A magnetic gold nanoparticle probe kit, its preparation method and application

Abstract

This invention discloses a magnetic gold nanoparticle probe kit, its preparation method, and its application, relating to biological detection. The magnetic gold nanoparticle probe is prepared by mixing Fe3O4@Au NPs with an ochratoxin monoclonal antibody solution at a ratio of 100 μg: 4 μL, with a pH of 7.4. The magnetic gold nanoparticle probe kit prepared using the magnetic gold nanoparticle probe includes a matching test strip and the magnetic gold nanoparticle probe. The test strip includes a backing plate, on which a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad are sequentially arranged along the sample flow direction. The nitrocellulose membrane is adhered to the center of the backing plate, and the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad overlap by 1-2 mm at their junctions. A detection line (T) and a control line (C) are provided on the nitrocellulose membrane. When the magnetic gold nanoparticle probe kit prepared in this application is applied to the detection of ochratoxin, it exhibits low antibody dosage, high sensitivity, and excellent anti-interference properties.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to a magnetic gold nanoparticle probe kit, its preparation method, and its application. Background Technology

[0002] Ochratoxin is a fungal toxin produced by certain molds in the genera *Aspergillus* and *Penicillium* that contaminates food. Crops can be contaminated by this fungal toxin due to improper storage during field growth or harvesting, as well as during rainy weather during transportation. Furthermore, ochratoxin is highly toxic and causes significant contamination, posing risks of nephrotoxicity, hepatotoxicity, immunotoxicity, carcinogenicity, teratogenicity, mutagenicity, and neurotoxicity; it is also slowly metabolized in the human body and difficult to eliminate. Therefore, rapid and immediate detection of ochratoxin is crucial. Currently, researchers have developed various analytical techniques for this fungal toxin, including high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS), capillary electrophoresis, electrochemical sensors, and enzyme-linked immunosorbent assay (ELISA). Although these methods are sensitive and reliable, the expensive instruments and complex operations are unsuitable for rapid on-site screening of agricultural products.

[0003] Immunochromatographic assays (ICA), also known as lateral flow biosensors, offer advantages such as good specificity, stability, speed, portability, ease of operation, and low cost, making them the most common point-of-care testing or rapid on-site screening method. Currently, ICA is widely used in the analysis of biomarkers, pathogenic microorganisms, toxins, drugs, and chemical contaminants. However, the low color intensity and weak colloidal stability of traditional colloidal gold result in low sensitivity of colloidal gold-based immunochromatographic test strips. Furthermore, the complex and diverse matrix of wheat and its products, along with the influence of sample processing procedures and the sample matrix, makes detection time-consuming and labor-intensive, with the most prominent drawback being relatively low analytical sensitivity. Wheat and its products contain abundant proteins, which may cause false positives in immunochromatographic assays, interfering with the results. Therefore, many reports both domestically and internationally have proposed using magnetic enrichment to improve capture efficiency and eliminate sample matrix interference. However, in competitive immunoassays of small molecules such as ochratoxin, many studies have used excessive amounts of labeled antibodies to enhance color development, increasing costs and wasting antibodies. Summary of the Invention

[0004] In view of this, the purpose of this invention is to address the shortcomings of the prior art by providing a magnetic gold nanoparticle probe kit for the detection of ochratoxin, which requires a small amount of antibody, has high sensitivity, and exhibits excellent anti-interference properties.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A magnetic gold nanoprobe is prepared by mixing Fe3O4@Au NPs with an ochratoxin monoclonal antibody solution at a ratio of 100 μg: 4 μL, with a pH of 7.4.

[0007] Furthermore, the gold nanoparticles in the Fe3O4@Au NPs have a particle size of 15-30 nm.

[0008] A method for preparing a magnetic gold nanoprobe includes the following steps:

[0009] (1) During the stirring process, 4 μL of 1 mg / mL ochratoxin monoclonal antibody was added to 1 mL of pH 7.4 100 μg / mL Fe3O4@Au NPs solution and stirred continuously for 30 min to obtain a mixture;

[0010] (2) Add 1% BSA to the above mixture and incubate at 4°C for 2 h; collect the product by centrifugation to remove excess BSA and obtain magnetic gold nanoparticle probe.

[0011] Furthermore, the Fe3O4@Au NPs in step (1) undergo surface modification, specifically as follows:

[0012] 1) A Fe3O4@Au NPs solution capped with oleylamine in hexane was mixed with an equal volume of 2 mM Pluronic F127 aqueous solution and sonicated for 30-50 minutes to obtain a homogeneous solution; the Fe3O4@Au NPs solution capped with oleylamine in hexane was prepared by dissolving 100 mg of Fe3O4@Au NPs in 10 mL of a mixed solvent of oleylamine and hexane, wherein the volume ratio of oleylamine to hexane in the mixed solvent was 3:1.

[0013] 2) Cover the flask with perforated aluminum foil and evaporate hexane under continuous magnetic stirring at 30-40°C, so that Pluronic F127 is adsorbed on the gold shell surface to obtain surface-modified Fe3O4@Au NPs.

[0014] A magnetic gold nanoparticle probe preparation kit includes a matching test strip and a magnetic gold nanoparticle probe. The test strip includes a backing plate, on which a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad are sequentially arranged along the sample flow direction. The nitrocellulose membrane is adhered to the middle of the backing plate. The sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad overlap by 1-2 mm at their junctions. A detection line (T) and a control line (C) are provided on the nitrocellulose membrane, and the distance between the detection line (T) and the control line (C) is 3 mm.

[0015] Furthermore, the amount of the magnetic gold nanoprobe is 5 μL.

[0016] A method for preparing a magnetic gold nanoparticle probe kit, wherein the test strip is prepared by the following steps:

[0017] 1) Two glass fiber membranes were soaked in PBS containing 2% BSA, 1% sucrose and 0.02% NaN3 for 30 min and dried at 37°C to form a sample pad and a conjugation pad.

[0018] 2) 0.6 mg / mL ochratoxin antigen and 1 mg / mL goat anti-mouse immunoglobulin were coated onto a nitrocellulose membrane at a streak rate of 0.5 μL / cm to serve as the detection line (T) and control line (C), respectively.

[0019] 3) Adhere the nitrocellulose membrane to the middle of the liner, then attach the conjugation pad and sample pad to its left side in sequence, and attach the absorbent pad to its right side, overlapping the junction by 1-2mm. Cut into 3mm wide test strips to obtain the product.

[0020] Application of a magnetic gold nanoparticle probe kit in the detection of ochratoxin.

[0021] The application method of the magnetic gold nanoparticle probe kit in the detection of ochratoxin includes the following steps:

[0022] (1) Add the magnetic gold nano probe to the test sample solution at a rate of 5 μL / mL (magnetic gold nano probe / test sample solution).

[0023] (2) After magnetic enrichment, the collected probe-target immune complex was dispersed in PBS buffer and detected using a test strip.

[0024] The beneficial effects of this invention are:

[0025] 1. This invention discloses a magnetic gold nanoparticle probe kit, comprising a matching test strip and a magnetic gold nanoparticle probe. The magnetic gold nanoparticle probe is a complex of Fe3O4@Au NPs and an ochratoxin monoclonal antibody solution. This magnetic gold nanoparticle probe can enrich the signal, and while ensuring the signal intensity, it can achieve controllable magnetic separation of ochratoxin in the sample. During detection, it can not only reduce the amount of ochratoxin monoclonal antibody used, but also greatly improve the detection sensitivity.

[0026] 2. The Fe3O4@Au NPs in this application are obtained by high-temperature pyrolysis of Fe3O4 magnetic nanoparticles, followed by in-situ reduction to obtain Fe3O4@Au NPs, and then surface modification and fixation using Pluronic F127. The preparation process is simple, and the prepared Fe3O4@Au NPs have controllable particle size and magnetic properties, strong stability, and can improve the sensitivity of the kit.

[0027] 3. The magnetic gold nanoparticle probe kit prepared in this application has a visual detection limit (VDL) of 1.5 ng / mL for ochratoxin and a threshold concentration of 1.5 ng / mL for the complete disappearance of the T line, which is twice as sensitive as the test strips currently on the market and has a wide detection range. In addition, the kit has good specificity for ochratoxin and excellent anti-interference performance.

[0028] 4. When applied to wheat sample detection, it exhibits high sensitivity, specificity, and stability, effectively meeting the need for rapid screening of ochratoxins in real samples. The development of this application opens up new avenues for signal amplification and performance improvement in immunochromatographic assays, which is beneficial for further expanding the application of immunosensors. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the structure of the test strip of the present invention;

[0030] Figure 2 This is a schematic diagram of the magnetic separation and enrichment structure of the present invention;

[0031] Figure 3 This is a graph showing the sensitivity detection results of the present invention;

[0032] Figure 4 This is a graph showing the specific detection results of this invention;

[0033] Figure 5 This is a graph showing the results of the application test.

[0034] In the diagram: 1-liner, 2-sample pad, 3-binding pad, 4-nitrocellulose membrane, 5-absorbent pad. Detailed Implementation

[0035] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0036] In the following examples, Fe3O4@Au NPs refers to magnetic iron tetroxide-gold nanocomposite particles, and BSA refers to bovine serum albumin. Example 1

[0037] A magnetic gold nanoparticle probe is prepared by mixing Fe3O4@Au NPs with an ochratoxin monoclonal antibody solution at a ratio of 100 μg: 4 μL, with a pH of 7.4; the gold nanoparticles in Fe3O4@Au NPs have a particle size of 15-30 nm.

[0038] The preparation method of magnetic gold nanoprobes includes the following steps:

[0039] (1) Preparation of magnetic Fe3O4:

[0040] 10.8 g FeCl3·6H2O and 36.5 g sodium oleate were dissolved in a mixed solution containing 80 mL ethanol, 60 mL water and 140 mL n-hexane. The solution was heated to 70 °C and refluxed for 4 hours. After separation, the upper organic phase was retained and washed several times with distilled water to remove salts. The solvent was removed by rotary evaporation to obtain iron oleate, a brownish-red waxy viscous liquid.

[0041] 4 mmol of ferric oleate was dissolved in a mixture of oleylamine and oleic acid (volume ratio 3:1) at 120°C and stirred for 1 hour. The mixture was then heated to 200°C and held for 2 hours. During heating, nitrogen was purged to remove trace amounts of hydrate vapor. The solution was then further heated to 300°C and held at that temperature for 2 hours. Fe3O4 nanoparticles were then precipitated by adding ethanol to the solution. The precipitate was washed alternately with hexane and ethanol to obtain brownish-black magnetic Fe3O4, which could be dispersed in hexane for storage.

[0042] (2) Preparation of core-shell structured Fe3O4@Au NPs:

[0043] 0.1 g of magnetic Fe3O4 was resuspended in 150 mL of water, and 2 mL of 1 wt% HAuCl4 solution was added. The mixture was stirred and sonicated for 30 min, followed by reaction at room temperature for 9 hours to obtain core-shell Fe3O4@Au NPs. The brownish-yellow Fe3O4@Au NPs were capped with oleylamine and dispersed in hexane for storage.

[0044] (3) Surface modification of Fe3O4@Au NPs:

[0045] 1) A stock solution of Fe3O4@Au NPs capped with oleylamine in hexane was mixed with an equal volume of 2 mM Pluronic F127 aqueous solution and sonicated for 30-50 minutes to obtain a homogeneous solution. Specifically, the Fe3O4@Au NPs solution capped with oleylamine in hexane was prepared by dissolving 100 mg of Fe3O4@Au NPs in 10 mL of a mixed solvent of oleylamine and hexane, wherein the volume ratio of oleylamine to hexane in the mixed solvent was 3:1.

[0046] 2) Cover the flask with perforated aluminum foil and evaporate hexane under continuous magnetic stirring within a temperature range of 30-40°C, so that Pluronic F127 is adsorbed on the gold shell surface, thereby realizing the transfer of the oil phase of Fe3O4@Au NPs to the aqueous phase, and obtaining surface-modified Fe3O4@Au NPs.

[0047] (4) Preparation of ochratoxin monoclonal antibodies:

[0048] First, the ochratoxin artificial antigen, i.e., the commercially available ochratoxin-bovine serum albumin conjugate, was diluted to 2 mg / mL with physiological saline and placed in a 2.5 mL syringe to immunize 6-8 week old female Balb / c mice. Multiple subcutaneous injections of 100 μg of immunogen per mouse were administered to induce the production of anti-ochratoxin antiserum. Booster immunizations were then performed every three weeks, with the dose halved to 50 μg each time, until the final sprint immunization. After the third immunization (second booster immunization), blood was collected from the tail vein of the mice, centrifuged, and the supernatant was obtained. The titer and inhibition of the mouse serum were simultaneously measured using an indirect competitive ELISA.

[0049] Subsequently, spleen cells from mice were fused with SP2 / 0 myeloma cells. Three to four days prior to fusion, the fused mice underwent a sprint immunization. The immunogen was diluted with physiological saline to 0.25 mg / mL, and 100 µL was injected intraperitoneally into the mice. Additionally, one week prior to fusion, frozen SP2 / 0 mouse myeloma cells were thawed and transferred to a 75 cm² culture medium. 2 The cells were cultured in culture flasks with 10% fetal bovine serum (FBS) medium. Once the tumor cells had reached confluence, they were passaged. The culture medium was replaced with fresh 10% FBS the day before fusion to ensure the cells were in the logarithmic growth phase and in good growth condition. The appropriate coating concentration and standard concentration were determined based on mouse serum ELISA results prior to fusion. The cell supernatant was analyzed one week after fusion using indirect ELISA and indirect competitive ELISA. Based on the results, 4-6 cell lines with high titers and good inhibition were selected as mother cells for subcloning. After the third subcloning, hybridoma cells with high titers, good inhibition, and single clusters in the cell wells were selected as mother cells for monoclonal antibody production. The hybridoma cells in the mother cell wells were transferred to 75 cm⁻¹ cells. 2 In culture flasks, hybridoma cells were expanded using 10% fetal bovine serum medium. When the hybridoma cells reached the bottom of the culture flask and were in logarithmic growth, the cells were collected into cryovials, an appropriate amount of cell cryopreservation solution was added, the flasks were labeled and sealed, and the cells were transferred to a cryopreservation box and placed at -80°C for one day. Then, the cells were transferred to liquid nitrogen for preservation.

[0050] The obtained cells were injected into the peritoneal cavity of female Balb / c mice. One week prior to injection, the mice were given 0.5 mL of Freund's incomplete adjuvant to suppress the immune response and accelerate and promote the growth of hybridoma cells in the mice. Seven to ten days after cell injection, numerous ascites tumors appeared in the peritoneal cavity of the mice. Once the peritoneum became swollen, the ascites was aspirated using a syringe, centrifuged at 5000 r / min for 10 min, and the supernatant was collected, transferred to centrifuge tubes, and stored at -20°C for later use.

[0051] The antibody was purified and lyophilized using the octanoic acid-ammonium sulfate method. The specific steps are as follows:

[0052] 1) Take out the frozen ascites fluid, thaw it, take 1 mL of mouse ascites fluid into a 2 mL centrifuge tube, add an equal volume of 0.06 M sodium acetate buffer solution at pH 4.4, mix well, and adjust the pH value to 4.4;

[0053] 2) While shaking, slowly add caprylic acid (33.3 µL caprylic acid per milliliter of ascites fluid) along the tube wall. After adding, place the tube on a horizontal shaker and shake for 30 min to precipitate the impurities.

[0054] 3) Centrifuge at 8000 r / min, 4°C for 10 min, discard the precipitate, transfer the supernatant to a new centrifuge tube, add an equal volume of saturated ammonium sulfate solution to make the ammonium sulfate concentration 50%, and shake at 4°C for 2 h.

[0055] 4) Centrifuge at 8000 r / min, 4°C for 10 min, discard the supernatant and collect the precipitate; dissolve the precipitate in 1 mL of 0.01 M PBS, then dialyze in 0.01 M PBS for three days, changing the dialysate every 12 h. After dialysis, determine the antibody concentration by ultraviolet spectrophotometry to obtain the ochratoxin monoclonal antibody. Dissolve a portion of the antibody at a concentration of 1 mg / mL in 10 mM phosphate buffer (PBS, pH 7.4) for later use.

[0056] (5) Preparation of magnetic gold nanoparticle probes:

[0057] 1) During stirring, add 4 μL of 1 mg / mL ochratoxin monoclonal antibody to 1 mL of pH 7.4 100 μg / mL Fe3O4@Au NPs solution (i.e., 100 μg Fe3O4@Au NPs dispersed in 1 mL of pH 7.4 PBS to form a pH 7.4 Fe3O4@Au NPs solution with a concentration of 100 μg / mL). Stir continuously for 30 min to allow the antibody to bind with the magnetic gold nanoparticles and obtain a mixture.

[0058] 2) Add 1% BSA to the above mixture and incubate at 4°C for 2 h to block excess sites on the surface of the magnetic gold nanoparticles; collect the product by centrifugation (10,000 rpm, 30 min) to remove excess BSA and prepare the magnetic gold nanoparticle probe; resuspend in 100 μL of a storage solution containing 2 mM borax, 0.1% PEG-20,000 and 0.02% NaN3.

[0059] A magnetic gold nanoparticle probe kit prepared using magnetic gold nanoparticle probes includes a matching test strip and magnetic gold nanoparticle probes, wherein the amount of magnetic gold nanoparticle probes is 5 μL / 1 ml of detection solution.

[0060] The structure of the test strip is as follows Figure 1 As shown, the device includes a liner 1. A sample pad 2, a conjugate pad 3, a nitrocellulose membrane 4, and an absorbent pad 5 are sequentially arranged on the liner 1 along the sample flow direction. The nitrocellulose membrane 4 is adhered to the middle of the liner 1. The sample pad 2, the conjugate pad 3, the nitrocellulose membrane 4, and the absorbent pad 5 overlap by 1.5 mm at their junctions. A detection line (T) and a control line (C) are provided on the nitrocellulose membrane 4, and the distance between the detection line (T) and the control line (C) is 3 mm.

[0061] The test strip is prepared by the following steps:

[0062] 1) Two glass fiber membranes were soaked in PBS containing 2% BSA, 1% sucrose and 0.02% NaN3 for 30 min to seal the micropores, and dried at 37°C to form sample pad 2 and conjugation pad 3.

[0063] 2) 0.6 mg / mL ochratoxin antigen and 1 mg / mL goat anti-mouse immunoglobulin were coated on nitrocellulose membrane 4 at a streak rate of 0.5 μL / cm, respectively, as the detection line (T) and control line (C).

[0064] 3) Adhere the nitrocellulose membrane 4 to the middle of the backing plate 1, then attach the conjugation pad 3 and sample pad 2 to its left side in sequence, and attach the absorbent pad 5 to its right side, with the overlap at the junction being 1.5mm. Cut it into 3mm wide test strips to obtain the product.

[0065] The application method of the magnetic gold nanoparticle probe kit for detecting ochratoxin in wheat products includes the following steps:

[0066] 1) A magnetic gold nanoparticle probe was added to the test sample solution at a concentration of 5 μL / 1 mL to rapidly and effectively capture the target analyte. Magnetic separation and enrichment were then performed using a magnet. The results are shown in [link to results]. Figure 2 As shown;

[0067] 2) After magnetic separation and enrichment, the collected probe-target immune complex was dispersed in 70 μL of PBS buffer (pH 7.4) and detected using a test strip.

[0068] 1. Performance evaluation of magnetic gold nanoparticle probe kit for detecting ochratoxin in wheat products

[0069] (1) Sensitivity detection

[0070] Ochratoxin standard was dissolved in 10 mM PBS (pH 7.4) and serially diluted to obtain test solutions with concentrations ranging from 5 to 0 ng / mL. PBS served as a blank control.

[0071] Take 100 μL of test solution and perform immunochromatographic detection using the magnetic gold nanoparticle probe kit prepared in Example 1 and the traditional test strip (the test strip prepared in Example 1, without magnetic gold nanoparticle probe). Observe the visual results within 15 min.

[0072] When the T-line is visually significantly lighter than the negative control line, the corresponding minimum ochratoxin concentration is defined as the visual detection limit (VDL). When the T-line completely disappears, the corresponding minimum concentration is considered as the threshold concentration. Results are shown in [Figure Number]. Figure 3 .

[0073] Figure 3 The first row shows the test results of the kit prepared in Example 1, and the second row shows the test results of the conventional test strip. The test results of the conventional test strip and the kit from Example 1 are presented by... Figure 3 As can be seen, the VDL of the traditional test strip and the kit of this application are 0.5 ng / mL and 0.25 ng / mL, respectively, indicating that the sensitivity of the kit of this application is improved by 2 times. The threshold concentrations for the complete disappearance of the T line are 4 ng / mL and 1.5 ng / mL, respectively. Therefore, it can be seen that the kit prepared in this application not only has high sensitivity but also a wide detection range.

[0074] (2) Specific detection

[0075] Blank PBS buffer and PBS buffer containing 20 ng / mL ochratoxin were used as negative and positive controls, respectively. Aflatoxin B1, vomitoxin, and zearalenone were used as interfering substances. Results of the specificity assessment experiment are as follows: Figure 4 As shown.

[0076] Depend on Figure 4 It can be seen that the T-line signal intensity of the interference group is similar to that of the negative group, indicating that the developed test strip does not cross-react with other structural analogs and exhibits good specificity for ochratoxin.

[0077] 2. Application Test

[0078] The magnetic gold nanoparticle probe kit prepared in Example 1 of this application was applied to the detection of ochratoxin in wheat samples.

[0079] Wheat was used as the actual sample. Ochratoxin was added to wheat samples identified by HPLC as negative, achieving a continuous concentration from 20 to 0 μg / kg. After thorough mixing, the spiked samples were incubated overnight at 4°C. 5 g of thoroughly ground wheat flour was ultrasonically dissolved in 10 mL of PBS to extract ochratoxin. After centrifugation at 10,000 rpm for 5 min, the collected supernatant was analyzed, with PBS as a blank control. The sample pad of the test strip was immersed in the test solution. Through capillary action, the sample flowed through the test strip, and visual results were obtained within 15 min. The results are shown in [Figure number missing]. Figure 5 .

[0080] Figure 5 The test results show that the VDL of the test strip in the wheat sample detection solution is 2.5 μg / kg. The detection sensitivity of the wheat sample, after conversion, is consistent with that of the standard ochratoxin solution, indicating that the designed kit has excellent sensitivity, specificity, and applicability. The developed ICA can well meet the needs of rapid screening for ochratoxins in real samples.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Any other modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention, as long as they do not depart from the spirit and scope of the technical solutions of the present invention, should be covered within the scope of the claims of the present invention.

Claims

1. A magnetic gold nanoprobe, characterized in that: It was prepared by mixing Fe3O4@Au NPs with ochratoxin monoclonal antibody solution at a ratio of 100 μg: 4 μL, with a pH of 7.4; The Fe3O4@AuNPs were prepared by the following steps: 0.1 g of magnetic Fe3O4 was resuspended in 150 mL of water, 2 mL of 1 wt% HAuCl4 solution was added, the mixture was stirred and sonicated for 30 min, and then reacted at room temperature for 9 hours to obtain core-shell Fe3O4@AuNPs. The brownish-yellow Fe3O4@AuNPs were capped with oleylamine and dispersed in hexane for storage. Surface modification of Fe3O4@AuNPs: 1) A Fe3O4@Au NPs solution capped with oleylamine in hexane was mixed with an equal volume of 2 mL MPluronic F127 aqueous solution and sonicated for 30-50 minutes to obtain a homogeneous solution; the Fe3O4@Au NPs solution capped with oleylamine in hexane was prepared by dissolving 100 mg of Fe3O4@Au NPs in 10 mL of a mixed solvent of oleylamine and hexane, wherein the volume ratio of oleylamine to hexane in the mixed solvent was 3:

1. 2) Cover the flask with perforated aluminum foil and evaporate hexane under continuous magnetic stirring at 30-40°C, so that Pluronic F127 is adsorbed on the gold shell surface to obtain surface-modified Fe3O4@Au NPs.

2. The magnetic gold nanoprobe according to claim 1, characterized in that: The gold nanoparticles in the Fe3O4@Au NPs have a particle size of 15-30 nm.

3. A method for preparing the magnetic gold nanoprobe according to claim 1, characterized in that: Includes the following steps: (1) During the stirring process, 4 μL of 1 mg / mL ochratoxin monoclonal antibody was added to 1 mL of pH 7.4 100 μg / mL Fe3O4@Au NPs solution and stirred continuously for 30 min to obtain a mixture; (2) Add 1% BSA to the above mixture and incubate at 4°C for 2 h; collect the product by centrifugation to remove excess BSA and obtain magnetic gold nanoparticle probe.

4. A magnetic gold nanoparticle probe kit prepared using the magnetic gold nanoparticle probe of claim 1, characterized in that: The test strip includes a matching test strip and a magnetic gold nanoparticle probe. The test strip includes a backing plate (1). A sample pad (2), a conjugate pad (3), a nitrocellulose membrane (4), and an absorbent pad (5) are arranged sequentially on the backing plate (1) along the sample flow direction. The nitrocellulose membrane (4) is adhered to the middle of the backing plate (1). The sample pad (2), the conjugate pad (3), the nitrocellulose membrane (4), and the absorbent pad (5) overlap by 1-2 mm at their junctions. A detection line (T) and a control line (C) are provided on the nitrocellulose membrane (4). The distance between the detection line (T) and the control line (C) is 3 mm.

5. The magnetic gold nanoparticle probe kit according to claim 4, characterized in that: The amount of the magnetic gold nanoprobe is 5 μL.

6. A method for preparing the magnetic gold nanoparticle probe kit according to claim 4, characterized in that: The test strip is prepared by the following steps: 1) Two glass fiber membranes were soaked in PBS containing 2% BSA, 1% sucrose and 0.02% NaN3 for 30 min and dried at 37°C to form sample pad (2) and conjugate pad (3). 2) 0.6 mg / mL ochratoxin antigen and 1 mg / mL goat anti-mouse immunoglobulin were coated on nitrocellulose membrane (4) at a streak rate of 0.5 μL / cm, respectively, as the detection line (T) and control line (C). 3) Adhere the nitrocellulose membrane (4) to the middle of the liner (1), then attach the conjugate pad (3) and sample pad (2) to its left side in sequence, and attach the absorbent pad (5) to its right side. Overlap the junction by 1-2 mm, and cut it into 3 mm wide test strips to obtain the product.

7. The application of the magnetic gold nanoparticle probe kit of claim 4 in the detection of ochratoxin.

8. The application according to claim 7, characterized in that: The application method includes the following steps: (1) Add the magnetic gold nanoparticle probe to the test sample solution at a concentration of 5 μL / mL; (2) After magnetic enrichment, the collected probe-target immune complex was dispersed in PBS buffer and detected using a test strip.

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