg / mL) solution in a 1:2 molar ratio, and incubated at room temperature for 60 min. Then, the supernatant was removed by magnetic separation again, and a resuspension solution (0.1 M PBS containing 1% BSA and 0.05% Tween-20) was added, to obtain the magnetic fluorescent signal probe.

[0033] Synthesis of the aptamer: a truncated aptamer with a 5' end modified biotin was synthesized by Shanghai Generay Biotech Co., Ltd.

[0034] First, the sample pad was soaked in buffer (0.05 M PBS containing 1% BSA, 0.5% Tween-20, and 0.8% NaCl, pH 7.4) for 30 min for pretreatment, and vacuum dried at 37°C overnight. Then, streptavidin was mixed with biotin-conjugated tetrodotoxin aptamer at a molar ratio of 1:5 at 4°C for 2 h. The obtained conjugate was sprayed on the nitrocellulose membrane at a speed of 0.6 μL / cm to form the test line. Another 0.2 mg / mL anti-Fab mouse secondary antibody was sprayed on the nitrocellulose membrane at a speed of 0.6 μL / cm to form the control line, and the distance between the test line and the control line was kept at 6 mm. Then, the sample to be detected was mixed with the fluorescent magnetic signal probe in a centrifuge tube, and incubated for 10 min. After magnetic separation, the sample was discarded, and the buffer was added again. After mixing, the sample was added to the sample pad. When the T line did not develop color and the C line developed yellow color, the result was negative. When the T line was yellow and the C line gradually weakened until it disappeared, the result was positive. After the color development was completed, the gradient change in fluorescence was observed under UV light. After the test strip was completely dried, the T value was detected by a reading instrument, and a standard curve was drawn, so that the content of tetrodotoxin in the sample could be quantitatively detected. Example

[0035] The specific steps are as follows: The magnetic fluorescent composite material was prepared by amide reaction. 5 mg / mL streptavidin magnetic beads were taken into a light-proof container, washed twice with 0.2 M PBS buffer to remove the surface protective liquid, separated on a magnetic stand and the supernatant was discarded, and then 50 μL of 0.2 M PBS buffer was added to resuspend the magnetic beads to a concentration of 1 mg / mL. Subsequently, NHS-Rhodamine B was dissolved in N,N-dimethylformamide to a concentration of 20 mM, and the solution was added to the pretreated magnetic bead solution at a molar ratio of 1:1, and then mixed by blowing and shaking in a low-speed shaking incubator at room temperature in the dark for 2 h. After the reaction was completed, 200 μL of 0.05 M amino compound solution (final concentration 10 mM) was added to the reaction system, and the reaction was terminated after incubation at room temperature in the dark for 20 min. The prepared mixture solution was placed on a magnetic stand, the supernatant was discarded by magnetic separation, and the washing buffer was used to wash for 3 times. Then, 0.2 M PBS buffer was added, and the sample was stored at 4°C in the dark.

[0036] The monoclonal antibody stored in the laboratory was dialyzed in enzyme digestion buffer (0.01 M PBS containing 0.2 mM EDTA, pH 7.2) overnight, and the solution was replaced 3 times during the dialysis. The enzyme digestion buffer was used to adjust the antibody concentration to 0.5 mg / mL. The papain freeze-dried powder was dissolved in the enzyme digestion buffer containing 1 mM cysteine (1 mg / mL), and activated at 37°C for 20 min. The activated papain solution was mixed with the monoclonal antibody solution at a ratio of 1:100, and reacted at 37°C for 8 h. Iodoacetamide was added and mixed to a final concentration of 10 mM, and incubated at 37°C for 60 min. The solution was centrifuged at 20000 ×g at 4°C for 15 min, and the supernatant was taken. The Fab fragment was separated and purified by Protein A / G affinity chromatography, the Fab-containing elution peak and washing liquid were combined, and the solution was concentrated to about 1 / 5 of the original volume by centrifugation at 4°C and 6000 ×g using a 35 kDa ultrafiltration tube, and the solvent was replaced with 0.1 M PBS (pH 7.2).

[0037] First, biotinylated Fab fragments were prepared. The Fab fragments were added dropwise to a solution of Sulfo-NHS-Biotin dissolved in ultrapure water at a molar ratio of 1:20, and reacted at room temperature in the dark for 45 min. Then, a stop solution was added to a final concentration of 60 mM to terminate the reaction for 15 min, and finally the free biotin and reaction byproducts were removed by dialysis against 0.2 M dialysis solution for 12 h to obtain the biotinylated Fab. Then, an appropriate amount of magnetic fluorescent material solution (5 mg / mL) was added, and after magnetic separation, 200 μL of deionized water was added to mix with the biotinylated Fab antibody fragments (0.2 mg / mL) at a molar ratio of 1:3, and incubated at room temperature for 90 min. Then, the sample was again subjected to magnetic separation, and a resuspension solution (0.2 M PBS containing 2% BSA and 0.1% Tween-20) was added to obtain the magnetic fluorescent signal probe.

[0038] Synthesis of the aptamer: a truncated aptamer with a 5' end modified with biotin was synthesized by Shanghai Shengong Biotechnology.

[0039] First, the sample pad was soaked in buffer (0.01 M PBS containing 0.5% BSA, 0.1% Tween-20, and 0.5% NaCl, pH 7.2) for 30 min for pretreatment, and then vacuum dried at 37 °C overnight. Then, streptavidin was mixed with biotin-conjugated tetrodotoxin aptamer at a molar ratio of 1:8 at 4 °C for 1 h, and the resulting conjugate was sprayed on the nitrocellulose membrane at a speed of 0.8 μL / cm to form the test line. Another 0.1 mg / mL anti-Fab mouse antibody was sprayed on the nitrocellulose membrane at a speed of 0.8 μL / cm to form the control line, and the distance between the test line and the control line was kept at 4 mm. Then, the sample to be detected was mixed with the fluorescent magnetic signal probe in a centrifuge tube, and after incubation for 20 min, the sample was subjected to magnetic separation to enrich the detection substance, and the supernatant was discarded. The buffer was added again, mixed uniformly, and then added dropwise to the sample pad. When the T line did not develop color and the C line developed yellow color, the result was negative. When the T line was yellow and the C line gradually weakened until it disappeared, the result was positive. After the color development was completed, the gradient change in fluorescence was observed under UV light. After the test strip was completely dried, the T value was detected by a reading instrument and a standard curve was drawn, so that the content of tetrodotoxin in the sample could be quantitatively detected.

[0040] The prepared immunochromatographic test strip for detecting tetrodotoxin has the characteristics of high accuracy, wide linear range, and low detection limit. At the same time, the detection results of actual samples show that the prepared immunochromatographic test strip has very good practical application value.

[0041] The above examples are only for illustrating the present application, but not for limiting the present application. Based on the above description, many improvements and changes can be made to the present application. Within the scope of the appended claims, the present application can have other implementations different from the above description, and the selection of other reagents, adjustment of dispersion time and other methods are within the scope of the present application.

Claims

1. A test strip for detecting tetrodotoxin based on a magnetic fluorescent signal probe and a Fab-tetrodotoxin-aptamer sandwich method, comprising the following steps: (1) Preparation of magnetic fluorescent composite material: Magnetic fluorescent composite material was prepared by amide reaction. Streptavidin magnetic beads were added to a light-proof container and washed 2-3 times with 0.1-0.2 M PBS buffer to remove the surface protective solution. The beads were then separated on a magnetic rack and the supernatant was discarded. A certain volume of 0.1-0.2 M PBS buffer was added to resuspend the magnetic beads to a concentration of 0.5-1.5 mg / mL. The esterified fluorescent dye was then dissolved in an anhydrous solvent to a concentration of 10-20 mM. This solution was added to the pretreated magnetic bead solution at a molar ratio of 1:1-2. After mixing by blowing, the mixture was reacted in a low-speed shaking incubator at room temperature in the dark for 1-2 h. After the reaction was completed, 50-250 μL of an amino compound solution with a concentration of 0.05-0.2 M (final concentration 10-50 mM) was added to the reaction system and incubated at room temperature in the dark for 15-30 min to terminate the amide reaction. Place the prepared mixture solution on a magnetic rack, magnetically separate and discard the supernatant, wash 3-5 times with washing buffer, add 0.1-0.2 M PBS buffer, and store at 2-8°C protected from light. (2) Preparation of magnetic fluorescent signal probe: First, biotinylated Fab fragments were prepared. Biotinylation reagent and Fab fragment were added dropwise to Fab solution at a molar ratio of 1:5-20. The reaction was carried out at room temperature in the dark for 30-60 min. Then, a stop solution with a final concentration of 50-100 mM was added to terminate the reaction for 10-20 min. Finally, the solution was placed in 0.1-0.2 M dialysate for 8-12 h to remove free biotin and reaction byproducts, thus obtaining biotinylated Fab. Then, take an appropriate amount of magnetic fluorescent material solution (5~10 mg / mL), magnetically separate and discard the supernatant, add a certain volume of deionized water, and mix with biotinylated Fab antibody fragment (0.5~2.0 mg / mL) solution at a molar ratio of 1:1~2. Incubate at room temperature for 60~90 min, then magnetically separate again and discard the supernatant. Add resuspension (0.1~0.2 M PBS containing 1~2% BSA and 0.05~0.1% Tween-20) to obtain the magnetic fluorescent signal probe. (3) Design and synthesis of the aptamer: First, the secondary and three-dimensional structural information of the initial aptamer was obtained through the online platform mfold, and the three-dimensional structural model was obtained using Pymol. Then, the tetrodotoxin was molecularly docked with the aptamer using Autodock to obtain the binding site. Subsequently, some redundant sequences that did not participate in the binding were truncated to obtain the truncated new aptamer sequence. The truncated aptamer with biotin modified at the 5' end was synthesized by Shanghai Sangon Biotech. (4) Preparation of sandwich immunochromatographic test strips for tetrodotoxin detection: First, the sample pad is pretreated by soaking in buffer solution (0.01-0.05 M PBS containing 0.5-1% BSA, 0.1-0.5% Tween-20, and 0.5-0.8% NaCl, pH 7.0-7.5) for 20-30 min, and then vacuum dried overnight at 20-40 °C. Subsequently, streptavidin and biotin-modified tetrodotoxin aptamers are mixed at a molar ratio of 1:5-10 and incubated at 2-8 °C for 1-2 h. The resulting conjugate is sprayed onto a nitrocellulose membrane at a rate of 0.6-1.2 μL / cm to form a test line. Separately, spray 0.1–0.5 mg / mL of anti-Fab mouse secondary antibody onto a nitrocellulose membrane at a rate of 0.6–1.2 μL / cm to form a control line, maintaining a distance of 3–8 mm between the test line and the control line. Then, mix the sample to be tested with a fluorescent magnetic signal probe in a centrifuge tube, incubate for 5–20 min, perform magnetic separation to enrich the analyte, discard the supernatant, add buffer solution again, mix thoroughly, and then drop the mixture onto a sample pad. When the T line is colorless and the C line is yellow, the result is negative; when the T line is yellow and the C line gradually weakens until it disappears, the result is positive. After the color development is complete, irradiate with UV light to observe a gradient of fluorescence. After the test strip is completely dry, use a reader to measure the T value and plot a standard curve to quantitatively detect the tetrodotoxin content in the sample.

2. The test strip preparation for detecting tetrodotoxin based on a magnetic fluorescent signal probe and a Fab-tetrodotoxin-aptamer sandwich method according to claim 1, characterized in that, In step (1), fluorescent groups are modified on the surface of streptavidin magnetic beads by amide reaction, while retaining the binding site of streptavidin to biotin, thus obtaining a material that combines magnetism and fluorescence.

3. The preparation of a test strip for detecting tetrodotoxin based on a magnetic fluorescent signal probe and a Fab-tetrodotoxin-aptamer sandwich method according to claim 1, characterized in that, In step (2), the magnetic fluorescent material prepared in step (1) is coupled with the biotinylated Fab fragment to prepare a magnetic fluorescent signal probe for sandwich test strip detection.

4. The preparation of a test strip for detecting tetrodotoxin based on a magnetic fluorescent signal probe and a Fab-tetrodotoxin-aptamer sandwich method according to claim 1, characterized in that, The truncated new adaptation sequence mentioned in step (3) is 5'-ATACCAGCTTATTCAATTTAATGCGGGGTGAGGCTCAATCAAGGAAAGATATAAGTAAGCAA-3'.

5. The preparation of a test strip for detecting tetrodotoxin based on a magnetic fluorescent signal probe and a Fab-tetrodotoxin-aptamer sandwich method according to claim 1, characterized in that, In step (4), a signal-enhancing sandwich test strip is constructed using a specific Fab antibody fragment of tetrodotoxin and a truncated aptamer. The color of the test line can be observed with the naked eye, and the higher the concentration of toxin, the higher the color intensity. Subsequently, the concentration of tetrodotoxin can be further quantified by detecting the fluorescence intensity with an instrument.

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