An immunoprobe for simultaneously determining two mycotoxins on the same detection line

Abstract

This application relates to the field of rapid food safety detection technology, specifically disclosing an immunoprobe that simultaneously detects two mycotoxins on the same detection line. It uses a gap-enhanced silver sphere-encased gold flower nanomaterial as a substrate, with two carefully selected different Raman signal molecules placed in the interlayer of the nanomaterial. Monoclonal antibodies against T-2 toxin and DON are respectively linked to the substrate surface. The prepared immunoprobe (P-GERT) is then used to detect these toxins. ATP @Ag-Ab T‑2 P-GERT NBT @Ag-Ab DON This method was applied to LFIA. The Raman characteristic peaks of ATP and NBT did not overlap on the same detection line. Utilizing the correlation between characteristic peak intensity and target concentration, rapid simultaneous detection of T-2 toxin and DON was achieved on the same detection line. The standard curve concentration ranges for T-2 toxin and DON detection were 0.0033 pg / mL–100 ng / mL and 0.0067 pg / mL–100 ng / mL, respectively, with IC50 values ​​of 0.35 ng / mL and 0.29 ng / mL, respectively. Other common fungal toxins had no effect on the detection of T-2 toxin and DON; indicating that the established SERS-LFIA method is not only highly sensitive but also highly specific.

Description

Technical Field

[0001] This application relates to the field of rapid food safety testing technology, and more specifically, to an immune probe that can simultaneously detect two mycotoxins on the same detection line. Background Technology

[0002] Mycotoxins are secondary metabolites produced by various fungi (including Aspergillus, Penicillium, Fusarium, and Alternaria) and are commonly found in moldy agricultural products such as grains, nuts, and fruits. Mycotoxin contamination of animal feed can lead to contamination of animal-derived foods such as meat, eggs, and dairy products. Most mycotoxins remain structurally stable at normal cooking temperatures, and their accumulation in the food chain can have serious toxic effects on human health, such as causing vomiting, anorexia, cytotoxicity, and reproductive toxicity. Multiple large-scale data surveys show that the mycotoxin contamination rate of grains exceeds 25%, causing significant economic losses. T-2 toxin and DON have similar structures (both containing trichothecene rings) and are major mycotoxins contaminating crops. Among trichothecene toxins, T-2 toxin is the most toxic, while DON is the most frequently detected mycotoxin. Furthermore, multiple international studies have shown that most tested agricultural products exhibit multiple mycotoxin contamination. Therefore, establishing a reliable rapid detection platform for the simultaneous detection of T-2 toxin and DON is of great significance for ensuring food security and protecting human health. The EU has set the following combined limits for T-2 and HT-2 toxins: less than 20 µg / kg in milled cereal products, and less than 10 µg / kg in infant formula and processed cereal products suitable for infants and young children. my country's national standard GB2761-2017 specifies limits for DON in food, stating that the DON content in cereals and their products, such as corn, cornmeal, barley, wheat, oatmeal, and wheat flour, should be less than 1000 µg / kg.

[0003] Several technologies have been developed for rapid screening, semi-quantitative analysis, and precise quantification of mycotoxins. High-performance liquid chromatography (HPLC) and high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS), as reliable instrumental analytical methods, can achieve precise quantification of mycotoxins. However, these instrumental analytical methods require expensive equipment, professionally trained operators, and complex sample pretreatment procedures, making them unsuitable for point-of-care testing (POCT) scenarios. Advances in immunology have spurred the development of several new analytical methods as simpler and more economical alternatives to instrumental analytical methods. However, traditional enzyme-linked immunosorbent assay (ELISA) is time-consuming and lacks stability, often leading to false positive results. Side-flow immunoassay (LFIA) is convenient and fast, and is often used for rapid on-site screening of large numbers of samples. However, traditional gold nanoparticle-based LFIA can only be interpreted visually, resulting in low sensitivity. Although fluorescent LFIA has higher sensitivity, it is susceptible to interference from background fluorescence and cannot simultaneously detect multiple targets on the same detection line, resulting in low detection efficiency.

[0004] Surface-enhanced Raman spectroscopy (SERS) has gradually developed into an important spectroscopic analysis technique due to its high sensitivity, fast analysis speed, and specific molecular fingerprints. SERS-LFIA combines the advantages of LFIA (simple, rapid, and inexpensive operation) with the high sensitivity of SERS, and has received considerable attention in the field of food safety supervision in recent years. Currently, LFIA mainly achieves simultaneous detection of multiple components by setting multiple parallel T lines on the test strip. The number of parallel T lines on a test strip of limited length is generally limited to 3-4; simply increasing the number of parallel T lines on the test strip leads to increased detection time and increased detection error. To fully utilize the strong multi-component detection capability of SERS-LFIA and improve quantitative detection efficiency, this invention discloses a SERS-LFIA method for simultaneously determining the content of T-2 toxin and DON on the same detection line. Summary of the Invention

[0005] This invention is a novel analytical method that combines SERS and LFIA to fully leverage the advantages of each. Using gap-enhanced silver sphere-encased gold flower nanomaterials as a substrate, and relying on the intensities of non-overlapping Raman characteristic peaks of the two Raman signal molecules for quantitative analysis, this highly sensitive and specific multi-component rapid detection method is based on the specific reaction between antibody and antigen.

[0006] The first objective of this invention is to provide a method for preparing a SERS immune probe for simultaneous detection of T-2 toxin and DON on the same detection line, comprising the following steps: Raman signal molecularly labeled gap-enhanced gold nanoflowers were mixed with CTAC in water, and silver nitrate and a reducing agent were added sequentially to react and obtain Raman signal molecularly labeled gap-enhanced silver sphere-encapsulated gold flower nanomaterials. The pH of the dispersion of the Raman signal molecularly labeled gap-enhanced silver sphere-wrapped gold flower nanomaterial was adjusted to 8.0. Monoclonal antibodies against T-2 toxin or DON were added, mixed evenly, and allowed to stand for 12 hours. BSA was then added and the reaction continued for 1 hour to achieve the purpose of blocking and obtain the immune probe.

[0007] In one embodiment of the present invention, the monoclonal antibody against T-2 toxin and the monoclonal antibody against DON are derived from hybridoma cell line T10H11, which was deposited at the China Center for Type Culture Collection on September 25, 2025, with accession number C2025301, and hybridoma cell line D9H2, with accession number C2025299.

[0008] In one embodiment of the present invention, the Raman signal molecularly labeled gap-enhanced gold nanoflowers are prepared by the following method: Mix HAuCl4 with CTAC, add sodium borohydride, mix until the solution turns brownish-yellow, incubate in the dark to obtain gold seeds; HAuCl4 and CTAC were mixed, and ascorbic acid solution and diluted gold seed dispersion were added. The mixture was stirred, allowed to stand for several days, and then centrifuged to prepare gold nanospheres.

[0009] Gold nanospheres were mixed with an ethanol solution of Raman signal molecules, subjected to ultrasonic reaction, and centrifuged to obtain gold nanospheres labeled with Raman signal molecules.

[0010] HAuCl4 and CTAC were mixed, and the gold nanospheres labeled with the above Raman signal molecules and ascorbic acid solution were added. The mixture was subjected to sonication and centrifugation to obtain Raman signal molecule-labeled gap-enhanced gold nanoflowers.

[0011] In one embodiment of the present invention, the Raman signaling molecule is p-aminothiophenol (ATP) or p-nitrothiophenol (NBT).

[0012] The second objective of this invention is to provide a SERS immune probe for detecting T-2 toxin and DON obtained by the preparation method described above.

[0013] A third objective of this invention is to provide a composition comprising the SERS immune probe for detecting T-2 toxin and DON.

[0014] A fourth objective of this invention is to provide a test strip comprising the SERS immune probes for detecting T-2 toxin and DON.

[0015] A fifth object of the present invention is to provide the use of the immune probe, the composition, or the test strip in the detection of T-2 toxin and DON.

[0016] In one embodiment of the invention, the T-2 toxin and DON are derived from grains or feed.

[0017] In one embodiment of the present invention, the grain is one or more of corn, wheat, barley, rice, millet, sorghum and oats.

[0018] In one embodiment of the present invention, the concentration of the T-2 toxin is 0.0033 pg / mL. -1 ~100ngmL -1 The concentration of DON is 0.0067 pg / mL. -1 ~100ngmL -1 .

[0019] In one embodiment of the present invention, the method for preparing the SERS immune probe for detecting T-2 toxin and DON specifically includes the following steps: (1) Preparation of gap-reinforced gold nanoflowers (P-GERTNPs) First, prepare a gold seed dispersion. Mix a certain amount of HAuCl4 with CTAC, then quickly add freshly prepared NaBH4 (ice). Stir the mixture vigorously for 2 minutes until the color of the solution changes from bright yellow to brownish-yellow. Immediately place the container containing the above mixture in a 30°C water bath in the dark for incubation until ready for use.

[0020] Take a glass bottle, add HAuCl4 and CTAC separately, mix well, then quickly add ascorbic acid (AA) solution and diluted gold seed dispersion. Stir the mixture gently for a few seconds and keep it undisturbed for several days to promote particle growth. Finally, centrifuge and wash the product, remove the supernatant to obtain gold nanospheres, and store at 4℃ for later use.

[0021] Take a glass bottle, add gold nanosphere dispersion and an ethanol solution of Raman signaling molecules (p-aminothiophenol ATP or p-nitrothiophenol NBT), sonicate, centrifuge, and obtain gold nanospheres labeled with Raman signaling molecules.

[0022] HAuCl4 and CTAC were mixed, and the gold nanospheres labeled with the above Raman signal molecules and ascorbic acid solution (AA) were added. The mixture was subjected to sonication and centrifugation to obtain Raman signal molecule-labeled gap-enhanced gold nanoflowers (P-GERTNPs).

[0023] (2) Preparation of silver sphere-coated gold nanoflowers (P-GERT@AgNPs) CTAC, silver nitrate (AgNO3) solution, and AA solution were mixed in a water bath at 70°C, followed by the addition of P-GERTNPs. After reacting for several hours, the reaction was terminated by placing the flask in ice water. The mixture was then centrifuged, washed, and stored at 4°C until use. The prepared P-GERTNPs were then... ATP @AgNPs are used as a base material for immune probes.

[0024] (3) Preparation of immune probes (P-GERT@AgNPs-Ab) Take P-GERT ATP Add @AgNPs to a centrifuge tube, add a small amount of H2O and mix well. Adjust the pH of the system to 8. At 4°C, add the anti-T-2 toxin monoclonal antibody, mix well, and let stand overnight. Add BSA to the reaction system and stir gently to achieve the blocking purpose. Finally, centrifuge the solution and separate the immunoprobe (P-GERT). ATP The @AgNPs-Ab was resuspended in an equal volume of pure water and stored at 4°C. The final immunoprobe solution was yellow.

[0025] Take P-GERT NBT Add @AgNPs to a centrifuge tube, add a small amount of H2O and mix well. Adjust the pH of the system to 8. At 4°C, add the anti-DON monoclonal antibody, mix well, and let stand overnight. Add BSA to the reaction system and stir gently to achieve the blocking effect. Finally, centrifuge the solution and separate the immunoprobe (P-GERT). NBT The @AgNPs-Ab was resuspended in an equal volume of pure water and stored at 4°C. The final immunoprobe solution was yellow.

[0026] In one embodiment of the present invention, the test strip comprises a sample pad, a nitrocellulose membrane (NC membrane), and an absorbent pad. These are all fixed together on a polyvinyl chloride (PVC) substrate. The NC membrane has detection lines (T lines) and control lines (C lines). Depending on the detection requirements, the target antigen or antibody is typically evenly coated on the T lines, while a secondary antibody (such as goat anti-mouse IgG) is coated on the C lines.

[0027] In one embodiment of the present invention, the test strip includes a test line, a control line, a sample pad, and an absorbent pad; the test line is coated with two coated antigens (a conjugate of the target substance and ovalbumin OVA), namely T-2-OVA and DON-OVA; the control line is covered with goat anti-mouse IgG; and an immunoprobe and a sample solution are added to the sample pad. The immunoprobe is a 3 μL GP-GERT. ATP @AgNPs-Ab and 6μLP-GERT NBT@AgNPs-Ab, the coating antigens T-2-OVA and DON-OVA have concentrations of 2 mg / mL. -1 and 1 mg / mL -1 During the preparation of the immune probe, the volumes of anti-T-2 toxin monoclonal antibody and anti-DON monoclonal antibody added are 3 μL and 1 μL, respectively.

[0028] This invention characterizes the obtained immune probes and, under the selected experimental conditions after optimization, establishes a SERS-LFIA for the simultaneous determination of T-2 toxin and DON content.

[0029] The mechanism of this invention is competitive immunoassay. T-2 toxin and DON in the standard solution (or sample) compete with the coating antigen for limited antibodies on the immunoassay probe. Higher concentrations of T-2 toxin and DON result in less immunoassay probe on the T line and a weaker SERS signal. Furthermore, the immunoassay probes used to detect T-2 toxin and DON concentrations are labeled with different Raman signal molecules (ATP and NBT), and the characteristic peaks of the two Raman signal molecules used for quantification do not overlap. Therefore, the content of T-2 toxin and DON can be analyzed simultaneously on the same detection line. A standard curve can be plotted based on the correspondence between T-2 toxin and DON content and the SERS signal, and the target analytes in the sample can be quantitatively determined.

[0030] The technical solution of the present invention has the following advantages over the prior art: (1) This invention successfully prepared gap-enhanced silver sphere-coated gold flower nanomaterials as the substrate material for immunoprobes, and established a surface-enhanced Raman spectroscopy side-flow immunochromatography method for simultaneously determining the content of T-2 toxin and DON on the same detection line.

[0031] (2) The gap-enhanced silver sphere-coated gold flower nanomaterial prepared by the present invention has a strong SERS enhancement effect and good storage stability.

[0032] (3) The two different Raman signal molecules (ATP, NBT) carefully selected by this invention are on the same T line. The Raman characteristic peaks of ATP and NBT do not overlap and do not interfere with each other, ensuring the smooth detection of the two components on the same T line.

[0033] (4) The method of the present invention has high sensitivity for detecting T-2 toxin and DON content, with a sensitivity IC50 of 0.35 ng / mL for each. -1 and 0.29 ng / mL -1 .

[0034] (5) The detection method of the present invention has high specificity and almost no cross-reaction with the several fungal toxins selected in the cross-reaction.

[0035] (6) The present invention has simple sample processing, short detection time and low testing cost. Attached Figure Description

[0036] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein... Figure 1 This is a schematic diagram of the preparation method of the immune probe in this invention; Figure 2 The images are TEM images of Aucore (gold core, A), P-GERTNPs (B), and P-GERT@AgNPs (C) in Example 1; where (A) is the TEM image of Aucore; (B) is the TEM image of P-GERTNPs; and (C) is the TEM image of P-GERT@AgNPs. Figure 3 The UV-Vis spectra of Aucore, P-GERTNPs and P-GERT@AgNPs in Example 1 are shown below. Figure 4 The images show TEM images of P-GERTNPs synthesized with different volumes of Raman signaling molecules added in Example 1; (A) is a TEM image of P-GERTNPs synthesized with 2 μL of Raman signaling molecules added; (B) is a TEM image of P-GERTNPs synthesized with 10 μL of Raman signaling molecules added; (C) is a TEM image of P-GERTNPs synthesized with 50 μL of Raman signaling molecules added; (D) is a TEM image of P-GERTNPs synthesized with 100 μL of Raman signaling molecules added; (E) is a TEM image of P-GERTNPs synthesized with 200 μL of Raman signaling molecules added; and (F) is a Raman spectrum corresponding to different types of P-GERTNPs. Figure 5 This is a schematic diagram of the surface-enhanced Raman spectroscopy side-flow immunochromatographic test strip used in Example 2 to simultaneously detect T-2 toxin and DON content; Figure 6 For example 2, SERS-LFIA was measured in 0 ng / mL under different conditions. -1 Spectra on the T line of the analyte: In A, (Ⅰ) P-GERT ATP @Ag-Ab was used as a probe, and T-2-OVA was dispersed on the T line; (II) P-GERT ATP @Ag-Ab was used as a probe, and DON-OVA was dispersed on the T line; (III) P-GERT ATP @Ag-BSA was used as a probe, and T-2-OVA was dispersed on the T line; (IV) P-GERT ATP@Ag-Ab was used as a probe, and OVA was dispersed on the T line; (V) P-GERT ATP @Ag-Ab was used as a probe, and Na2CO3 and NaHCO3 buffer solutions were dispersed on line T; in B, (Ⅰ) P-GERT NBT @Ag-Ab was used as a probe, and DON-OVA was dispersed on the T line; (II) P-GERT NBT @Ag-Ab was used as a probe, and T-2-OVA was dispersed on the T line; (III) P-GERT NBT @Ag-BSA was used as a probe, and DON-OVA was dispersed on the T line; (IV) P-GERT NBT @Ag-Ab was used as a probe, and OVA was dispersed on the T line; (V) P-GERT NBT @Ag-Ab was used as a probe, and Na2CO3 and NaHCO3 buffer solutions were dispersed on the T line; Figure 7 This is a graph showing the results of the optimized experimental conditions in Example 2; where (A) represents the B0 / B ratio corresponding to different concentrations of T-2 toxin-coated antigen. 0.1 (B0 and B) 0.1 The SERS signal intensities corresponding to 0 ng / mL and 0.1 ng / mL; B0 / B 0.1 (B) The larger the size, the better the competitive inhibition effect); (C) Immune probe P-GERT ATP B0 / B corresponding to different T-2 toxin antibody volumes during @Ag-Ab preparation process 0.1 (C) Immunoprobe P-GERT ATP @Ag-Ab dosage corresponding to B0 / B 0.1 (D) B0 / B corresponding to different DON-coated antigen concentrations 0.1 (E) Immunoprobe P-GERT NBT B0 / B0 corresponding to different DON antibody volumes during @Ag-Ab preparation process 0.1 (F) Immunoprobe P-GERT NBT @Ag-Ab dosage corresponding to B0 / B 0.1 ; Figure 8 This is a diagram illustrating the feasibility verification of using immune probes containing different Raman signal molecules for two-component detection in Example 2. Figure 9The following is a SERS-LFIA standard curve for the simultaneous detection of T-2 toxin and DON in Example 2: (A) SEM image of the test strip after SERS-LFIA in a negative case; (B) Images of the test strip after SERS-LFIA in different cases; (C) SEM image of the test strip after SERS-LFIA in a strongly positive case; (D) SERS spectrum of Raman signal molecules on the T line after standard concentration determination; (E) SERS-LFIA standard curve for detecting T-2 toxin; (F) SERS-LFIA standard curve for detecting DON. Figure 10 The figure shows the reproducibility test results of the prepared test strips in Example 3. The figure also shows the Raman spectra obtained by randomly selecting five points on the T line after SERS-LFIA when the concentrations of the T-2 toxin and DON mixed solution were 0 ng / mL, 0.1 ng / mL, and 1 ng / mL, respectively. The Raman spectrum at 1136 cm⁻¹ is shown in the figure. -1 and 1333 cm -1 The specific signal value at that location; Figure 11 The diagram shows the cross-reaction detection structure in Example 2; (A) is the SERS spectrum on the T line after SERS-LFIA using PBST, 100 ng / mL T-2 toxin and DON standard solution and other 100 ng / mL cross-reactants; (B) is the bar chart of the calculated cross-reaction rate. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0038] Example 1: Preparation of Immunoprobes (1) Preparation of gap-reinforced gold nanoflowers (P-GERTNPs) First, prepare the gold seed dispersion as follows: Add 0.258 mL of HAuCl4 (4.86 mmol / L) to the dispersion. -1 ) and 5 mL CTAC (0.1 mol / L) -1 Mix, then quickly add 0.225 mL of freshly prepared NaBH4 (0.02 mol / L) with ice. -1 Stir the mixture vigorously for 2 minutes, until the color of the solution changes from bright yellow to brownish-yellow. Immediately place the bottle containing the above mixture in a 30°C water bath and incubate for 2 hours until ready for use.

[0039] Take a reaction flask and add 1.03 mL of HAuCl4 (4.86 mmol / L) to each flask. -1) and 40 mL CTAC (0.1 mol / L) -1 Mix thoroughly, then quickly add 0.15 mL of ascorbic acid (AA) solution (0.04 mol / L). -1 ) and 0.1 mL of gold seed dispersion diluted 10 times. The mixture was gently stirred for a few seconds and kept undisturbed at room temperature for 7 days to promote particle growth. Finally, the product was centrifuged at 11000 rpm for 10 min, the supernatant was removed, and the precipitate was resuspended in 20 mL of CTAC (0.02 mol / L). -1 The gold nanospheres (Aucore) were obtained and stored at 4℃ for later use.

[0040] Take a centrifuge tube and add 2 mL of gold nanosphere dispersion and an ethanol solution (10 mmol / L) of Raman signaling molecules (p-aminothiophenol ATP or p-nitrothiophenol NBT). -1 The mixture was sonicated for 30 minutes, centrifuged at 10,000 rpm for 10 minutes, the supernatant was removed, and the precipitate was resuspended in 1 mL of CTAC (0.05 mol / L). -1 Raman-labeled gold nanospheres were obtained and stored at 4°C for later use.

[0041] Add 0.96 mL of HAuCl4 (4.86 mmol / L) -1 ) and 10 mL CTAC (0.05 mol / L) -1 Mix the above mixture with 2.5 mL of Raman-labeled gold nanospheres and 0.48 mL of AA solution (0.04 mol / L). -1 The mixture was sonicated for 15 min, centrifuged at 6000 rpm for 10 min, washed three times with water to remove the supernatant, and the precipitate was resuspended in 5 mL of CTAC (0.05 mol / L). -1 ( ), and interstitial enhanced gold nanoflowers with Raman signal molecular labels were obtained and stored at 4℃ for later use.

[0042] (2) Preparation of silver ball-encased gold flower nanomaterial (P-GERT@AgNPs) 5 mL of CTAC (0.025 mol / L) was added to a water bath at 70 °C. -1 ), 0.8 mL AgNO3 (0.01458 mol / L) -1 ) and 3.75 mL AA (0.04 mol / L) -1 Mix the ingredients, then add 2 mL of the above-mentioned P-GERTNPs. After reacting for 3 hours, place the bottle in ice water to terminate the reaction. Centrifuge at 4000 rpm for 10 min, wash three times with ultrapure water, remove the supernatant, and resuspend the precipitate in 4 mL of pure water to obtain yellow P-GERT@AgNPs. Store at 4℃ for later use.

[0043] (3) Preparation of immune probes (P-GERT@AgNPs-Ab) Take 0.4 mL of LP-GERTATP@AgNPs into a centrifuge tube, adjust the pH of the system to 8.0, and then add 3 μL of monoclonal antibody against T-2 toxin (0.5 mg / mL) at 4°C. -1 The mixture was left to stand overnight. Then, 2 μL of LBSA (5%) was added to the mixture, and it was left to stand for 1 hour to achieve blocking. Finally, the resulting solution was centrifuged at 2500 rpm for 10 minutes at 4°C, and then the immunoprobe (P-GERT) was... ATP The @AgNPs-Ab was resuspended in an equal volume of pure water and stored at 4°C. The final immunoprobe solution was yellow-green in color.

[0044] Take 0.3mLP-GERT NBT @AgNPs were placed in a centrifuge tube, the pH of the system was adjusted to 8.0, and then 1 μL of anti-DON monoclonal antibody (1 mg / mL) was added at 4°C. -1 The mixture was left to stand overnight. Then, 2 μL of LBSA (5%) was added to the mixture, and it was left to stand for 1 hour to achieve blocking. Finally, the resulting solution was centrifuged at 2500 rpm for 10 minutes at 4°C, and then the immunoprobe (P-GERT) was... NBT The @AgNPs-Ab was resuspended in an equal volume of pure water and stored at 4°C. The final immunoprobe solution was yellow-green in color.

[0045] TEM images of Aucore (gold core), P-GERTNPs, and P-GERT@AgNPs are shown below. Figure 2 As shown, (A) is a TEM image of the Au nucleus; (B) is a TEM image of p-GERTNPs; and (C) is a TEM image of p-GERT@AgNPs. (From...) Figure 2 TEM images revealed that the Au cores had a uniform size of approximately 20 nm. The P-GERT@AgNPs had a size of approximately 50 nm, with multiple nano-interstic gaps forming around the gold core, resulting in numerous hot spots and thus better Raman enhancement. P-GERT@AgNPs generated after adding silver growth solution had a size of approximately 100 nm and a relatively uniform shape. Furthermore, Ag nanoparticles exhibited stronger SERS enhancement properties than Au nanoparticles. While Ag nanoparticles, due to their oxidizing properties, might have resulted in less stable nanoparticles, the P-GERT@AgNPs prepared using this method not only showed good SERS enhancement but also demonstrated good stability.

[0046] Figure 3 The UV-Vis spectra of Aucore, P-GERTNPs, and P-GERT@AgNPs are given; Figure 3 It was found that the LSPR peak of Aucore was located at 540 nm, while the LSPR peak of P-GERTNPs was located at 590 nm with a wider peak width. This may be due to the petal-like structure formed during the synthesis process and the larger particle diameter. The two LSPR peaks of P-GERT@AgNPs were located at 450 nm and 620 nm, which belong to the silver shell and gold core in the particle structure, respectively.

[0047] To obtain substrate materials with better Raman enhancement effects, the synthesis process of P-GERTNPs was optimized. Figure 4 TEM images (A-E) and corresponding Raman spectra (F) of P-GERTNPs@Ag synthesized by adding different volumes of Raman signal molecules (2 μL, 10 μL, 50 μL, 100 μL, 200 μL) during the synthesis of P-GERTNPs; Figure 4 It was found that as the amount of Raman molecules added gradually increased, more and more petal-like structures formed on the outside of the gold core, and the particle size also increased to a certain extent. Raman spectra showed that the SERS signal intensity of P-GERTNPs initially increased and then decreased with increasing Raman signal molecule addition. Through comparison, P-GERTNPs synthesized with a Raman signal molecule addition of 100 μL were finally selected for subsequent experiments.

[0048] Example 2: Surface-enhanced Raman spectroscopy side-flow immunochromatographic test strip for simultaneous detection of T-2 toxin and DON content. In this embodiment, a surface-enhanced Raman spectroscopy lateral flow immunochromatographic test strip is used for the simultaneous detection of T-2 toxin and DON content, as shown in the example. Figure 5 The LFIA test strip consists of the following components: a sample pad, a nitrocellulose membrane (NC membrane), and absorbent paper. These are all fixed together on a polyvinyl chloride (PVC) substrate. The NC membrane has a test line (Tline) and a control line (Cline). The Tline on the NC membrane is uniformly coated with 5 μL (2 mg / mL) of sample. -1 T-2-OVA and 5μL 1mg / mL -1 For DON-OVA, evenly coat 5 μL of goat anti-mouse IgG (dilution ratio 1:30) with the C line. Attach the sample pad and absorbent paper to both ends of the NC membrane, ensuring the overlap does not exceed 2 mm. After assembly, allow to air dry at room temperature for 1 hour, then store the LFIA test strip in a sealed bag at 4℃ for later use.

[0049] The obtained immune probes were characterized, and under the selected experimental conditions after optimization, a SERS-LFIA method for simultaneously determining the content of T-2 toxin and DON was established.

[0050] During the SERS signal measurement experiment, a mixture of two immune probes (3 μL P-GERT) was first added to the sample pad. ATP @AgNPs-Ab and 6μLP-GERT NBT (@AgNPs-Ab), then the edge of the sample pad of the test strip is immersed in 200 μL of sample solution. With the help of absorbent paper, the sample solution carries the immunoprobe towards the absorbent paper, flowing sequentially through the T and C lines of the test strip. The test strip exhibits a stable color change within 15 minutes. SERS signals were measured at 10 random points on the T line using a portable Raman spectrometer. To ensure accuracy, the final results were averaged.

[0051] The relevant detection parameters are as follows: the excitation source is adjusted to 785nm, the laser power is 200mW, and the typical integration time is 5s.

[0052] To verify the application of the SERS-LFIA method in real samples, corn and wheat were randomly purchased from the local market for spiking experiments. Taking corn as an example, four clean 5mL centrifuge tubes were prepared. The corn was thoroughly ground and homogenized, sieved, and 0.5g of corn flour was accurately weighed and placed into the centrifuge tubes. Then, 0μL, 12μL, 120μL, and 1200μL of acetonitrile solution (0.5ng / mL) containing T-2 toxin and DON were added respectively. -1 T-2 toxin and 0.5 ng / mL -1 DON). After mixing thoroughly, add 2 mL of methanol / water (4:1, v / v) to each test tube, vortex vigorously for 10 min, centrifuge at 10000 rpm for 10 min, and transfer the supernatant to 4 new centrifuge tubes. Next, dry the solvent with nitrogen gas in a 60°C water bath. Finally, use 600 μL of PBST buffer (0.01 mol / L). -1 The sample solution is reconstituted and used as the sample solution in the LFIA process. The unspecified sample solution serves as a blank control, and the other operating procedures are the same as for the spiked sample. The recovery rates of T-2 toxin and DON are calculated as follows: Recovery rate (%) = [(Measured T-2 toxin or DON concentration - Blank control) / Spiked T-2 toxin or DON concentration] × 100%.

[0053] 1) Solution preparation Sodium carbonate-sodium bicarbonate buffer solution Weigh 2.606g Na₂CO₃·10H₂O and 3.434g NaHCO₃, dissolve them in 800mL of ultrapure water, adjust the pH, and add water to 1L to prepare a 0.05mol / L solution. -1 Sodium carbonate-sodium bicarbonate buffer solution with a pH of 9.6; Phosphate buffer (stock solution, PBS × 10) Weigh 21.961g Na₂HPO₄·12H₂O, 6.031g NaH₂PO₄·2H₂O, and 87.666g NaCl, add 800mL of ultrapure water, mix, and heat to dissolve; use 1mol / L -1 Adjust the pH to 7.4 with NaOH, add ultrapure water to 1L, and prepare a solution containing 0.15 mol / L NaOH. -1 NaCl, pH 7.4, 0.1 mol / L -1 Phosphate buffer (stock solution); Phosphate buffer-Tween stock solution (0.1 mol / L containing 1% Tween 20) -1 Phosphate buffer stock solution, PBST×10, pH 7.4); 2) Main instruments Transmission electron microscope: Tecnai G220, FEI Corporation, USA; Portable surface-enhanced Raman spectrometer: RamTracer-200-HAS, Opto-Nano Technology Co., Ltd.; Ultraviolet spectrophotometer: UV-2300, Shanghai Tianmei Scientific Instruments Co., Ltd.

[0054] 3) SERS-LFIA nonspecific adsorption test Before performing the LFIA procedure, this application requires verification against non-specific adsorption interference to prevent false positives caused by other non-specific substances, which could affect the accuracy and sensitivity of the detection. In the experiment, the LFIA procedure was performed under the following five conditions to verify the specific recognition of the two prepared probes on their corresponding antigens (T-2 toxin and DON standard solution concentration of 0 ng / mL). -1 Taking the T-2 toxin probe as an example: (I) P-GERT ATP @Ag-Ab was used as a probe, and T-2-OVA was dispersed on the T line; (II) P-GERT ATP @Ag-Ab was used as a probe, and DON-OVA was dispersed on the T line; (III) P-GERT ATP @Ag-BSA was used as a probe, and T-2-OVA was dispersed on the T line; (IV) P-GERT ATP @Ag-Ab was used as a probe, and OVA was dispersed on the T line; (V) P-GERT ATP @Ag-Ab was used as a probe, and Na2CO3 and NaHCO3 buffer solutions were dispersed on the T line. Experimental results are as follows: Figure 6 As shown, by Figure 6 It can be seen that only T-2-OVA and P-GERT are present on the T line of the NC membrane. ATP @Ag-Ab exhibits specific binding with no other non-specific adsorption; similar experiments demonstrate that only DON-OVA and P-GERT show specific binding.NBT @Ag-Ab exhibits specific binding and no other non-specific adsorption occurs.

[0055] 4) Optimization of SERS-LFIA experimental conditions This application optimizes the volume of the antibody linked to the immune probe, the concentration of the coated antigen, and the volume of the immune probe, etc., with the optimization results as follows: Figure 7 As shown.

[0056] Depend on Figure 7 It can be seen that the optimal experimental conditions for the simultaneous determination of T-2 toxin and DON using this method are: the amount of antibody used in the preparation of the two probes is 3 μL and 0.5 mg / mL, respectively. -1 T-2-Ab and 1 μL 1 mg / mL -1 The DON-Ab coating antigen applied to the T line was 5 μL (2 mg / mL). -1 T-2-OVA and 5 μL 1 mg / mL -1 The DON-OVA sample pad had a 3 μL LP-GERT immunoprobe added to it. ATP @AgNPs-Ab and 6μLP-GERT NBT @AgNPs-Ab.

[0057] 5) ELISA standard curve and sensitivity To quantitatively detect two targets on the same T-line, the characteristic peaks of the two Raman signal molecules must not overlap to avoid mutual interference. For P-GERT... ATP @AgNPs colloidal solution, P-GERT NBT The Raman signals of colloidal solutions of @AgNPs and mixtures thereof were measured, and the experimental results are as follows: Figure 8 As shown in the figure, the characteristic Raman peak of ATP is at 1136 cm⁻¹, and the characteristic Raman peak of NBT is at 1333 cm⁻¹. -1 P-GERT ATP @AgNPs and P-GERT NBT The characteristic peaks of the two Raman signal molecules in the mixed solution of @AgNPs do not overlap. Therefore, the correspondence between the intensity of the characteristic peaks of ATP and NBT Raman spectra and the concentration of the target analytes can be used to quantitatively detect T-2 toxin and DON, respectively.

[0058] Prepare a mixed standard solution of T-2 toxin and DON (0 ng / mL) -1 10-5 ng / mL -1 10⁻⁴ ng / mL -1 10-3 ng / mL -1 10-2 ng / mL -1 10⁻¹ ng / mL-1 1.0 ng / mL -1 10 ng / mL -1 and 100 ng / mL -1 This is used in the SERS-LFIA procedure. After adding 200 μL of the mixed standard solution to the edge of the sample pad, the solution flows through the test strip. After 15 minutes, the color of the T and C lines can be seen to stabilize with the naked eye. As the concentration of the mixed standard solution gradually increases, the color of the T line gradually lightens, and the number of immune probes captured on the T line decreases. Figure 9 (A-C in the diagram). Subsequently, a portable Raman spectrometer was used to measure the SERS signal intensity of the immune probes captured by the antigen on the corresponding T-line. Figure 9 D in the middle). The standard curves for SERS-LFIA detection of T-2 toxin and DON are plotted in the form of B / B0×100%vs.lgC ( Figure 9 In the equation (E, F), B and B0 represent the SERS intensities measured at the standard concentration and zero concentration, respectively. Figure 9 It can be seen that the IC50 values ​​for detecting T-2 toxin and DON can be calculated using the standard curve to be 0.35 ng / mL. -1 and 0.29 ng / mL -1 Based on 3 standard deviations (SD), the limit of detection (LOD) was estimated to be 0.0033 pg / mL. - 1 and 0.0067 pg / mL -1 .

[0059] Example 3: Reproducibility of SERS-LFIA Three sets of mixed standard solutions with different concentrations were prepared, each containing 0 ng / mL. -1 0.1 ng / mL -1 and 1.0 ng / mL -1 In these three cases, SERS-LFIA was performed, and the SERS signal was measured at several different points randomly selected on the T-line. Figure 10 This will test the precision of the method. For example... Figure 10 As shown, at 0 ng / mL -1 0.1 ng / mL -1 1.0 ng / mL -1 The relative standard deviation (RSD) values ​​of the SERS intensity at the location ranged from 1.58% to 3.27%, indicating that the method has good reproducibility.

[0060] Example 4: Specificity of SERS-LFIA SERS-LFIA specificity can be expressed as cross-reactivity rate. Cross-reactivity rate (CR%) = (IC50 of T-2 toxin or DON / IC50 of the cross-substance) × 100%. The lower the cross-reactivity rate, the higher the specificity of SERS-LFIA.

[0061] In this invention, several common mycotoxins were selected, namely aflatoxin B1 (AFB1), ochratoxin A (OTA), patulin (PAT), zearalenone (ZEN), and fumonisin (FB1), to detect the specificity of SERS-LFIA. Figure 10 It can be seen that these common fungal toxins have almost no overlap, indicating that the constructed detection method has high specificity.

[0062] Example 5: SERS-LFIA determination of T-2 toxin and DON content in real samples Two actual samples (corn flour and wheat flour) were purchased from a local market to verify the practicality of the SERS-LFIA method, using a spiked recovery approach. Different amounts of a mixed standard solution of T-2 toxin and DON were added to the actual samples. After sample pretreatment, the eluent was subjected to the SERS-LFIA procedure, and the contents of T-2 toxin and DON in the spiked samples were calculated using a standard curve. The experimental results are shown in Table 1. The recoveries of T-2 toxin and DON in the spiked samples ranged from 89.7% to 114.6%, with relative standard deviations (RSDs) ranging from 2.32% to 9.09% (n=3).

[0063] Table 1. T-2 toxin and DON content in real samples determined by SERS-LFIA. T-2 toxin corn 0.01 <![CDATA[(1.04±0.06)×10 -2 ]]> 6.00 104.2 T-2 toxin corn 0.1 <![CDATA[(10.06±0.51)×10 -2 ]]> 5.04 100.6 T-2 toxin corn 1 1.09±0.07 6.07 109.2 T-2 toxin wheat 0.01 <![CDATA[(0.98±0.03)×10 -2 ]]> 3.47 98.3 T-2 toxin wheat 0.1 <![CDATA[(8.97±0.22)×10 -2 ]]> 2.48 89.7 T-2 toxin wheat 1 1.06±0.06 5.42 105.6 DON corn 0.01 <![CDATA[(0.98±0.09)×10 -2 ]]> 9.09 97.9 DON corn 0.1 <![CDATA[(10.78±0.25)×10 -2 ]]> 2.32 107.8 DON corn 1 1.03±0.06 5.78 103.4 DON wheat 0.01 <![CDATA[(1.08±0.04)×10 -2 ]]> 3.34 108.3 DON wheat 0.1 <![CDATA[(11.4±0.46)×10 -2 ]]> 4.06 114.0 DON wheat 1 1.15±0.05 4.52 114.6 Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An immunoprobe for simultaneous determination of two mycotoxins on the same detection line, characterized in that, The preparation of the immune probe includes the following steps: Raman-labeled gap-enhanced gold nanoflowers were mixed with hexadecyltrimethylammonium chloride in water, and silver nitrate and a reducing agent were added sequentially to react and obtain Raman-labeled gap-enhanced silver sphere-encapsulated gold flower nanomaterials. The pH of the obtained Raman-labeled gap-enhanced silver sphere-encapsulated gold flower nanomaterial dispersion was adjusted to 8.0, and monoclonal antibodies against T-2 toxin or DON were added. The mixture was mixed evenly and allowed to stand for 12 hours. Bovine serum albumin was added and the reaction was continued for 1 hour to achieve the purpose of blocking, thus obtaining an immune probe.

2. The immunoprobes for simultaneous determination of two mycotoxins on the same detection line according to claim 1, characterized in that: The Raman signal molecularly labeled gap-enhanced gold nanoflower material was prepared by the following method: Mix HAuCl4 with CTAC, add sodium borohydride, mix until the solution turns brownish-yellow, incubate in the dark to obtain gold seeds; HAuCl4 was mixed with CTAC, and ascorbic acid solution and diluted gold seed dispersion were added and stirred. The mixture was allowed to stand for several days and then centrifuged to obtain gold nanospheres. Gold nanospheres were mixed with an ethanol solution of Raman signal molecules, subjected to ultrasonic reaction, and centrifuged to obtain gold nanospheres labeled with Raman signal molecules. HAuCl4 and CTAC were mixed, and Raman-labeled gold nanospheres and ascorbic acid solution were added. The mixture was subjected to sonication and centrifugation to obtain Raman-labeled gap-enhanced gold nanoflowers.

3. The immunoprobes for simultaneous determination of two mycotoxins on the same detection line according to claim 1, characterized in that: The Raman signal molecule is p-aminothiophenol or p-nitrothiophenol.

4. A composition characterized in that: An immune probe comprising any one of claims 1-3 for simultaneously detecting two fungal toxins on the same detection line.

5. A test strip, characterized by: An immune probe comprising any one of claims 1-3 for simultaneously detecting two fungal toxins on the same detection line.

6. A surface-enhanced Raman spectroscopy side-flow immunochromatographic method for the simultaneous detection of T-2 toxin and deoxynivalenol on the same detection line, characterized in that, The immune probe for simultaneously detecting two fungal toxins on the same detection line as described in any one of claims 1-3, or the composition described in claim 4, or the test strip of claim 5 may be used.

7. The application of the surface-enhanced Raman spectroscopy side-flow immunochromatography method according to claim 6 in the simultaneous detection of T-2 toxin and DON.

8. Use according to claim 7, characterized in that: The T-2 toxin and DON are derived from grains or feed.

9. Use according to claim 8, characterized in that: The grains are one or more of the following: corn, wheat, barley, rice, millet, sorghum, and oats.

10. The SERS-LFIA method for simultaneously determining two mycotoxins on the same detection line according to claim 1, characterized in that: The concentrations of the T-2 toxin and DON were 0.0067 pg / mL to 100 ng / mL and 0.0033 pg / mL to 100 ng / mL, respectively.

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