A test strip for rapid limit determination of vomitoxin and its application

Abstract

The present application relates to the technical field of immunochromatographic detection, and particularly relates to a test strip for rapid limit determination of vomitoxin and application thereof, the test strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent paper, an immunological probe is prepositioned on the conjugate pad, the probe comprises a core-shell structure nanoparticle with physical coloration and enzyme-like catalytic activity and a recognition molecule modified on the surface of the nanoparticle, the nitrocellulose membrane is provided with a detection line coated with a vomitoxin-carrier protein conjugate and a quality control line coated with a quality control antibody; by adjusting the modification amount of the recognition molecule and the shell structure parameters, the judgment threshold values of the physical coloration and the catalytic coloration are accurately corresponding to 1000 µg / kg and 5000 µg / kg respectively. The present application does not need external instruments and is suitable for rapid screening in grain storage and processing sites.

Description

Technical Field

[0001] This invention relates to the field of immunochromatographic detection technology, and in particular to a test strip for rapid determination of vomitoxin limits and its application. Background Technology

[0002] Deoxynivalenol (DON), commonly known as vomitoxin, is a trichothecene compound produced by Fusarium fungi, primarily contaminating cereal crops such as wheat, corn, and barley. DON exhibits extremely high chemical and thermal stability, making it difficult to degrade using conventional food processing methods. Globally, the detection rate of DON in grains remains high. Once absorbed into the body through the food chain, DON can inhibit protein synthesis and induce toxic reactions such as vomiting, diarrhea, and immunosuppression, posing a serious threat to human health and livestock development.

[0003] Lateral flow immunoassay (LFIA) technology has become the mainstream tool for on-site screening of fungal toxins due to its advantages such as ease of operation, low cost, rapid response, and no need for complex instruments. Traditional lateral flow immunoassay strips mostly use colloidal gold nanoparticles (AuNPs) as markers. The presence or intensity of the physical color on the test line is observed with the naked eye and compared with a preset single threshold to obtain qualitative or semi-quantitative results.

[0004] Chinese Patent Application Publication No. CN104345145A, published on February 11, 2015, discloses a test strip for detecting vomitoxin and its application. The test strip includes a sample absorption pad, a conjugate release pad, a reaction membrane, an absorbent pad, and a base plate. The reaction membrane has a detection line coated with a vomitoxin hapten-carrier protein conjugate and a control line coated with goat anti-mouse antibody. The conjugate release pad is coated with a vomitoxin monoclonal antibody-colloidal gold label. This invention also provides a method for detecting vomitoxin residues in grains and feed using the above-mentioned vomitoxin test strip. However, the following problems still exist: First, colloidal gold can only provide a single colorimetric signal, and its color intensity can only reflect the competitive inhibition result at a single threshold concentration, making it difficult to simultaneously determine two different concentration limits on the same test strip. When faced with the dual requirement of determining the food limit of 1000 µg / kg and the feed limit of 5000 µg / kg, it is necessary to either prepare two test strips with different sensitivities for separate detection, or to perform gradient dilution of the sample extract before secondary detection, which is cumbersome and prone to introducing errors.

[0005] Second, this type of technology only uses colloidal gold as a passive chromogenic label, without utilizing functional signal modulation methods other than colloidal gold. The chromogenic sensitivity relies entirely on empirical matching of the amount of gold-labeled antibody and the antigen coating concentration, lacking a mechanism for precise and independent control of the detection threshold. When it is necessary to adjust the limit standard, all preparation parameters need to be systematically optimized again, resulting in insufficient versatility and flexibility.

[0006] Third, as the concentration of DON in the sample increases, the color of the detection line gradually fades until it disappears. The naked eye has limited ability to distinguish color changes in lighter areas, which can easily lead to interpretation errors near the threshold concentration. When the sample concentration is exactly around 1000 µg / kg, it is difficult for the operator to accurately determine whether the detection line has completely disappeared, increasing the risk of false positives or false negatives. In recent years, researchers have attempted to introduce nanozymes into lateral chromatography systems, utilizing their peroxidase-like activity to catalyze substrate color development and achieve signal amplification. However, existing nanozyme lateral chromatography techniques still mainly focus on improving detection sensitivity through signal enhancement, achieving only a single detection limit lower than traditional colloidal gold, and have not yet broken through the limitations of single threshold detection. This cannot meet the rapid grading requirements of the two step limit standards of 1000 µg / kg and 5000 µg / kg in the detection of vomitoxin. Summary of the Invention

[0007] Therefore, the present invention provides a test strip for rapid determination of vomitoxin limits and its application, in order to solve the technical problems in the prior art where colloidal gold test strips can only provide a single colorimetric signal, lack a dual threshold independent control mechanism, and have low accuracy of visual interpretation near the threshold, resulting in the inability to achieve rapid classification and determination of dual limits of vomitoxin on a single test strip.

[0008] On the one hand, the present invention provides a test strip for rapid determination of vomitoxin limits, the test strip comprising a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper; The binding pad is pre-positioned with an immune probe, which comprises Au@Pd core-shell nanoparticles and an anti-vomiting toxin monoclonal antibody modified on the surface of the nanoparticles. The core of the Au@Pd core-shell nanoparticles is a gold nanoparticle, and the outer shell is a palladium shell. The thickness of the palladium shell is obtained by controlling the final concentration of sodium tetrachloropalladium to 1.0 mM during the preparation process. The amount of anti-vomiting toxin monoclonal antibody modified on the surface of the nanoparticles is obtained by controlling the antibody addition amount to 0.75 µL / 1 mL of nanoparticle solution during the preparation process. The nitrocellulose membrane is provided with a detection line and a control line. The detection line is coated with a vomitoxin-carrier protein conjugate, and the control line is coated with a goat anti-mouse IgG polyclonal antibody. The carrier protein is bovine serum albumin. When the test strip is used for the detection of vomitoxin, the physical colorimetric visual judgment threshold corresponds to the first limit value, and the catalytic colorimetric visual judgment threshold corresponds to the second limit value.

[0009] Furthermore, the coating concentration of the vomitoxin-bovine serum albumin conjugate on the detection line is 0.25 mg / mL to 1.5 mg / mL, and the coating concentration of the goat anti-mouse IgG polyclonal antibody on the quality control line is 0.25 mg / mL to 1.5 mg / mL.

[0010] Furthermore, the coating concentration of the vomitoxin-bovine serum albumin conjugate on the detection line is 1.0 mg / mL, and the coating concentration of the goat anti-mouse IgG polyclonal antibody on the quality control line is 0.5 mg / mL.

[0011] Furthermore, the core-shell structured nanoparticles are prepared by a seed growth method. In a gold nanoparticle seed solution, ascorbic acid is used as a reducing agent to reduce the sodium tetrachloropalladium precursor. The shell thickness is adjusted by controlling the amount of sodium tetrachloropalladium.

[0012] On the other hand, the present invention provides a rapid detection kit for determining the limit of vomitoxin, the kit comprising the above-mentioned test strip and a catalytic substrate solution; the catalytic substrate solution comprises 0.03% hydrogen peroxide and 3% 3-amino-9-ethylcarbazole.

[0013] In another aspect, the present invention provides the application of the above-mentioned test strip in the preparation of products for rapid determination of vomitoxin limits.

[0014] Furthermore, the test strip determines whether the concentration of vomitoxin exceeds a first limit value and a second limit value through two-step readings; the first limit value is 1000µg / kg, and the second limit value is 5000µg / kg.

[0015] Furthermore, the two-step reading includes: first, without adding an exogenous catalytic substrate, determining whether the concentration of vomitoxin in the sample exceeds the first limit value by observing the physical color development result of the detection line; second, after completing the first step reading, applying a catalytic substrate solution to the test strip, and determining whether the concentration of vomitoxin in the sample exceeds the second limit value by observing the catalytic color development result of the detection line.

[0016] Compared with existing technologies, the advantages of this invention are as follows: This invention, for the first time, introduces core-shell structured nanoparticles with both physical colorimetric and enzyme-like catalytic activities as immunoprobes into a lateral chromatography test strip. Through a single chromatography step-by-step reading, it achieves rapid classification and determination of dual limits for vomitoxin, effectively solving the technical problem that traditional colloidal gold test strips can only perform single-threshold detection. This invention achieves separate calibration of two detection thresholds through the synergistic regulation of antibody modification amount and shell thickness; wherein the antibody modification amount mainly controls the physical colorimetric sensitivity, and the shell thickness mainly controls the catalytic colorimetric sensitivity, and both can be adjusted separately through orthogonal optimization, so that the two thresholds accurately correspond to 1000µg / kg and 5000µg / kg, respectively. Simultaneously, by introducing catalytic colorimetric signal amplification on the basis of physical colorimetry, this invention effectively improves the accuracy of interpretation near the threshold, solving the problem that traditional colloidal gold test strips are difficult to accurately interpret near the threshold concentration with the naked eye, and reducing the risk of false positives and false negatives.

[0017] Furthermore, the test strip provided by this invention does not require external instruments or sample dilution steps; detection can be completed in two steps with a single sample addition, making it suitable for rapid on-site screening scenarios such as grain storage and processing. By changing the recognition molecule and capture reagent, it can be extended to the dual-limit detection of other fungal toxins, demonstrating good versatility.

[0018] Furthermore, specificity experiments showed that the test strip of the present invention has high specificity for vomitoxin and is not affected by cross-interference from common fungal toxins such as zearalenone, aflatoxin B1, fumonisin B1, penicillin, and T-2 toxin; it has good intra-batch and inter-batch reproducibility, and its performance does not significantly decrease after 10 weeks of storage at room temperature; actual sample testing confirmed that the test results were in complete agreement with the LC-MS / MS confirmatory values. Attached Figure Description

[0019] Figure 1 The image shows the characterization of the gold nanoparticle core of the present invention; where a is a photograph of the gold nanoparticle solution, b is a UV-Vis absorption spectrum, c is a transmission electron microscope image, and d is a particle size distribution statistical diagram. Figure 2 Characterization diagrams of Au@Pd core-shell structured nanoparticles with different shell thicknesses according to the present invention are shown below; where a is a transmission electron microscope image, b is a particle size distribution statistical diagram, c is an energy dispersive X-ray spectrum, d is a UV-Vis absorption spectrum, and e is a palladium / gold elemental ratio diagram determined by inductively coupled plasma mass spectrometry. Figure 3 This is a graph verifying the enzyme catalytic activity of Au@Pd core-shell nanoparticles of the present invention; where a to h are kinetic curves of Au@Pd core-shell nanoparticles with different shell thicknesses under different hydrogen peroxide concentrations, i is a summary graph of the maximum reaction rate Vmax corresponding to different shell thicknesses, and j and k are reaction rate graphs. Figure 4 The diagram shows the optimization results of the preparation conditions of the immune probe and test strip of the present invention; where a is the optimization result of potassium carbonate dosage, b is the optimization result of bovine serum albumin blocking concentration, c is the optimization result of Au@Pd core-shell nanoparticle labeling concentration, d is the optimization result of control line coating concentration, and e is the optimization result of detection line coating concentration. Figure 5 This is a dual-parameter optimization matrix diagram of the immune probe of the present invention; where a is the physical colorimetric result diagram of different antibody amounts and shell thicknesses at 1000µg / kg, b is the catalytic colorimetric result diagram of different antibody amounts and shell thicknesses at 5000µg / kg, and c is the grayscale value analysis matrix diagram. Figure 6 This is a diagram illustrating the effect of the dual-limit detection method under optimal conditions according to the present invention; Figure 7 This is a diagram showing the specificity test results of the test strip of the present invention; Figure 8 This is a graph showing the results of intra-batch and inter-batch reproducibility tests for the test strips of this invention; Figure 9 This is a graph showing the storage stability test results of the test strips of this invention; Figure 10 This is a graph showing the experimental results of the test strip of the present invention on the spiked recovery of different grain matrices; Figure 11 This is a diagram showing the actual sample test results of the test strip of this invention; Figure 12 This is a comparative experimental result diagram of the test strip of the present invention and commercially available test strips; Figure 13 This is a schematic diagram illustrating the structure of the test strip of the present invention and the principle of dual-limit detection. Detailed Implementation

[0020] To make the objectives and advantages of the present invention clearer, the present invention will be further described below with reference to embodiments; it should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0021] Example 1: Preparation and characterization of gold nanoparticle cores Measure 150 mL of ultrapure water into a three-necked flask, set the oil bath temperature to 120 °C, and the stirring speed to 1500 rpm. After boiling for 1 hour, add 348 µL of 127 mM chloroauric acid hydrate, and after a 2-minute interval, quickly add 1 mL of 224 mM anhydrous trisodium citrate, and continue the oil bath for 1 hour. Then, lower the oil bath temperature to 90 °C and maintain it for 30 minutes.

[0022] Seed growth was then performed in four cycles: each time, 660 µL of 180 mM anhydrous trisodium citrate was added first, followed by 660 µL of 75 mM chloroauric acid after 2 minutes, with heating for 40 minutes per cycle. After four cycles, heating was stopped, and the mixture was cooled before adding 750 µL of 100 mg / mL BSPP (bis(p-sulfonylphenyl)phenylphosphine dihydrate) as a stabilizer. After standing overnight, the mixture was centrifuged for purification and then diluted to volume with 1 mg / mL BSPP solution to obtain the gold nanoparticle solution.

[0023] The prepared gold nanoparticles were characterized by UV-Vis absorption spectroscopy and transmission electron microscopy. UV-Vis absorption spectroscopy showed that the gold nanoparticle solution exhibited a sharp characteristic absorption peak at 520 nm, corresponding to the localized surface plasmon resonance effect of the gold nanoparticles. Figure 1 (b)). Transmission electron microscopy images show that the gold nanoparticles have a regular spherical structure with clear edges and good dispersion. Figure 1 (c) has an average particle size of 27.73 ± 0.13 nm. Figure 1 (d) The prepared gold nanoparticle solution was a uniform wine-red color. Figure 1 (a) shows that no precipitation or aggregation was observed after storage at room temperature in the dark for one month, indicating that the colloidal gold system has good dispersibility and stability. These results demonstrate that this embodiment successfully prepared gold nanoparticles with uniform size and good dispersibility, which can be used as ideal seed crystals for subsequent palladium shell growth.

[0024] Example 2: Preparation and characterization of Au@Pd core-shell nanoparticles with different shell thicknesses Taking the preparation of a 5 mL solution of Au@Pd core-shell nanoparticles as an example, the gold nanoparticle seed solution prepared in Example 1 with a final concentration of 5 nM, anhydrous trisodium citrate with a final concentration of 3 mM, and different volumes of 100 mM sodium tetrachloropalladium solution were added sequentially to a 10 mL round-bottom flask to achieve final concentrations of 0.2 mM, 0.5 mM, 1.0 mM, 1.4 mM, 2.0 mM, 4.0 mM, and 6.0 mM, respectively. Water was then added to a total volume of 5 mL. After stirring in an ice-water bath for 30 minutes, ascorbic acid with a final concentration of 30 mM was added dropwise as a reducing agent, and stirring was continued for 45 minutes. After the reaction was completed, BSPP was added to a final concentration of 0.5 mg / mL, and the mixture was incubated overnight. Then, the mixture was concentrated by centrifugation at 8000 r / min for 6 minutes and stored in a 4 °C refrigerator to obtain a series of Au@Pd core-shell nanoparticles with different shell thicknesses.

[0025] Au@Pd core-shell nanoparticles with different shell thicknesses were characterized by transmission electron microscopy, energy-dispersive X-ray spectroscopy, ultraviolet-visible absorption spectroscopy, and inductively coupled plasma mass spectrometry.

[0026] Transmission electron microscopy images showed that when the sodium tetrachloropalladium concentration was below 0.5 mM, no obvious shell formed on the surface of the gold nanoparticles; at a concentration of 1.0 mM, a smooth and continuous thin shell was observed on the surface of the gold nanoparticles, indicating good monodispersity; as the sodium tetrachloropalladium concentration gradually increased from 1.0 mM to 6.0 mM, the shell surface gradually transformed from a smooth thin layer to a polycrystalline or branched structure, and the particle diameter gradually increased from 27.85 ± 0.19 nm to 36.9 ± 0.16 nm. Figure 2 (a) and (b) in the text). Energy-dispersive X-ray spectroscopy further confirmed the simultaneous presence of gold and palladium. Figure 2 (c) In the text, UV-Vis absorption spectroscopy shows that as the thickness of the palladium shell increases, the characteristic absorption peak near 520 nm gradually broadens and redshifts. Figure 2 (d)). Inductively coupled plasma mass spectrometry results showed that the elemental ratio of palladium to gold gradually increased with increasing sodium tetrachloropalladium precursor concentration. Figure 2 (e)). The above results demonstrate that this embodiment successfully prepared a series of Au@Pd core-shell structured nanoparticles with different shell thicknesses, achieving precise control over the shell structure parameters.

[0027] Example 3: Verification of the enzyme-like catalytic activity of Au@Pd core-shell structured nanoparticles Take 1 mL of Au@Pd core-shell nanoparticle solutions with different shell thicknesses and absorbance of 1, corresponding to final sodium tetrachloropalladium concentrations of 0.2 mM, 0.5 mM, 1.0 mM, 1.4 mM, 2.0 mM, 4.0 mM, and 6.0 mM. Add 1 mM of 3,3',5,5'-tetramethylbenzidine and different concentrations of hydrogen peroxide, with final hydrogen peroxide concentrations of 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, and 40 mM. After mixing and reacting for 10 minutes, scan the absorbance at 652 nm over time using a UV spectrophotometer. Calculate the initial reaction rate based on the rate of change of absorbance per unit time, and calculate the maximum reaction rate Vmax for each material using the Michaelis-Menten equation.

[0028] The results show that Au@Pd core-shell nanoparticles with different shell thicknesses all exhibit typical Michaelis kinetics, and the reaction rate changes with substrate concentration according to the Michaelis equation ( ). Figure 3 (a)-(h), (j), and (k)). With increasing sodium tetrachloropalladium precursor concentration, the catalytic activity exhibits a trend of first increasing and then decreasing. The catalytic activity reaches its peak at a sodium tetrachloropalladium concentration of 2.0 mM, with Vmax of 0.244 ± 0.026 µM·s. -1When the concentration was further increased to 6.0 mM, the catalytic activity decreased. Figure 3 (i)). This result shows that the enzyme-like catalytic activity of Au@Pd core-shell nanoparticles can be precisely tuned by adjusting the amount of precursor in the shell, providing an experimental basis for adjusting the catalytic colorimetric sensitivity through shell structure parameters.

[0029] Example 4: Optimization of preparation conditions for immunoprobes and test strips 4.1 Coupling pH Optimization Take 1 mL of Au@Pd core-shell nanoparticle solution with absorbance of 1, and add different volumes of 0.1 M potassium carbonate solution to make final potassium carbonate concentrations of 0 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, and 1.0 mM, respectively. Then add 1 µL of anti-vomiting toxin monoclonal antibody. After standing for 60 minutes, add 100 µL of 10% bovine serum albumin solution to each vial, mix well, and let stand for 30 minutes. Centrifuge at 5000 r / min for 4 minutes, discard the supernatant, and resuspend in 100 µL of reconstitution solution containing 10 mM Tris-HCl, 10% sucrose, 1% bovine serum albumin, and 0.05% Tween-20. Evenly drop the resuspended immunoprobe solution onto the conjugate pad, dry at 37°C for 2.5 hours, and assemble test strips for detection.

[0030] The results showed that the color development of both the test line and the control line was weak without the addition of potassium carbonate; the test line and control line bands were clear and uniform with the best color development effect when the final concentration of potassium carbonate was 0.2 mM; with further increasing the amount of potassium carbonate, the color development of the test line and control line gradually weakened. Figure 4 (a)). Therefore, a final potassium carbonate concentration of 0.2 mM was chosen as the optimal coupling pH condition.

[0031] 4.2 Optimization of Bovine Serum Albumin Blocking Concentration Take 1 mL of Au@Pd core-shell nanoparticle solution with absorbance of 1, add 2 µL of 0.1 M potassium carbonate solution to adjust the pH, and then add 1 µL of anti-vomiting toxin monoclonal antibody. After standing for 60 minutes, add 100 µL of bovine serum albumin solutions with mass fractions of 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, and 4.0% respectively for blocking, mix well, and let stand for 30 minutes. Subsequent processing is the same as in Section 4.1, assemble the test strips for detection.

[0032] The results showed that when the bovine serum albumin concentration was below 1.0%, the release of the immune probe was hindered, the detection line color was weak, and the detection line color could still be observed in positive samples, indicating non-specific adsorption; when the bovine serum albumin concentration was 2.0%, the immune probe was released completely and non-specific adsorption was effectively suppressed, and the detection line and control line color were clear. Figure 4 (b) In this context, when the concentration exceeds 4.0%, the increased viscosity of the system leads to a decrease in the chromatography rate, and uncontrolled aggregation of the immunoprobe occurs. Therefore, a bovine serum albumin concentration of 2.0% by mass was selected as the optimal blocking concentration.

[0033] 4.3 Optimization of Immune Probe Dosage The absorbance A520 of the Au@Pd core-shell nanoparticle solution was diluted to six gradients: 0.25, 0.5, 0.75, 1.0, 1.25, and 1.5. Immunoprobes were prepared and test strips were assembled according to the optimal conditions determined in Sections 4.1 and 4.2 for detection.

[0034] The results showed that when the absorbance was 0.25 and 0.5, the detection line and control line were lightly colored and difficult to interpret with the naked eye; when the absorbance was 1.0, the background was clean and the detection line and control line were clear and sharp, with the best color development effect. Figure 4 (c) In the section on absorbance, when the absorbance is 1.25 and 1.5, excessive nanomaterials cause background residue during chromatography, interfering with result interpretation. Therefore, an absorbance of 1.0 was selected as the optimal amount of immunoprobe.

[0035] 4.4 Optimization of Coating Concentration for Quality Control Line Goat anti-mouse IgG polyclonal antibody at concentrations of 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1.0 mg / mL, 1.25 mg / mL, and 1.5 mg / mL was coated at the control line, and 0.5 mg / mL of vomitoxin-bovine serum albumin conjugate was coated at the control line. The strips were dried at 37°C for 2.5 hours and then assembled for testing.

[0036] The results showed that the color intensity of the control line increased with increasing goat anti-mouse IgG coating concentration. At a coating concentration of 0.25 mg / mL, the color was too faint to serve as an effective quality control reference; at a coating concentration of 0.5 mg / mL, the color was clear and uniform, and did not affect the visual interpretation of the test line. Figure 4 (d) When the concentration is higher than 1.0 mg / mL, it will competitively bind too much of the immune probe, leading to weakened color development of the detection line. Therefore, 0.5 mg / mL was selected as the optimal coating concentration of goat anti-mouse IgG for the quality control line.

[0037] 4.5 Optimization of detection line coating concentration The test strips were coated with 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1.0 mg / mL, 1.25 mg / mL and 1.5 mg / mL of vomitoxin-bovine serum albumin conjugate, respectively, and the control strips were coated with 0.5 mg / mL of goat anti-mouse IgG polyclonal antibody. The strips were dried at 37°C for 2.5 hours and then assembled for testing.

[0038] The results showed that the detection line was too faint at a coating concentration of 0.25 mg / mL, making it difficult to visually observe negative results; while the detection line was vibrant and easy to interpret visually at a coating concentration of 1.0 mg / mL, and also exhibited sufficient competitive sensitivity to both limit concentrations. Figure 4 (e) When the coating concentration is higher than 1.25 mg / mL, too many antigen sites are immobilized on the detection line, resulting in weak competitive inhibition and decreased sensitivity. Therefore, 1.0 mg / mL was selected as the optimal coating concentration for detecting the vomitoxin-bovine serum albumin conjugate.

[0039] Example 5: Dual-parameter regulation—determination of optimal antibody labeling amount and optimal shell thickness This embodiment systematically examines the effects of combinations of Au@Pd core-shell nanoparticles with different shell thicknesses and different amounts of recognition molecule modifications on the physical and catalytic colorimetric thresholds by constructing a multidimensional orthogonal optimization matrix. The aim is to determine the optimal parameter combination that allows the physical colorimetric visual judgment threshold to correspond to the first limit value of 1000 µg / kg and the catalytic colorimetric visual judgment threshold to correspond to the second limit value of 5000 µg / kg.

[0040] Take 1 mL of Au@Pd core-shell nanoparticle solutions with different shell thicknesses and absorbance of 1. The shell thicknesses correspond to final sodium tetrachloropalladium concentrations of 0.2 mM, 0.5 mM, 1.0 mM, 1.4 mM, and 2.0 mM, respectively. Add 2 µL of 0.1 M potassium carbonate solution to adjust the pH, and then add 0.25 µL, 0.5 µL, 0.75 µL, 1.0 µL, 1.25 µL, 1.5 µL, 1.75 µL, 2.0 µL, 2.25 µL, and 2.5 µL of anti-vomiting toxin monoclonal antibody, respectively. After standing for 60 minutes, add 100 µL of 2.0% bovine serum albumin solution to each vial, mix well, and let stand for 30 minutes. Centrifuge at 5000 r / min for 4 minutes, discard the supernatant, resuspend in 100 µL of reconstitution solution, and evenly drop the resuspended immunoprobe solution onto the conjugate pad. Dry at 37°C for 2.5 hours and assemble the test strip.

[0041] Immunochromatographic detection was performed using standard solutions of vomitoxin at concentrations of 1000 µg / kg and 5000 µg / kg, respectively. The gray values ​​of the detection lines were read, and a result analysis matrix was plotted. Figure 5(a)-(c)). The figure uses different shades of color to represent different ranges of grayscale values, showing the changing trend of the dual-indicator control.

[0042] The results showed that the amount of antibody modification modulates the physical colorimetric sensitivity: when the amount of antibody added was low, the binding force between the probe and the antigen on the detection line was insufficient, and the color of the detection line was faint; as the amount of antibody increased, the color intensity of the detection line tended to stabilize. Figure 5 (a)). Meanwhile, Au@Pd core-shell nanoparticles with different shell thicknesses exhibit varying affinities for antibodies; thicker shells result in better affinity and correspondingly increased physical colorimetric sensitivity. Shell thickness modulates catalytic colorimetric sensitivity: under the same antibody modification, thicker shells lead to higher catalytic activity and deeper color after catalytic colorimetry. Figure 5 (b) in the figure. The grayscale value analysis matrix further verifies the changing trend of the above dual-indicator regulation. Figure 5 (c) in the middle.

[0043] Considering both visually apparent colorimetric depth and catalytic effect, Au@Pd core-shell nanoparticles prepared with 1.0 mM sodium tetrachloropalladium and 0.75 µL of antibody labeling were selected as the optimal detection system. With this combination, the visual threshold for physical colorimetry precisely corresponds to the first limit of 1000 µg / kg, and the visual threshold for catalytic colorimetry precisely corresponds to the second limit of 5000 µg / kg.

[0044] It should be noted that although the optimization of parameters such as pH, BSA blocking concentration, and probe dosage in Examples 4.1-4.3 was performed using an antibody addition of 1 µL, these parameters are mainly determined by the properties of the nanoparticles themselves: pH optimization depends on the matching relationship between the isoelectric point of the antibody and the surface charge of the nanoparticles; when the antibody addition is adjusted from 1 µL to 0.75 µL (a change of 25%), the optimal pH condition does not shift; the BSA blocking concentration depends on the total specific surface area of ​​the nanoparticles and is not directly related to the antibody modification amount; the probe dosage (absorbance A520 = 1.0) is an optimization of the nanomaterial's own concentration and is independent of the antibody modification amount. Furthermore, the multidimensional orthogonal optimization matrix of Example 5 shows that, at the optimal antibody amount of 0.75 µL, using the pH, BSA blocking concentration, and probe dosage conditions determined in Example 4, both limit thresholds accurately correspond to the target values, verifying that the above parameters are still applicable at the optimal antibody amount.

[0045] Example 6: Complete process and effect demonstration of the dual-limit detection method Under the optimized conditions determined in Example 5, namely Au@Pd core-shell nanoparticles prepared with 1.0 mM sodium tetrachloropalladium, 0.75 µL of antibody labeling, a detection line coating concentration of 1.0 mg / mL, and a control line coating concentration of 0.5 mg / mL, a series of vomitoxin standards of different concentrations were tested to verify the dual-limit detection effect of the present invention.

[0046] Sample pretreatment: Accurately weigh 5.0 g of the pulverized grain sample into a 50 mL centrifuge tube, add 25 mL of sample diluent containing 10 mM phosphate buffer and 0.05% Tween-20 (v / v), and vortex for 1–2 minutes. Take 1 mL of the mixture and centrifuge at 4000 r / min for 5 minutes, then collect the supernatant for analysis.

[0047] Detection Procedure: Add 60 µL of the supernatant to the sample pad of the test strip. The liquid will migrate forward under capillary action. After 10 minutes of chromatography, take the first reading. Directly observe the color of the test line to determine if the concentration of vomitoxin in the sample exceeds the first limit of 1000 µg / kg. After completing the first reading, add a catalytic substrate solution to the sample pad of the same test strip. The catalytic substrate solution contains 0.03% hydrogen peroxide and 3% 3-amino-9-ethylcarbazole. After 15 minutes, take the second reading and observe if the test line shows catalytic color development to determine if the concentration of vomitoxin in the sample exceeds the second limit of 5000 µg / kg.

[0048] Comprehensive judgment criteria: If the physical color development of the first step reading detection line is visible, the concentration of vomitoxin in the sample is determined to be less than 1000 µg / kg; if the physical color development of the first step reading detection line disappears but the catalytic color development of the second step reading detection line is visible, the concentration of vomitoxin in the sample is determined to be between 1000 µg / kg and 5000 µg / kg; if neither the first nor the second step reading detection lines show color development, the concentration of vomitoxin in the sample is determined to be not less than 5000 µg / kg.

[0049] The results showed that when the concentration of vomitoxin was in the range of 0 µg / kg to 900 µg / kg, the physical color development of the detection line was clearly visible; when the concentration reached 1000 µg / kg, the physical color development basically disappeared; when the concentration was in the range of 1000 µg / kg to 4000 µg / kg, the physical color development disappeared, but the detection line was clearly brown after catalytic color development; when the concentration was in the range of 4000 µg / kg to 5000 µg / kg, the physical color development disappeared, and the catalytic color development was still visible but gradually weakened; when the concentration reached 5000 µg / kg and above, the catalytic color development also disappeared. Figure 6The results show that the two critical points of physical color disappearance and catalytic color disappearance correspond to the first limit value of 1000µg / kg and the second limit value of 5000µg / kg, respectively, and the interpretation boundaries at the two limit points are clear, proving that the test strip of the present invention can achieve rapid classification and determination of dual limits for vomitoxin on a single test strip.

[0050] Example 7: Test strip performance verification 7.1 Specificity Test Common mycotoxins found in grains were selected as interfering agents, including zearalenone, aflatoxin B1, fumonisin B1, penicillin, and T-2 toxin. The concentration of each interfering agent was set to 50,000 µg / kg using sample diluent. A 5,000 µg / kg vomitoxin standard was used as a positive control, and a sample diluent containing no toxins was used as a negative control. 60 µL of each sample solution was added to the sample pad of the test strip, and the colorimetric results were observed after 10 minutes of chromatography.

[0051] The results showed that, except for the positive control group for vomitoxin where the color development of the test line was completely inhibited, the test lines of all other interfering groups showed clear purplish-red bands that were indistinguishable from those of the negative control group. Figure 7 This indicates that the test strip of the present invention has high specificity for vomitoxin and no cross-reactivity with common fungal toxins such as zearalenone, aflatoxin B1, fumonisin B1, penicillin, and T-2 toxin.

[0052] 7.2 Reproducibility Test The vomitoxin standard was diluted to six concentration gradients: 0 µg / kg, 500 µg / kg, 1000 µg / kg, 2000 µg / kg, 4000 µg / kg, and 7500 µg / kg, using sample diluent. Test strips were randomly selected from the same batch and tested at each concentration, with three replicates per concentration to assess intra-batch reproducibility. Test strips were also randomly selected from two different batches and tested using the same procedure to assess inter-batch reproducibility.

[0053] The results showed that, at the same spiked concentration, the color depth of the detection lines in each parallel test group was highly consistent, especially at the two key judgment thresholds of 1000 µg / kg and 5000 µg / kg, where all replicate samples showed consistent interpretation results. Figure 8 This indicates that the test strips of the present invention exhibit good intra-batch and inter-batch reproducibility, and that the preparation process is stable and reliable.

[0054] 7.3 Stability Testing Test strips prepared in the same batch were sealed in aluminum foil bags containing desiccant and stored at room temperature (25°C). The test strips were removed at 0, 2, 4, 6, 8, and 10 weeks of storage, and tested for vomitoxin standards at four concentrations: 0 µg / kg, 750 µg / kg, 3750 µg / kg, and 7500 µg / kg. Each concentration was tested in triplicate.

[0055] The results showed that during the 10-week observation period, the color intensity of the test line on the test strips did not differ significantly from the initial value, the background remained clean, and no nonspecific color development occurred. Figure 9 This indicates that the antibody in the test strip of the present invention is tightly coupled with the Au@Pd core-shell structured nanoparticles, and its biological activity is effectively maintained during storage, demonstrating good shelf-life stability.

[0056] 7.4 Matrix Spike Recovery Experiment Three representative grains—corn, barley, and wheat—were selected as blank matrices. 5.0 g of the pulverized sample was accurately weighed into a 50 mL centrifuge tube, and vomitoxin standard was added to achieve final concentrations of 0 µg / kg, 750 µg / kg, 3750 µg / kg, and 7500 µg / kg, respectively. 25 mL of sample diluent was added, and the mixture was vortexed for 1–2 minutes. 1 mL of the mixture was centrifuged at 4000 rpm for 5 minutes, and the supernatant was collected for analysis according to the procedure in Example 6. Three replicates were performed for each concentration.

[0057] The results showed that the negative sample backgrounds of all three matrices were clean, with no significant matrix interference. At the same spiked concentration, the colorimetric intensities of the test and control lines were essentially the same for all three matrices, and the colorimetric gradients perfectly matched the theoretical spiked concentrations. Figure 10 This indicates that the test strip of the present invention has excellent matrix compatibility, can overcome matrix interference from a variety of common grains, and can achieve accurate detection of vomitoxin in different grains without complicated purification steps.

[0058] 7.5 Actual Sample Testing To verify the accuracy of the test strip of the present invention in practical applications, low-concentration positive samples (444.18±83.51µg / kg) and high-concentration positive samples (3987.76±781.60µg / kg) confirmed by LC-MS / MS were selected for testing. The sample pretreatment and detection steps were the same as in Example 6. Each sample was tested three times, and the results were compared with those of vomitoxin standards at 1000µg / kg and 5000µg / kg.

[0059] The results showed that physical color development was visible in low-concentration positive samples, indicating a vomitoxin concentration below 1000 µg / kg; physical color development disappeared in high-concentration positive samples, but catalytic color development was visible, indicating a vomitoxin concentration between 1000 µg / kg and 5000 µg / kg. Figure 11 The above interpretation results are in complete agreement with the LC-MS / MS confirmatory values, proving that the test strip of the present invention has good accuracy and practicality in actual sample testing.

[0060] Example 8: Assembly and preparation method of test strips This embodiment provides a complete method for assembling lateral chromatography test strips.

[0061] Sample pads and absorbent paper were cut to 15mm×300mm and 20mm×300mm dimensions, respectively. A glass fiber membrane was cut to 8mm×300mm as a binding pad, and a nitrocellulose membrane was cut to 25mm×300mm. The nitrocellulose membrane was adhered to the central area of ​​a PVC substrate. Detection lines and control lines were set on the nitrocellulose membrane, with a 4mm spacing between them. The detection lines were coated with 1.0 mg / mL of vomitoxin-bovine serum albumin conjugate, and the control lines were coated with 0.5 mg / mL of goat anti-mouse IgG polyclonal antibody. Streaking was performed using a three-dimensional gold sputtering apparatus at a speed of 1µL / cm, followed by drying at 37℃ for 2.5 hours.

[0062] The immunoprobe solution prepared according to the optimized conditions determined in Example 5 was adjusted to the working concentration with a reconstitution solution and uniformly dropped onto a glass cellulose membrane. The amount of immunoprobe loaded per centimeter of the conjugate pad was 50 µL, calculated based on absorbance A520 = 1.0. The membrane was then dried at 37°C for 2.5 hours to obtain the conjugate pad. The sample pad was then soaked in a sample pad treatment solution and dried at 37°C for 12 hours for later use. The sample pad treatment solution contained 10 mM phosphate buffer, 2% bovine serum albumin (w / w), and 0.05% Tween-20 (v / v).

[0063] On a PVC base plate, the sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper are sequentially overlapped from bottom to top along the sample flow direction, with each component overlapping by 1mm to 2mm to ensure the continuity of the chromatography solvent flow. The assembled test strip is then cut into 4mm wide strips using a microcomputer-controlled automatic cutter and sealed in an aluminum foil bag containing desiccant for storage.

[0064] When using the test strip prepared according to the above method, 60 µL of sample extract is added to the sample pad, and physical color development is observed after chromatography for 10 minutes. After completing the first step reading, the catalytic substrate solution is added, and catalytic color development is observed after several minutes. The results of the two readings are used to comprehensively determine whether the concentration of vomitoxin in the sample exceeds the two limits of 1000 µg / kg and 5000 µg / kg. Vomitoxin standards with concentrations of 0 µg / kg, 800 µg / kg, 900 µg / kg, 1000 µg / kg, 1100 µg / kg, 1200 µg / kg, 4000 µg / kg, 4500 µg / kg, 5000 µg / kg, 5500 µg / kg, and 6000 µg / kg in Example 6 were tested, and a comparative experiment was conducted between the test strip of this invention and commercially available test strips. Figure 12 The results showed that all concentration gradients could be clearly distinguished, and the test strip of the present invention was superior to commercial test strips in terms of dual limit interpretation, indicating that the test strip prepared in this embodiment has good application performance.

[0065] In summary, such as Figure 13 As shown, the test strip of this invention is composed of a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper stacked sequentially. An immunoprobe pre-placed on the conjugate pad competitively reacts with vomitoxin in the sample during chromatography. Results are interpreted through color changes at the detection line T and the control line C. In the two-step reading mode, physical color development corresponds to a first limit of 1000 µg / kg, and catalytic color development corresponds to a second limit of 5000 µg / kg, thus enabling dual-limit classification of vomitoxin concentration on a single test strip.

[0066] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

Claims

1. A test strip for rapid determination of vomitoxin limits, characterized in that, The test strip includes a sample pad, a conjugate pad, a nitrocellulose membrane, and absorbent paper; The binding pad is pre-positioned with an immune probe, which comprises Au@Pd core-shell structured nanoparticles and an anti-vomiting toxin monoclonal antibody modified on the surface of the nanoparticles. The core of the Au@Pd core-shell structured nanoparticles is a gold nanoparticle, and the outer shell is a palladium shell. The thickness of the palladium shell is obtained by controlling the final concentration of sodium tetrachloropalladium to be 0.2 mM to 2.0 mM during the preparation process. The amount of anti-vomiting toxin monoclonal antibody modified on the surface of the nanoparticles is obtained by controlling the amount of antibody added to be 0.25 µL to 2.5 µL / 1 mL of nanoparticle solution during the preparation process. The nitrocellulose membrane is provided with a detection line and a control line. The detection line is coated with a vomitoxin-carrier protein conjugate, and the control line is coated with a goat anti-mouse IgG polyclonal antibody. The carrier protein is bovine serum albumin. When the test strip is used for the detection of vomitoxin, the physical colorimetric visual judgment threshold corresponds to the first limit value, and the catalytic colorimetric visual judgment threshold corresponds to the second limit value.

2. The test strip for rapid determination of vomitoxin limits according to claim 1, characterized in that, The coating concentration of the vomitoxin-bovine serum albumin conjugate on the detection line is 0.25 mg / mL to 1.5 mg / mL, and the coating concentration of the goat anti-mouse IgG polyclonal antibody on the quality control line is 0.25 mg / mL to 1.5 mg / mL.

3. The test strip for rapid determination of vomitoxin limits according to claim 2, characterized in that, The coating concentration of the vomitoxin-bovine serum albumin conjugate on the detection line is 1.0 mg / mL, and the coating concentration of the goat anti-mouse IgG polyclonal antibody on the quality control line is 0.5 mg / mL.

4. The test strip for rapid determination of vomitoxin limits according to claim 1, characterized in that, The core-shell structured nanoparticles were prepared by a seed growth method. In a gold nanoparticle seed solution, ascorbic acid was used as a reducing agent to reduce the sodium tetrachloropalladium precursor. The shell thickness was adjusted by controlling the amount of sodium tetrachloropalladium.

5. A rapid detection kit for determining the limit of vomitoxin, characterized in that, The kit comprises the test strip as described in any one of claims 1 to 4, and a catalytic substrate solution; The catalytic substrate solution contains 0.03% hydrogen peroxide and 3% 3-amino-9-ethylcarbazole.

6. The use of the test strip according to any one of claims 1 to 4 in the preparation of a product for the rapid determination of vomitoxin limits.

7. The application according to claim 6, characterized in that, The test strip determines whether the concentration of vomitoxin exceeds a first limit and a second limit through two-step readings; the first limit is 1000 µg / kg and the second limit is 5000 µg / kg.

8. The application according to claim 7, characterized in that, The two-step reading includes: The first step is to determine whether the concentration of vomitoxin in the sample exceeds the first limit value by observing the physical color development results of the detection line without adding exogenous catalytic substrate. The second step involves applying a catalytic substrate solution to the test strip after completing the reading in the first step. By observing the catalytic color development result of the detection line, it is determined whether the concentration of vomitoxin in the sample exceeds the second limit value.

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