Magnetic nanoprobe and method for detecting fungaltoxin by using critical offset magnetic particle spectrum technology of magnetic nanoprobe

Abstract

The invention discloses a magnetic nanoprobe and a method for detecting fungaltoxin by a critical offset magnetic particle spectroscopy, the magnetic nanoprobe is formed by modifying the surface of a Fe3O4 magnetic nanoparticle with an antibody capable of specifically recognizing fungaltoxin, the antibody is coupled with an aldehyde group through an amino group modified on the surface of the Fe3O4 nanoparticle, and the magnetic nanoprobe is formed by detecting the fungaltoxin. Stable covalent linkage is formed, and targeted recognition and combination of the aflatoxin B1 and the deoxynivalenol are achieved. The magnetic nanoprobe adopted by the invention can realize detection and quantification of the target toxin through a critical offset magnetic particle spectrum technology under an unmarked condition, the nonlinear response of the Fe3O4 nanoparticles is excited through a bias magnetic field, and by measuring the magnetic phase change generated before and after the Fe3O4 nanoparticles are combined with the toxin, the detection and quantification of the target toxin can be realized. The sensitive detection on the target toxin is realized. The method has the advantages of high detection sensitivity, no elution operation, rapidness, low cost and the like.

Description

Technical Field

[0001] The present invention belongs to the field of food safety, and in particular relates to a method for detecting mycotoxins using a magnetic nanoprobe and a critical shift magnetic particle spectroscopy technique thereof. Background Art

[0002] Mycotoxins are toxic secondary metabolites secreted by fungi that can cause serious diseases such as carcinogenesis, mutagenesis, and teratogenesis in humans and animals. Aflatoxin B1 (AFB1) and deoxynivalenol (DON) are two common toxic metabolites produced by Aspergillus flavus and Fusarium spp., respectively, and widely contaminate food and its products. Both are not only highly toxic and carcinogenic, but can also cause serious damage to the human liver, immune system, and nervous system. Long-term ingestion poses a serious threat to human health. Because their concentrations in samples are usually low and the sample matrix is complex, the detection process often faces problems such as insufficient sensitivity and cumbersome operation. Therefore, establishing an efficient, rapid, and easy-to-operate detection method is of great significance for ensuring food safety.

[0003] Currently, the main methods for detecting AFB1 and DON in samples include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS / MS), and enzyme-linked immunosorbent assay (ELISA). HPLC offers high separation power and sensitivity, making it suitable for quantitative analysis of a wide range of toxins. However, its complex workflow, tedious sample pretreatment steps, and long detection cycles make it unsuitable for rapid, large-scale screening. LC-MS / MS offers higher sensitivity and selectivity, enabling accurate qualitative and quantitative analysis of multiple toxins simultaneously. However, its expensive equipment, high maintenance costs, and high operator skill requirements limit its widespread adoption in grassroots testing and field applications. ELISA is widely used due to its ease of operation, rapid detection, and suitability for initial screening of large numbers of samples. However, its results are susceptible to antibody cross-reactivity and sample matrix interference, its semi-quantitative nature, and relatively poor stability and accuracy make it unsuitable for final confirmatory analysis. Therefore, developing a detection method with high sensitivity, simple pretreatment and high detection efficiency is of great significance for food safety monitoring. Summary of the Invention

[0004] Purpose of the invention: In response to the problems of complex operation, strong equipment dependence, and difficulty in field application in existing detection technologies for fungal toxins, the present invention provides an Fe3O4 magnetic nanoprobe, as well as a method for rapid detection of toxins based on magnetic nanoprobes and critical shift magnetic particle spectroscopy technology. The present invention utilizes Fe3O4 nanoparticles modified with specific antibodies on the surface to efficiently identify and magnetically separate target toxins, and directly reads the changes in magnetic signals through a critical shift magnetic particle spectroscopy detection system without the need for complex labeling or elution processes, thereby enabling rapid and sensitive detection of trace toxins in complex samples. Compared with traditional detection methods that rely on large instruments or immunoreagents, the magnetic nanoprobe and its detection method of the present invention significantly improve detection flexibility and portability, reduce detection costs, and have broad application prospects in the field of food safety.

[0005] The present invention also provides a method for preparing an Fe3O4 nanoprobe whose surface is modified with specific antibodies. The probe can achieve selective enrichment and magnetic separation of aflatoxin B1 and deoxynivalenol, is suitable for use in a critical shift magnetic particle spectral detection system, and is used to construct a high-sensitivity and high-specificity fungal toxin detection platform.

[0006] Technical solution: In order to achieve the above-mentioned purpose, the present invention provides a magnetic nanoprobe, which is composed of Fe3O4 magnetic nanoparticles with antibodies that can specifically recognize fungal toxins modified on the surface. The antibodies are coupled through the amino and aldehyde groups modified on the surface of the Fe3O4 nanoparticles to form a stable covalent connection.

[0007] The Fe3O4 magnetic nanoparticles are synthesized by a co-precipitation method and have a particle size of 15-25 nm.

[0008] Wherein, the antibody is an antibody that targets and recognizes and binds to aflatoxin B1 or deoxynivalenol.

[0009] The method for preparing the magnetic nanoprobe of the present invention comprises the following steps:

[0010] (1) Preparation of Fe3O4 magnetic nanoparticles;

[0011] (2) Preparation of Fe3O4 magnetic nanoparticles with surface modified aldehyde functional groups: amino groups are modified on the surface of Fe3O4 magnetic nanoparticles using a silane reagent, and then glutaraldehyde is mixed with the surface modified amino group Fe3O4 magnetic nanoparticles, thereby grafting the aldehyde functional groups onto the surface modified amino group Fe3O4 magnetic nanoparticles;

[0012] (3) Preparation of Fe3O4 magnetic nanoparticle probes: Take the nanoparticles prepared in step (2), add antibodies and reaction solvents, and react; after the reaction is completed, discard the remaining antibodies under the action of magnetism, and then encapsulate with bovine serum albumin. After the reaction is completed, discard the remaining bovine serum albumin under the action of magnetism to obtain Fe3O4 magnetic nanoparticle probes.

[0013] The method comprises the following steps: dissolving FeCl3 in double-distilled water, adding Na2SO3 to react, further adding NH3·H2O to react, washing with double-distilled water until the suspension is neutral, dissolving the product in deionized water, and drying in a vacuum drying oven.

[0014] Wherein, the silane reagent in step (2) is 3-aminopropyltriethoxysilane, the reaction temperature is 70-80°C, the reaction time is 10-12h, and the reaction temperature of glutaraldehyde and Fe3O4 nanoparticles is room temperature, and the reaction time is 1-2h.

[0015] Wherein, the concentration of the antibody in step (3) is aflatoxin B1 antibody is 10-20 μg / mL, the volume is 20-30 μL, the coupling temperature is room temperature, and the time is 2-3 h; the concentration of the antibody is deoxynivalenol antibody is 40-50 μg / mL, the volume is 20-30 μL, the coupling temperature is room temperature, and the time is 2-3 h; the concentration of bovine serum albumin is 1-5%, the encapsulation temperature is room temperature, and the time is 1-2 h.

[0016] The magnetic nanoprobe of the present invention is used in detecting toxins through critical shift magnetic particle spectroscopy under label-free conditions.

[0017] The detection method comprises mixing the magnetic nanoprobe with a sample, recording the phase response of the nanoparticles by a critical shift magnetic particle spectrometer, and calculating the concentration of the target toxin based on the phase change.

[0018] Among them, by applying an alternating magnetic field and a bias magnetic field, the nonlinear magnetic response phase difference before and after the magnetic probe is connected to the fungus toxin is detected, thereby realizing label-free, real-time quantitative analysis of the fungus toxin in the liquid sample.

[0019] The critical shift magnetic particle spectroscopy detection platform based on surface functionalized Fe3O4 magnetic nanoparticles described in the present invention is characterized in that it includes the magnetic nanoprobe and critical shift magnetic particle spectrometer described in claim 1, and uses the magnetic phase difference generated before and after the immune reaction as a detection signal to achieve highly sensitive and quantitative detection of toxins.

[0020] The present invention provides a critical shift magnetic particle spectroscopic detection system for detecting aflatoxin B1 and deoxynivalenol, based on Fe3O4 nanoprobes. The probes are composed of superparamagnetic Fe3O4 nanoparticles, with reactive groups introduced through chemical modification on their surfaces. Antibodies that specifically recognize aflatoxin B1 and deoxynivalenol are further coupled to form functionalized nanoprobes with excellent dispersibility and targeted recognition capabilities. In the critical shift magnetic particle spectroscopic detection system, after binding to the target toxin in a sample, the probes can detect the phase change of their third harmonic under a specific bias magnetic field, thereby achieving rapid and sensitive quantitative detection of the toxins.

[0021] The method for detecting aflatoxin B1 and deoxynivalenol based on Fe3O4 nanoprobe critical shift magnetic particle spectroscopy technology of the present invention comprises the following steps:

[0022] Step 1: Prepare Fe3O4 nanoparticles and disperse them in deionized water.

[0023] Step 2: Use silane reagent to modify amino groups on the surface of Fe3O4 nanoparticles, then use glutaraldehyde to introduce aldehyde groups, and disperse them in PBS.

[0024] Step 3: The modified Fe3O4 nanoparticles are coupled with antibodies, then encapsulated with bovine serum albumin, and finally placed in a critical shift magnetic particle spectrometer to measure its third harmonic signal.

[0025] Step 4: The Fe3O4 nanoparticles coupled with the antibody react with the toxin, and then are placed in a critical shift magnetic particle spectrometer to measure the third harmonic signal again.

[0026] Preferably, in step 1, the Fe3O4 nanoparticles are prepared by co-precipitation method and have a particle size of 15 to 25 nm.

[0027] Preferably, in step 2, the silane reagent is 3-aminopropyltriethoxysilane, the reaction temperature is 70°C, and the reaction time is 12 hours. The reaction temperature of glutaraldehyde and Fe3O4 nanoparticles is room temperature, and the reaction time is 2 hours. The pH of the PBS buffer is 7.4.

[0028] Preferably, in step three, the concentration of the aflatoxin B1 antibody is 10 μg / mL, the volume is 20 μL, the coupling temperature is room temperature, and the time is 2 hours; the concentration of the deoxynivalenol antibody is 40 μg / mL, the volume is 20 μL, the coupling temperature is room temperature, and the time is 2 hours; the concentration of bovine serum albumin is 1%, the encapsulation temperature is room temperature, and the time is 1 hour. The concentration of the magnetic nanoprobe modified with the antibody detected in the critical shift magnetic particle spectrometer is 3 mg / mL, the volume is 100 μL, and the temperature is room temperature.

[0029] Preferably, in step 4, the antibody and antigen immune reaction time is 25 minutes, the added toxin volume is 10 μL, and the reaction temperature is room temperature. The particle concentration detected in the critical shift magnetic particle spectrometer is about 3 mg / mL, the volume is 110 μL, and the temperature is room temperature.

[0030] The present invention provides an application of Fe3O4 nanoprobe-based critical shift magnetic particle spectroscopy technology in detecting aflatoxin B1 and deoxynivalenol.

[0031] The present invention first prepares Fe3O4 nanoparticles, then modifies the surface of the particles with functional groups such as amino groups and aldehyde groups, then couples the particles with antibodies to form probes that specifically recognize toxins, and finally places the particles in a critical shift magnetic particle spectrometer to detect their phase change values, which can be used to detect AFB1 and DON in actual samples.

[0032] Specifically, the present invention couples Fe3O4 nanoparticles modified with aldehyde functional groups on the surface with antibodies, and through covalent binding, obtains particles that can specifically recognize AFB1 and DON, and uses critical offset magnetic particle spectroscopy to detect their initial magnetization response values. Subsequently, AFB1 and DON standards are added to the system, and AFB1 and DON specifically bind to the antibodies on the surface of the Fe3O4 nanoprobe, resulting in an increase in the hydrated particle size of the Fe3O4 nanoparticles, causing the critical offset magnetic particle spectrometer response value to decay. By analyzing the difference in critical offset magnetic particle spectral response values before and after the addition of toxins, a quantitative relationship between the response difference and the toxin concentration can be obtained. The Fe3O4 nanoparticles are prepared by a coprecipitation method using 2 mol / L ferric chloride, 1 mol / L sodium sulfite, and 0.85 mol / L ammonia water.

[0033] Design mechanism: The present invention constructs a critical shift magnetic particle spectroscopy detection platform based on surface functionalized Fe3O4 magnetic nanoparticles, which can be used to identify a variety of small molecule toxins such as AFB1 or DON. The principle is: First, Fe3O4 magnetic nanoparticles with good dispersibility are synthesized by coprecipitation method, and then covalently coupled with antibodies after aldehyde modification to construct specific recognition probes. When the target fungal toxin is present in the system, the specific binding of the antibody-antigen will cause changes in the surface charge distribution and hydration particle size of the Fe3O4 magnetic nanoparticles, resulting in changes in the third harmonic signal. By detecting and collecting the magnetic signals of the Fe3O4 magnetic nanoparticles before and after the change using a critical shift magnetic particle spectrometer, a linear relationship between the fungal toxin content and the change value can be obtained, thereby realizing the quantitative detection of fungal toxins. By replacing the modified antibodies, the platform can achieve specific detection of AFB1 or DON in different experiments, respectively, and has good versatility and selectivity.

[0034] The magnetic nanoprobes employed in this study can detect and quantify target toxins under label-free conditions using critical shift magnetic particle spectroscopy. The detection principle involves stimulating the nonlinear response of Fe₃O₄ nanoparticles with a bias magnetic field and measuring the magnetic phase changes produced before and after binding to the toxin, enabling sensitive detection of the target toxin. This method offers the advantages of high sensitivity, no elution steps, rapidity, and low cost. Compared to traditional enzyme-linked immunosorbent assays (ELISAs), it significantly improves operational simplicity and real-time detection capabilities, making it suitable for the rapid screening and quantitative analysis of mycotoxins in grains, food, and feed.

[0035] The present invention is based on a phase detection mechanism of nanoparticle rotational retardation after immune recognition: after an immune response occurs, the target toxin (such as AFB1) binds to the surface antibody, causing an increase in the hydrodynamic diameter of the particle and a change in the surface tension, thereby affecting the particle's response phase in an AC magnetic field. This change is most sensitive under a critical bias magnetic field and is the core source of the signal used for detection in the present invention. In the present invention, based on COMPASS detection of surface-functionalized Fe3O4 nanoparticles, the detection system parameters are specifically optimized to match the immune particle response characteristics: to match the response behavior of the immune-modified Fe3O4 particles, the present invention adjusts the AC magnetic field frequency, amplitude, and DC bias magnetic field in the critical offset magnetic particle spectroscopy system, so that the phase response curve has linear discernibility within the target concentration variation range, thereby improving the quantitative ability. Preferably, the excitation frequency is 1000 Hz, the amplitude is 0.1, the current is 10A, and the voltage is 3.5V.

[0036] The present invention proposes for the first time the introduction of Fe3O4 magnetic nanoparticles modified with surface aldehyde groups into the COMPASS detection platform for the detection of biological molecules (such as toxins). For the first time, the "phase change induced by immune recognition" mechanism was systematically used for the signal output of the COMPASS platform, and a quantitative analysis method was established. For the first time, a biological detection system with antibody-functionalized Fe3O4 as the core and combined with bias magnetic field modulation was constructed to achieve sensitive detection of small molecule toxins such as AFB1. Compared with existing toxin detection technologies, the present invention combines functionalized Fe3O4 magnetic nanoparticles with COMPASS phase detection technology for the first time to construct a label-free, wash-free, and highly sensitive magnetic immunoassay platform suitable for the rapid quantitative detection of small molecule toxins such as AFB1.

[0037] The detection of the present invention does not require complex labeling or enzyme reactions, and is simple to operate and fast to detect. Compared with enzyme-linked immunosorbent assays such as ELISA, the present invention is based on magnetic response phase changes, does not require fluorescence, enzyme or gold nanoparticle labels, and does not require washing steps. The detection can be completed through only one sample addition and response collection, shortening the detection time to 15-30 minutes.

[0038] The detection sensitivity of the present invention is high and is suitable for small molecule toxins. Small molecule toxins such as AFB1 are difficult to identify by traditional immune sandwich methods due to their small structure. However, the present invention sensitively captures nanomolar (ng / mL or even pg / mL) concentration changes through changes in the magnetic response of particles triggered by surface antibody binding, achieving highly sensitive quantitative detection.

[0039] The signal of the present invention has strong specificity and high anti-interference ability. The present invention uses phase difference as the core detection signal. Compared with traditional amplitude signals or color comparison, it has higher stability and resolution, and is particularly suitable for samples with strong background interference in complex matrices (such as grain extracts).

[0040] The platform constructed by the present invention is highly versatile and can be expanded to detect a variety of toxins or protein markers. It can be used for the detection of other small molecules (such as aflatoxin B2, etc.) by simply replacing the coupled antibodies. It has high modularity and platform potential.

[0041] This paper establishes an immunoassay system that combines surface aldehyde-functionalized Fe₃O₄ magnetic nanoparticles with critical shift magnetic particle optics (COMPASS) technology. This system, for the first time, utilizes the magnetic phase difference generated before and after the immune reaction (rather than the traditional amplitude change) as the detection signal, achieving highly sensitive and quantitative detection of toxins. This system represents a leap from traditional physical parameter detection to biomolecule detection, with potential for platform-based applications and can be extended to the detection of other small molecule toxins, proteins, and pathogenic markers.

[0042] The present invention prepares Fe3O4 magnetic nanoparticles with surface aldehyde-modified materials. The material has good dispersibility, functionalization activity and magnetic response performance, and is a basic carrier for achieving efficient antibody coupling and stable phase response. Specific antibodies (such as AFB1 or DON monoclonal antibodies), and their ability to bind to toxin molecules directly determine the specificity and sensitivity of the detection. In the present invention, the coupling reaction between the antibody and the aldehyde-modified Fe3O4 ensures a stable probe structure and sensitive response through the controllable covalent binding of the aldehyde group to the antibody amino group. Setting and control of the critical bias magnetic field: Only under specific bias conditions can a significant phase change be caused before and after the binding of the target toxin, ensuring that the signal difference is detectable.

[0043] In the experiment of the present invention, by setting different bias magnetic fields, the magnetic phase response curves of the functionalized Fe3O4 magnetic particles before and after binding with toxins were measured, and it was found that there was a stable and quantifiable difference in the phase before and after binding (such as a phase difference of >5°); direct detection of small molecule toxins such as AFB1 and DON was achieved under label-free and non-elution conditions, and the results were stable, indicating that the magnetic phase change is sufficient as a quantitative analysis indicator. Figure 10 and 11Phase difference data diagram, the present invention shows a linear relationship between the response and the toxin concentration, proving the quantification ability; linear regression results (R 2 >0.96) and LOD can further illustrate the detection sensitivity.

[0044] The present invention achieved a minimum detection limit of 0.008 ng / mL for AFB1 in experiments, as demonstrated by standard curve data; the RSD (relative standard deviation) was generally below 10%, meeting the repeatability standard for sensitive detection. Furthermore, unlike the enzyme-linked immunosorbent assay (ELISA) method, which requires repeated washing and incubation steps, the present method requires only mixing the reaction and directly detecting the phase response. The entire process can be completed within 30 minutes, significantly simplifying the steps compared to ELISA and enabling rapid detection without elution.

[0045] The Fe3O4 magnetic nanoparticles used in the present invention can be obtained by self-production under laboratory conditions through a modified co-precipitation method. The reaction temperature does not exceed 50°C. No toxic solvents or high-pressure equipment are required during the reaction. The process is mild and safe, and has low requirements for the experimental environment. The chemicals used in the preparation process are ferric chloride, sodium sulfite and ammonia water, which are all conventional chemicals in the laboratory, and the amount synthesized once can be used for half a year. The antibody consumption in the present invention is extremely low (only 0.2μg per tube). Calculated at 2000 yuan / mg, the single reagent cost is about 0.8 yuan; no expensive reagents such as markers, eluents, enzyme substrates, etc. are required, and the cost of magnetic particles is much lower than that of commercial test kits. While the cost of each sample of ELISA is about 5 to 10 yuan or more, the material cost of this method is less than 2 yuan.

[0046] In actual detection, the present invention has good spiked recoveries of AFB1 and DON in rice, wheat, and corn samples (68.6% to 108.6%), with RSDs less than 15%, indicating that the platform can stably function in complex food matrices and has strong universality.

[0047] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0048] (1) By introducing aldehyde groups on the surface of Fe3O4 nanoparticles, directional coupling of antibody molecules is achieved, forming a recognition interface with spatial configuration matching and chemical environment stability, significantly improving the specific recognition ability of AFB1 and DON and avoiding nonspecific adsorption interference.

[0049] (2) The present invention is based on critical shift magnetic particle spectroscopy technology, which indirectly reflects the toxin concentration by measuring the phase change before and after the antibody-modified particles bind to the toxin. It does not require fluorescent / enzyme labels, nor does it require toxin elution during the detection process, thus simplifying the operation process.

[0050] (3) Different from the traditional molecular imprinting technology that relies on toxin standards to prepare the imprinting layer, the present invention uses antibodies instead of templates, avoiding the safety and environmental risks of using highly toxic templates such as AFB1, making the operation safer and the material preparation greener.

[0051] (4) The Fe3O4 nanoparticles of the present invention can be obtained by self-production, and the preparation process is mild and has low equipment requirements. The equipment cost is low (<50,000 yuan), the detection cost is low (about 2 yuan / sample), and the overall platform material cost is much lower than that of commercial ELISA kits. It is suitable for promotion to large-scale sample screening or on-site rapid detection scenarios.

[0052] (5) The self-made Fe3O4 particles of the present invention have good magnetic properties and can achieve rapid directional aggregation under an external magnetic field. Combined with high-affinity antibodies, they can achieve rapid enrichment of target molecules, thereby improving the efficiency and sensitivity in actual sample detection.

[0053] (6) The present invention is the first to construct a label-free toxin detection platform based on the combination of a specific functionalized nanomagnetic probe and critical offset magnetic particle spectroscopy technology. This platform can achieve highly sensitive, rapid, and quantitative toxin detection by monitoring the phase response changes caused by the binding of target toxins to nanoprobes without the need for fluorescence, enzymes, or metal labels. Compared with existing detection methods (such as ELISA, colloidal gold, LC-MS, etc.), the detection method of the present invention is label-free, wash-free, and easy to operate. After the magnetic nanoparticles are modified with antibodies, they directly interact with the toxins in the sample without the need for cumbersome labeling steps or washing processes. The entire detection process is more suitable for on-site and rapid screening applications. The detection sensitivity is high and is suitable for the quantification of small molecule toxins. Small molecule toxins are difficult to detect by sandwich method in immunoassays. The present invention performs physical detection by affecting the phase response of magnetic particles. It has extremely high sensitivity under critical bias magnetic fields and significant signal changes. The detection range of DON is 0.01-1000 ng / mL, which is 3 orders of magnitude higher than the ELISA method. Phase signals offer excellent stability and anti-interference capabilities. Unlike optical color comparison or fluorescence intensity, this method utilizes phase changes under an AC magnetic field as a detection readout signal, resulting in minimal background impact, a high signal-to-noise ratio, and excellent reproducibility. Nanomagnetic probes offer platform-wide scalability, allowing them to be used to detect different target toxins or biomarkers simply by replacing the conjugated antibody, demonstrating excellent platform versatility and potential for technological transfer. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Schematic diagram of the principle of detecting aflatoxin B1 and deoxynivalenol based on Fe3O4 nanoprobe critical shift magnetic particle spectroscopy technology in the present invention

[0055] Figure 2 is a transmission electron microscope image of Fe3O4 nanoparticles;

[0056] Figure 3 Spectra of Fe3O4 nanoparticles with different volumes and concentrations in critical offset magnetic particle spectroscopy test

[0057] Figure 4 Infrared spectra of Fe3O4 and modified groups

[0058] Figure 5 The third harmonic amplitude of Fe3O4 nanoparticles changes with time

[0059] Figure 6 The optimization results of AFB1-Ab related conditions are as follows: (a) is the optimization result of AFB1-Ab concentration; (b) is the optimization result of AFB1-Ab volume; (c) is the optimization result of AFB1-Ab and AFB1 immune reaction time;

[0060] Figure 7 The optimization results of the relevant conditions of DON-Ab, (a) is the optimization result of DON-Ab concentration; (b) is the optimization result of DON-Ab and DON immune reaction time; (c) is the optimization result of DON-Ab and Fe3O4-CHO coupling time;

[0061] Figure 8 It is the specific response of Fe3O4-AFB1-Ab to the target AFB1 and other toxins;

[0062] Figure 9 It is the specific response of Fe3O4-DON-Ab to the target DON and other toxins;

[0063] Figure 10 This is the standard curve for AFB1 detection based on critical shift magnetic particle spectroscopy;

[0064] Figure 11 This is the standard curve for DON detection based on critical shift magnetic particle spectroscopy;

[0065] Figure 12 The amplitude comparison of Fe3O4 synthesized by solvothermal method and co-precipitation method;

[0066] Figure 13 Comparison of the amplitude stability of Fe3O4 modified with carboxyl and aldehyde groups;

[0067] Figure 14 Schematic diagram of the constructed critical shift magnetic particle spectrometer. DETAILED DESCRIPTION

[0068] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0069] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0070] Aflatoxin B1-conjugated antigen monoclonal antibody (AFB1-Ab), product number: LD013, was purchased from Shandong Landu Biotechnology Co., Ltd.

[0071] Deoxynivalenol antibody (DON-Ab conjugated toxin antigen monoclonal antibody, product number: LD006) was purchased from Shandong Landu Biotechnology Co., Ltd.

[0072] Aflatoxin B1 (AFB1), deoxynivalenol DON, AFB2, AFG1, FB1, OTA, and ZEN are all commercially available; specification: ≥95%.

[0073] The critical offset magnetic particle spectrometer was constructed according to the literature Critical Offset Magnetic PArticleSpectroScopy for rapid and highly sensitive medical point-of-carediagnostics.Nat Commun 13,7230(2022), and its construction schematic is referenced Figure 14 , or the existing critical shift magnetic levitation particle spectrometer can be used.

[0074] Example 1

[0075] Synthesis of functionalized Fe3O4 nanoparticles

[0076] 1. Preparation of Fe3O4 nanoparticles:

[0077] Fe3O4 MNPs were prepared by co-precipitation method: 2mol / LFeCl3 was prepared, and 3mL was taken into a conical flask containing 10.33mL of double-distilled water. 1mol / LNa2SO3 was prepared, and 2mL was slowly added to the above conical flask under stirring. When it was just added, the color of the solution gradually changed from light yellow to red. When it turned back to light yellow, 80mL of 0.85mol / LNH3·H2O was added to the conical flask and stirred. The color of the solution quickly turned black and a precipitate was produced. After continuing the reaction for 30 minutes, the precipitate was washed with double-distilled water until the suspension was neutral (pH 7.0±0.1, confirmed by pH test paper). After washing with anhydrous ethanol three times, the product was dispersed in deionized water and dried in a vacuum drying oven to obtain Fe3O4 nanoparticles. The transmission electron microscopy results are shown as follows: Figure 2 As shown, the particles are uniform in size, with a particle diameter of about 15-25 nm.

[0078] 2. Construction of amino-functionalized Fe3O4 nanoparticles

[0079] Take 100mL of 5mg / mL Fe3O4 nanoparticle aqueous solution, mix it after ultrasonication for 5min, pour it into a three-necked flask, fix it on a mechanical stirrer, and place it in a constant temperature water bath at 70℃. While stirring, add 1mL of APTES to the suspension, and then stir it at 400r / min for 12h. The product is washed three times with ethanol and water, magnetically separated, and finally redispersed in an aqueous solution to obtain Fe3O4-NH2 solution. Take a small part to measure its solid content and calculate the solution concentration.

[0080] 3. Construction of aldehyde-functionalized Fe3O4 nanoparticles

[0081] Take 5 mL of 3 mg / mL Fe3O4-NH2 aqueous solution in a PE test tube, and add 0.4 mL of 50% glutaraldehyde. Oscillate and cross-link at room temperature for 2 hours. The product is magnetically separated and washed three times with PBS buffer solution (pH = 7.4). Finally, it is dispersed in PBS solution to obtain Fe3O4-CHO solution. Take a small part to measure its solid content and calculate the solution concentration.

[0082] 4. Optimization of Fe3O4 nanoparticle volume

[0083] Prepare the 6 mg / mL Fe3O4 nanoparticle solution in step 1, take 12.5, 25, 50, 100, and 200 μL of the solution and add them to 5 test tubes respectively. After uniform ultrasonic oscillation, use a critical shift magnetic particle spectrometer to detect the third harmonic signal, where the excitation frequency is 1000 Hz, the amplitude is 0.1, the current is 10 A, and the voltage is 3.5 V. The effect of the volume of the Fe3O4 nanoparticle solution on the level and stability of the third harmonic signal can be observed. Figure 3 As can be seen in a, the signal intensity and signal-to-noise ratio increase with increasing volume. Saturation is reached after 100 μL, so 100 μL is selected as the detection volume.

[0084] 5. Optimization of Fe3O4 nanoparticle concentration

[0085] The concentrations of the Fe3O4 nanoparticle solution prepared in step 1 were configured to 0.5, 1, 2, 3, and 4 mg / mL, respectively. After ultrasonic oscillation, 100 μL of the solution was taken into 5 test tubes. The third harmonic signal was detected using a critical shift magnetic particle spectrometer with an excitation frequency of 1000 Hz, an amplitude of 0.1, a current of 10 A, and a voltage of 3.5 V. The effect of the Fe3O4 nanoparticle solution concentration on the level and stability of the third harmonic signal was observed. Figure 3 As can be seen in b, the third harmonic signal intensity of the particles increases with the increase of concentration. Considering that high-concentration particles are prone to agglomeration, 3 mg / mL was selected as the detection concentration.

[0086] 6. Infrared characterization of functionalized Fe3O4 nanoparticles

[0087] The functionalized Fe3O4 nanoparticles synthesized in step 3 were characterized by infrared spectroscopy. Figure 4 580cm -1 The Fe-O stretching vibration peak near 1625cm is always present, which indicates that the structure of Fe3O4 is not destroyed during the reaction. -1 and 3422cm -1 Corresponding to the stretching vibration of OH on the Fe3O4 surface; in curve b, 1000cm -1 The absorption peak at 2925 cm is caused by the stretching vibration of the Si-O-Si bond of the APTES functional group. -1 The vibration peak of the CH bond was also observed at 1717 cm -1 The peak corresponding to the stretching vibration of the C=O bond is 2858 cm -1 The stretching vibration similar to alkyl CH also appeared, and the surface aldehyde group was successfully modified on the Fe3O4 surface.

[0088] 7. Stability analysis of third harmonic amplitude

[0089] Prepare the 3 mg / mL Fe3O4 nanoparticle solution synthesized in step 1, take 100 μL of each and add it to five PE test tubes. Use a critical offset magnetic particle spectrometer to measure at 0, 10, 20, 30, 40, 50, and 60 minutes, where the excitation frequency is 1000 Hz, the amplitude is 0.1, the current is 10 A, and the voltage is 3.5 V. Record the response value of the third harmonic. Observe the change of the third harmonic response value and error over time. The results are as follows: Figure 5 ,Within one hour, the third harmonic amplitude of the particles is stable, and the standard deviation is no more than 4 degrees,,proving that the constructed Fe3O4 nanoparticles have small errors and the toxin determination results are more accurate.

[0090] Example 2

[0091] The surface aldehyde-modified Fe3O4 nanoparticles prepared in Example 1 were used as carriers, and AFB1-Ab was coupled on their surfaces. The specific steps were as follows:

[0092] 1. Optimization of AFB1-Ab concentration

[0093] Prepare 3mg / mL Fe3O4-CHO, take 100μL and add it to six groups of 18 PE test tubes, and sonicate for 1 minute. Add 30μL of AFB-Ab (0, 0.1, 1, 10, 20, 40μg / mL) prepared in advance to each test tube, and place it in a shaker for coupling at room temperature and 160rpm for 2h. After the reaction is completed, use a magnet to remove the supernatant and wash twice with PBS buffer. Then add 100μL of 1% bovine serum albumin for encapsulation, place it in a shaker, and couple at room temperature at 160rpm for 1h. After the reaction is completed, use a magnet to remove the supernatant and wash twice with PBS buffer. Finally, add 100μL PBS (pH7.4) to obtain Fe3O4-CHO-Ab solution. The tubes were placed in a critical shift magnetic particle spectrometer and their initial values were measured and recorded. The excitation frequency was 1000 Hz, the amplitude was 0.1, the current was 10 A, and the voltage was 3.5 V. Subsequently, 10 μL of a 100 ng / mL AFB1 solution was added to each tube and the tubes were placed in a shaker at room temperature and 160 rpm for half an hour. The third harmonic response value of each tube was again measured and recorded. The results are shown in Figure 1. Figure 6 As shown in Figure a, the phase difference gradually increases with increasing AFB1-Ab concentration, reaching its maximum at 40 μg / mL. Although the phase difference increases slightly at 20 μg / mL and 40 μg / mL, the increases are limited, and the antibody consumption increases significantly, leading to a significant increase in cost. Therefore, 10 μg / mL was selected as the optimal concentration for AFB1-Ab use.

[0094] 2. Optimization of AFB1-Ab volume

[0095] Prepare 3mg / mL Fe3O4-CHO solution, take 100μL and add it to six groups of 18 PE test tubes, and ultrasonicate for 1min. Prepare the concentration of AFB1-Ab according to the results in Example 2, add different volumes of AFB1-Ab (0, 10, 20, 30, 40, 50μL) respectively, and place it in a shaking table for coupling at room temperature and 160rpm for 2h. After the reaction is completed, the supernatant is removed by magnetic adsorption and washed twice with PBS buffer, then 100μL of 1% bovine serum albumin is added for encapsulation, placed in a shaking table, and coupled at room temperature at 160rpm for 1h. After the reaction is completed, the supernatant is removed by magnetic adsorption and washed twice with PBS buffer, and finally 100μL PBS is added. Place it in a critical shift magnetic particle spectrometer, measure its initial value and record it, wherein the excitation frequency is 1000Hz, the amplitude is 0.1, the current is 10A, and the voltage is 3.5V. Then, add 10 μL of 100 ng / mL AFB1 solution to each tube and place it in a shaker at room temperature and 160 rpm for half an hour. Measure and record the third harmonic response value of each tube again. Figure 6 As shown in b, as the antibody volume increases, the phase difference value increases, but the growth rate slows down after 20 μL and has reached a saturation trend. Therefore, 20 μL is selected as the optimized antibody volume.

[0096] 3. Optimization of AFB1 binding time

[0097] Prepare 1.6mL of Fe3O4-CHO solution with a concentration of 3mg / mL, and carry out a coupling reaction with AFB1-Ab of the concentration and volume determined in Example 2 at room temperature and 160rpm for 2 hours. After the reaction is completed, use a magnet to adsorb and remove the supernatant, then wash twice with PBS buffer, then add 1.6mL of 1% bovine serum albumin for encapsulation, place in a shaker, and couple at room temperature at 160rpm for 1h. After the reaction is completed, use a magnet to remove the supernatant, wash twice with PBS buffer, and finally resuspend in 1.6mL PBS. Take 100μL of the above solution and add it to two groups of 6 polyethylene (PE) test tubes, 3 test tubes in each group. Place the test tube in a critical offset magnetic particle spectrometer to measure and record the initial signal value, where the excitation frequency is 1000Hz, the amplitude is 0.1, the current is 10A, and the voltage is 3.5V. Then, 10 μL of 100 ng / mL AFB1 solution was added to one group of test tubes, and 10 μL of PBS was added to the other group as a control group. The signal value of each test tube was measured at 5, 15, 25, and 40 minutes after the coupling reaction. Figure 6 As shown in Figure c, as the immune response time increases, the phase difference decreases. Therefore, 25 minutes is selected as the optimal immune response time.

[0098] Example 3

[0099] The optimization method of Example 3 is the same as that of Example 2, except that the antibody used is DON-Ab, which replaces AFB1-Ab to optimize the binding time with magnetic nanoparticles.

[0100] Figure 7 a is the optimization result of DON-Ab concentration. As the antibody concentration increases, the phase difference value also increases, and at 40 μg / mL, the signal intensity is still significantly improved. Therefore, 40 μg / mL is selected as the optimal antibody concentration; Figure 7 b is the optimization result of the immune response time. As the binding time increases, the phase difference of the third harmonic gradually increases, reaching the maximum at about 25 minutes, and then gradually decreases. This shows that at 25 minutes, the binding of DON and Fe3O4 MNPs reaches a saturated state and forms a stable complex. Finally, 25 minutes is selected as the optimal immune response time. Figure 7c shows the optimized binding time of DON-Ab and magnetic nanoparticles. As time increases, the third harmonic phase difference first increases and then decreases, indicating that extending the reaction time does not significantly improve the signal but instead increases experimental error. 2 h was selected as the optimal binding time for DON-Ab and magnetic nanoparticles. Fe3O4-CHO-Ab bound to DON-Ab was prepared according to the above optimized conditions.

[0101] Example 4

[0102] The Fe3O4 nanoparticle probe prepared in Example 2 was used as the material to investigate and analyze its specificity.

[0103] First, 1.8 mL of the 3 mg / mL Fe3O4-CHO-Ab solution prepared in Example 2 was taken, and 100 μL of the suspension was added to six groups of 18 test tubes, and then the initial magnetization response value was measured and recorded using a critical offset magnetic particle spectrometer. After the measurement was completed, 10 μL of 100 ng / mL AFB1, AFB2, AFG1, FB1 and pure methanol (as a control) were added to each group of test tubes, and the coupling reaction was carried out at room temperature for 25 minutes at a speed of 180 rpm. After the reaction was completed, the critical offset magnetic particle spectrometer was used again to measure the response value. Figure 8 The Fe3O4-CHO-coupled AFB1-Ab specifically recognized and reacted with AFB1, while showing no significant response to other target molecules. Furthermore, no significant cross-reactivity was observed between AFB1 and other toxin molecules. Therefore, the Fe3O4-CHO-coupled AFB1-Ab demonstrated highly specific recognition of AFB1, while other target molecules failed to elicit a similar response.

[0104] Example 5

[0105] The analysis method of Example 5 is the same as that of Example 4, except that the Fe3O4-CHO-Ab prepared in Example 3 combined with DON-Ab was used, and the toxins were DON, AFB1, OTA, and ZEN.

[0106] Depend on Figure 9 It can be seen that by comparing the signal differences between the four toxins and the control group, it can be clearly seen that the third harmonic signal difference of Fe3O4MNPs in the DON group is the largest before and after the reaction, which shows that the DON-Ab coupled with Fe3O4 MNPs can specifically recognize and react to DON molecules, and there is no obvious cross-reaction between DON molecules and the other three types of toxin molecules. This method has good specificity for detecting DON.

[0107] Example 6

[0108] Based on the Fe3O4-CHO-Ab prepared in Example 2, AFB1 was detected. The specific detection process is as follows:

[0109] (1) Dissolve 1 mg of AFB1 powder in 1 mL of methanol and disperse evenly by ultrasonication. Then dilute with pure methanol to obtain standard solutions of 0.0001 ng / mL, 0.001 ng / mL, 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, and 1000 ng / mL, respectively. Set aside for later use.

[0110] (2) Take the 3mg / mL Fe3O4-CHO-Ab solution prepared in Example 2. Divide it into 9 groups of 27 test tubes in total, add 100μL of Fe3O4-CHO-Ab solution to each test tube, and then put it into the critical offset magnetic particle spectrum to measure its initial value, wherein the excitation frequency is 1000Hz, the amplitude is 0.1, the current is 10A, and the voltage is 3.5V. Take 10μL of AFB1 prepared in advance in step (1) and add it to the test tube. The blank group is replaced with pure methanol and coupled with Fe3O4-CHO-Ab at room temperature for 25min at a speed of 180rpm. After the reaction is completed, the signal value is measured again. Figure 10 As shown in Figure 2, with the increase of AFB1 standard solution concentration, the phase difference of Fe3O4MNPs gradually increases. With the magnetic phase difference as the ordinate and the standard concentration as the abscissa, the obtained standard curve is y=1.12908·lg x+8.0412, and the linear correlation coefficient (R 2 ) was 0.996, the detection range was 0.01-100 ng / mL, and the detection limit was 0.008 mg / mL.

[0111] Example 7

[0112] The experimental process of Example 7 is the same as that of Example 6, except that the toxin to be detected is DON, and the detection material is the Fe3O4-CHO-Ab combined with DON-Ab prepared in Example 3.

[0113] With the increase of DON standard solution concentration, the phase difference of Fe3O4 MNPs showed a trend of increasing gradually. With the magnetic phase difference as the ordinate and the standard concentration as the abscissa, the obtained standard curve was y=1.3945lg x+5.2299, and the linear correlation coefficient (R 2 ) is 0.96, the detection range is 0.01-1000 ng / mL, and the detection limit is 0.012 ng / mL. Figure 11 shown.

[0114] Example 8

[0115] Based on the Fe3O4-CHO-Ab prepared in Example 2, the spiked recovery rate of AFB1 in actual samples was detected.

[0116] 1. Sample pretreatment

[0117] First, pulverize the rice, wheat, and corn samples separately. Then, weigh 5 g of each sample, for a total of four aliquots, and place them into 50 mL centrifuge tubes. Dilute the AFB1 stock solution to 100 ng / mL with methanol. Then, add 0 mL, 0.025 mL, 0.25 mL, and 2.5 mL of each aliquot to the four replicates, and adjust the volume to 10 mL with methanol. Vortex the sample to thoroughly mix with the toxin and place it in a fume hood to allow the methanol to evaporate to dryness.

[0118] 2. Sample extraction

[0119] After the methanol has completely evaporated, take out the 25mL 60% methanol extract prepared in advance and weigh 1g of NaCl at the same time. Add both to the conical flask and shake gently to mix evenly. Then, use a homogenizer to homogenize the mixture at high speed for about 60 seconds. After the treatment, place the conical flask in a shaker at 25°C and extract at 180rpm for about 30 minutes. After the extraction is completed, the extract is preliminarily filtered with a rapid qualitative filter paper, and then filtered multiple times with a 0.22μm organic filter head. The filtered filtrate is centrifuged at 10,000r / min for 10 minutes in a high-speed centrifuge, and the supernatant is collected and stored in the refrigerator.

[0120] 3. Recovery determination

[0121] The steps adopted when making the standard curve according to Example 6 were used, and these spiked samples were measured to obtain the spiked recovery data of this method. According to the calculation formula of recovery: recovery=(sample recovery detection value-blank recovery detection value) / standard detection value×100%, as shown in Table 1, the spiked recovery of rice was between 87.19%±13.4%% and 102.36%±11.93%, the spiked recovery of wheat was between 68.6%±8.65% and 96.71%±1.99%, the spiked recovery of corn was between 87.29%±11.95% and 101.71%±0.91%, and the overall spiked recovery was between 68.6%±8.65% and 102.36%±11.93%.

[0122] Table 1 Recovery of spiked aflatoxin B1

[0123]

[0124] Example 9

[0125] The experimental procedures of Example 9 are the same as those of Example 8, except that the toxin detected is DON.

[0126] According to Table 2, the spiked recovery of rice was between 75% ± 9.26% and 80.68% ± 27.31%, the spiked recovery of wheat was between 72.93% ± 13.1% and 104.68% ± 11.06%, and the spiked recovery of corn was between 71.87% ± 8.75% and 108.62% ± 16.2%. The overall spiked recovery was between 71.87% ± 8.75% and 108.62% ± 16.2%.

[0127] Table 2 Recovery of spiked deoxynivalenol

[0128]

[0129]

[0130] As can be seen from Examples 8 and 9, using the functionalized Fe3O4 magnetic nanoparticles and critical offset magnetic particle spectroscopy detection platform constructed by the present invention, spike recovery verification experiments were carried out on AFB1 and DON in various food matrices such as rice, wheat and corn. The results show that the spike recovery rates in various matrices are generally in the range of 68.60% to 108.62%; the relative standard deviation (RSD) of most tests is less than 15%, and some are better than 5%, showing good repeatability and accuracy. The above experimental results fully verify the effectiveness of the detection method of the present invention in the label-free, wash-free, high-throughput detection of small molecule toxins, and it has strong matrix adaptability and potential for promotion and application, providing a stable and reliable solution for the rapid on-site detection of toxins in agricultural products.

[0131] Example 10

[0132] In the screening stage, the present invention compares the effects of different synthesis methods (solvothermal method, coprecipitation method) and different types of magnetic particles (carboxyl modification, aldehyde modification) on the antibody modification efficiency and the critical offset magnetic particle spectrometer signal.

[0133] Fe3O4 MNPs were prepared by solvothermal method. The specific process is as follows:

[0134] (1) Preparation of oil-phase Fe3O4 magnetic nanoparticles:

[0135] Weigh 0.706g of ferric acetylacetonate into a 100mL beaker. Then, add 2.4mL of oleic acid and 38mL of anhydrous ethanol and stir for 10 minutes. Pour the mixed solution into a 50mL polytetrafluoroethylene reactor and react in a forced air drying oven at 180°C for 12 hours. After the reaction is complete, allow the reactor to cool to room temperature.

[0136] The solution was evenly divided into four 50 mL centrifuge tubes, and anhydrous ethanol was added dropwise to the centrifuge tubes, wherein V 乙醇 :V 溶液 =3:1. Centrifuge the solution (10 minutes, 8000 rpm), remove the small nanoparticles from the supernatant, and retain the precipitate. Add 5 mL of n-hexane to the precipitate, sonicate for 10 minutes, and centrifuge (10 minutes, 4000 rpm). Retain the supernatant to remove the large nanoparticles precipitated during the reaction, and then add ethanol. Repeat this process three times to remove both large and small Fe3O4 MNPs and excess oleic acid from the surface. Finally, dry the resulting brown-black product in a vacuum oven at 60°C for 4 hours to obtain dry oil-phase Fe3O4 MNPs.

[0137] (2) Oil-phase Fe3O4MNPs are converted to water-phase Fe3O4MNPs:

[0138] Weigh 0.2 g of trisodium citrate dihydrate and add it to 50 mL of deionized water. Stir to dissolve the trisodium citrate dihydrate. Add 0.5 mmol of oil-phase Fe3O4 nanoparticles (0.116 g) to the trisodium citrate dihydrate solution. After ultrasonic oscillation for 5 hours, use a magnet to collect the aqueous phase Fe3O4MNPs by magnetic adsorption, wash them three times with water and ethanol, and dry them in a vacuum drying oven.

[0139] The results are as follows Figure 12 As shown in Figure 3, the Fe3O4 MNPs prepared by the co-precipitation method have a higher and more stable third harmonic signal compared with the solvothermal method.

[0140] Carboxyl-functionalized Fe₃O₄ nanoparticles were constructed using the same method as in Example 1, except that 10 mg of COOH-PEG-COOH was dissolved in 2.5 mL of MES (0.1 mol / L, pH 6). EDC (0.0192 g, 0.1 mmol) was added and sonicated for 30 minutes. NHS (0.0115 g, 0.1 mmol) was then added and sonicated for 30 minutes. The mixture was then placed on a shaker at 140 rpm for 2 hours to activate the mixture. 2.5 mL of 5 mg / mL Fe₃O₄-NH₂ was immersed in the above solution, sonicated for 30 minutes, and then placed on a shaker at 140 rpm for overnight reaction. The mixture was placed on a magnet to allow adsorption and precipitation, and the supernatant was discarded. The mixture was washed three times with deionized water and finally dispersed in PBS (pH 7.4). A small portion was collected to measure the solids content and calculate the solution concentration.

[0141] The results are as follows Figure 13 As shown, it shows that the aldehyde-modified magnetic particles have higher binding efficiency and more stable third harmonic signal.

[0142] In summary, the use of aldehyde-functionalized magnetic particles and the stable coupling method of antibodies are key factors in achieving the detection effect of the present invention. The above comparative experiments and control variable analysis fully demonstrate the core role of these technical elements in the present invention.

[0143] Example 11

[0144] Comparisons of Examples 6 and 7 of the present invention with AFB1 and DON in the prior art are shown in Tables 3 and 4.

[0145] Table 3 Comparison of AFB1 related methods

[0146]

[0147] Table 4 Comparison of DON related methods

[0148]

[0149]

[0150] Among them, SPR 1 Sensitive Competitive Aptamer Surface Plasmon ResonanceSensor for Aflatoxin B1 Using Streptavidin as a Signal Enhancer[J].ACSPhysical ChemistryAu,2022.

[0151] Microfluidic paper device (μPAD) for rapid detection of aflatoxin B1 using an aptamer-based colorimetric assay[J].RSCAdvances,2020.

[0152] MRS technology is Magnetic relaxation switching immunoassay based on "limited-magnitude" particles for sensitive quantification of aflatoxin B1[J]. AnalyticaChimicaActa, 2023.

[0153] SPR 2Intramolecular charge transfer synergistic LSPR effect enhanced electrochemiluminescence platform for deoxynivalenol detection[J]. Sensors and Actuators B: Chemical, 2025.

[0154] Immunochromatography is Fluorescent microsphere immunochromatographic sensor for ultrasensitive monitoring deoxynivalenol in agricultural products[J]. Microchemical Journal, 2021.

[0155] Chemiluminescent immunosensor for rapid and quantitative determination of deoxynivalenol in cereal through the combination of magnetic solid-phase extraction and optical fiber-based homogeneous chemiluminescence immunosensor[J].Food Chemistry,2022.

[0156] In summary, it can be seen from Tables 3 and 4 above that the platform and method of the present invention show great advantages in detection cost (about 2 yuan / sample), detection time (about 2 minutes) and detection effect (detection limit).

Claims

1. A magnetic nanoprobe, characterized in that: The magnetic nanoprobe is composed of Fe3O4 magnetic nanoparticles with antibodies that can specifically identify fungal toxins modified on their surfaces. The antibodies are coupled through amino groups and aldehyde groups modified on the surfaces of the Fe3O4 nanoparticles to form a stable covalent connection.

2. The magnetic nanoprobe according to claim 1, characterized in that The Fe3O4 magnetic nanoparticles are synthesized by a co-precipitation method and have a particle size of 15-25 nm.

3. The magnetic nanoprobe according to claim 1, characterized in that The antibody is an antibody that targets, recognizes and binds to aflatoxin B1 or deoxynivalenol.

4. A method for preparing a magnetic nanoprobe, characterized in that: The steps include: (1) Preparation of Fe3O4 magnetic nanoparticles; (2) Preparation of Fe3O4 magnetic nanoparticles with surface modified aldehyde functional groups: amino groups are modified on the surface of Fe3O4 magnetic nanoparticles using a silane reagent, and then glutaraldehyde is mixed with the surface modified amino group Fe3O4 magnetic nanoparticles, thereby grafting the aldehyde functional groups onto the surface modified amino group Fe3O4 magnetic nanoparticles; (3) Preparation of Fe3O4 magnetic nanoparticle probes: Take the nanoparticles prepared in step (2), add antibodies and reaction solvents, and react; after the reaction is completed, discard the remaining antibodies under the action of magnetism, and then encapsulate with bovine serum albumin. After the reaction is completed, discard the remaining bovine serum albumin under the action of magnetism to obtain Fe3O4 magnetic nanoparticle probes.

5. The preparation method according to claim 4, characterized in that FeCl3 was dissolved in double-distilled water, and Na2SO3 was added to react. NH3·H2O was further added to react. The suspension was washed with double-distilled water until it became neutral. After washing, the product was dissolved in deionized water and dried in a vacuum drying oven.

6. The preparation method according to claim 4, characterized in that In step (2), the silane reagent is 3-aminopropyltriethoxysilane, the reaction temperature is 70-80°C, the reaction time is 10-12h, and the reaction temperature of glutaraldehyde and Fe3O4 nanoparticles is room temperature, and the reaction time is 1-2h.

7. The preparation method according to claim 4, characterized in that The antibody in step (3) is aflatoxin B1 antibody at a concentration of 10-20 μg / mL, the volume is 20-30 μL, the coupling temperature is room temperature, and the time is 2-3 h; the antibody is deoxynivalenol antibody at a concentration of 40-50 μg / mL, the volume is 20-30 μL, the coupling temperature is room temperature, and the time is 2-3 h; the bovine serum albumin concentration is 1-5%, the encapsulation temperature is room temperature, and the time is 1-2 h.

8. Use of the magnetic nanoprobe according to claim 1 in detecting toxins by critical shift magnetic particle spectroscopy under label-free conditions.

9. The use according to claim 8, characterized in that The phase response of the nanoparticles is recorded by a critical shift magnetic particle spectrometer, and the nonlinear magnetic response phase difference before and after the magnetic probe is connected to the fungus toxin is detected, thereby realizing label-free, real-time quantitative analysis of the fungus toxin in the liquid sample.

10. A critical shift magnetic particle spectroscopy detection platform based on surface functionalized Fe3O4 magnetic nanoparticles, characterized in that: Preferably, the magnetic nanoprobe and critical shift magnetic particle spectrometer according to claim 1 are included, and the magnetic phase difference generated before and after the immune reaction is used as a detection signal to achieve the detection of toxins.

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