CeO2 (at) Ru nanoparticle carrier, chemiluminescence signal probe, test strip and application

Abstract

The invention discloses a CeO2 (at) Ru nanoparticle carrier, a chemiluminescence signal probe, a test strip and application. The chemiluminescence signal probe comprises a signal carrier and a monoclonal antibody which is electrostatically adsorbed with the signal carrier. The particle size of the signal carrier CeO2 (at) Ru nanospheres (CeO2 (at) RuNSs) is 151.8 + / -9.1 nm, and the concentration of the OTA monoclonal antibody (OTA-mAb) is 1 mg / mL. According to the invention, a novel CL probe is prepared by using a CeO2 (at) RuNSs labeled antibody in the detection of a chemiluminescent test strip (CLICA) for the first time, and a luminol-H2O2 system is efficiently catalyzed. Benefited from the synergistic effect of CeO2 and Ru, the chemiluminescence efficiency is remarkably improved, and high-sensitivity visual detection of CLICA is realized. The lowest detection limit of the CeO2 (at) RuNSs-CLICA is 0.06 ng / mL and is far lower than that of traditional colorimetric ICA (0.23 ng / mL) based on gold nanoparticles, and an innovative thought is provided for development of a real-time detection technology.

Description

Technical Field

[0001] The present invention belongs to the field of immunoassay detection, and relates to a chemiluminescence (CL) signal probe, a chemiluminescent immunoassay strip (CLICA) for detecting ochratoxin A (OTA) and applications thereof, and particularly relates to a CeO2@Ru nanoparticle carrier, a chemiluminescence signal probe, a test strip and applications thereof. Background Art

[0002] OTA is a class of trace small molecule secondary metabolites produced by fungi, which are widely distributed in foods such as grains, traditional Chinese medicines, fruits and vegetables, and are the most serious pollutants in crops. Long-term intake can cause chronic diseases such as mutations, deformities and hormonal disorders, posing a serious threat to human health. Therefore, it is crucial to invent a rapid and accurate high-sensitivity detection method for OTA. ICA has great potential in the field of food safety due to its advantages such as simple operation, rapidity and low cost. However, the traditional AuNPs-ICA has the problem that the sensitivity cannot meet the detection requirements due to insufficient signal intensity, which hinders its development in trace analysis.

[0003] In recent years, with the help of the excellent physical / chemical properties of nanomaterials, a variety of new signal modes have been established, such as fluorescence, surface-enhanced Raman scattering, magnetism, photothermal, electrochemistry and chemiluminescence, which have greatly improved the sensitivity and accuracy of ICA. Among them, chemiluminescent immunoassay (CLICA) based on chemiluminescence has excellent signal-to-noise ratio, signal intensity and sensitivity, and does not require external light source interference, and has become one of the most promising biosensors.

[0004] At present, horseradish peroxidase (HRP)-luminol-H2O2 is one of the most commonly used CL systems. Among them, HRP, as a classic ROS trigger, significantly improves the CL intensity and realizes high-sensitivity CLICA detection. Unfortunately, the inherent defects of natural enzymes, including high cost, poor environmental tolerance and low chemical stability, seriously hinder their practical applications in CLICA.

[0005] Nanomaterial-based artificial enzymes have the characteristics of simple synthesis, low cost and excellent stability, and have become attractive alternatives to natural enzymes. Thanks to these advantages, various nanozymes have been invented, such as noble metals, metal oxides, metal-organic frameworks and single-atom catalysts, etc., to efficiently trigger the CL reaction and construct biosensors with good sensitivity. Among them, CeO2, as the most abundant rare earth metal oxide, has a variety of enzyme-like catalytic activities due to the reversible Ce 3+ / Ce 4+Redox pairs have made remarkable progress in the field of CL catalysis. However, compared with natural enzymes, their catalytic activity is not ideal, resulting in the lack of exploration of their application in CLICA. In addition, most existing CLICAs rely on cutting the detection line and placing it in the luminol-H2O2 system for solution detection, which cannot achieve the visualization of CLICA and hinders the further development of CLICA. Summary of the Invention

[0006] Aiming at the problems of insufficient CL efficiency and lack of CLICA visualization, the present invention provides a CeO2@Ru nanoparticle carrier, a chemiluminescence signal probe, a test strip and their applications for highly sensitive visual detection of OTA in wheat and coix seeds.

[0007] To achieve the purpose of the present invention, the following technical solutions are adopted:

[0008] A CeO2@Ru nanoparticle carrier, comprising spherical CeO2, with Ru loaded outside the CeO2 sphere; the particle size of the CeO2 sphere is 142.2 ± 7.1 nm, and the particle size of the CeO2@Ru nanoparticle carrier is 151.8 ± 9.1 nm;

[0009] The carrier is prepared by a metal self-deposition method from CeO2 and RuCl3.

[0010] Optionally, the preparation of the CeO2@RuNSs carrier includes:

[0011] CeO2 is dissolved in water. After ultrasonic treatment, the RuCl3 solution is slowly added to the CeO2 solution, and stirred at room temperature. The solution turns black-gray, and the precipitate is collected by centrifugation to obtain CeO2@Ru nanoparticles;

[0012] The concentration of the CeO2 solution is 1 mg / mL, the ultrasonic time is 30 min, the stirring time is 12 h, and the concentration of RuCl3 is 0.2 mM.

[0013] Optionally, PVP K30 and cerium nitrate are dissolved in ethylene glycol, and HCl solution is added under vigorous stirring;

[0014] When the solution is stirred until it becomes clear, it is transferred to a polytetrafluoroethylene-lined autoclave, heated at 160 °C, cooled to room temperature, centrifuged and washed multiple times, and the gray precipitate is collected to obtain CeO2;

[0015] The dosage of PVP K30 is 0.8 g, cerium nitrate is 2.0 g, ethylene glycol is 60 mL, HCl is 4 mL with a concentration of 1 M, and the heating time is 3 h.

[0016] A chemiluminescence signal probe, which includes the carrier and its electrostatically adsorbed monoclonal antibody;

[0017] The carrier described above is the CeO2@Ru nanoparticle carrier according to any one of the present invention;

[0018] The monoclonal antibody is OTA-mAb with a concentration of 1 mg / mL.

[0019] Optionally, the preparation method is as follows:

[0020] Dissolve the CeO2@Ru nanoparticles in water, incubate with OTA-mAb at room temperature, and block with bovine serum albumin, then centrifuge to obtain the CL signal probe;

[0021] The CeO2@Ru nanoparticle solution is 1 mL with a concentration of 1 mg / mL, OTA-mAb is 8 μL with a concentration of 1 mg / mL, the incubation time is 30 min, and the blocking time is 30 min.

[0022] The chemiluminescence signal probe described in the present invention is used for constructing a chemiluminescence test strip for detecting OTA in grains.

[0023] Optionally, the grains include wheat and / or coix seed.

[0024] A chemiluminescence test strip for detecting OTA in grains, adding the chemiluminescence signal probe described in the present invention into the sample to be tested, and preparing the test strip and the CL substrate solution;

[0025] This test strip includes attaching a nitrocellulose membrane to the corresponding position of a polyvinyl chloride base plate, pasting a blotting paper on the upper part of the nitrocellulose membrane, pasting a sample pad at the bottom end, and cutting and storing it sealed;

[0026] The preparation method of the nitrocellulose membrane: Both OTA-BSA and goat anti-mouse secondary antibody are sprayed on the nitrocellulose membrane at a scribing rate of 0.9 μL / cm, which are defined as the test line and the quality control line respectively, and dried overnight at 37 °C;

[0027] The concentration of OTA-BSA is 0.8 mg / mL, and the concentration of goat anti-mouse secondary antibody is 0.5 mg / mL;

[0028] The overlapping width of the blotting paper and the sample pad with the nitrocellulose membrane is 1.5 mm;

[0029] The preparation method of the sample pad: Put the glass fiber membrane into the blocking solution until it is completely soaked, and dry it overnight at 37 °C;

[0030] The cutting width is 3.2 mm.

[0031] Optionally, the CL substrate solution includes luminol, H2O2 and Tris-HCl buffer solution, and is used after mixing evenly;

[0032] The luminol concentration is 0.06 M, the H2O2 concentration is 0.25 M, and the Tris-HCl concentration is 0.4 M;

[0033] In the CL substrate solution, the volume ratio of luminol, H2O2, and Tris-HCl is 1:2:4.

[0034] Optionally, the detection method includes:

[0035] Insert the chemiluminescence test strip for detecting OTA in grains into the detection solution. After the immunoreaction, use a reader to obtain the colorimetric result, and then place it in a drying oven to fix the CeO2@Ru nanoparticles;

[0036] Then, drop the CL substrate solution onto the nitrocellulose membrane of the dried colorimetric test strip, collect the signal, and perform quantitative analysis using ImageJ software;

[0037] The immunoreaction time is 15 min, the drying time is 3 h, and the volume of the CL substrate solution is 10 μL.

[0038] Compared with the prior art, its advantages and positive effects are as follows:

[0039] (1) Novel CL signal probe: A novel CL probe is prepared by CeO2@RuNSs for the first time in immunochromatographic test strip detection. This probe has excellent catalytic activity and can significantly enhance the CL signal in the luminol-H2O2 system, improving the sensitivity and accuracy of detection.

[0040] (2) Visual detection: In the present invention, by adjusting the ratio and concentration of the CL substrate solution and dropping it onto the colorimetric ICA, the CL signal visualization is achieved.

[0041] (3) High sensitivity: The lowest detection limit of CeO2@RuNSs-CLICA prepared in the present invention for OTA is 0.06 ng / mL, which is much lower than that of the traditional gold nanoparticle-based ICA (0.23 ng / mL), and can be used as a general method for rapid and portable detection of the residue of mycotoxins in grains.

[0042] (4) Strong specificity: CeO2@RuNSs-CLICA prepared in the present invention has high specificity for OTA and its structural analogs ochratoxin B and ochratoxin C, and has no specificity for other mycotoxins.

[0043] (5) Good practical application: The present invention can detect OTA in wheat and coix seeds, has good practicability, and provides innovative ideas for the development of timely detection technologies. Brief Description of the Drawings

[0044] Figure 1 is the synthesis method of CeO2@RuNSs prepared in the present invention;

[0045] Figure 2 It is the characterization of CeO2@RuNSs material prepared by the present invention;

[0046] Figure 3 It is the characterization of the catalytic mechanism and antibody binding ability of CeO2@RuNSs prepared by the present invention;

[0047] Figure 4 This is the CeO2@RuNSs-luminol-H2O2 free radical scavenging experiment prepared by the present invention;

[0048] Figure 5 is the principle diagram of CeO2@RuNSs-CLICA prepared by the present invention;

[0049] Figure 6 It is the optimization result of CeO2@RuNSs-CLICA prepared by the present invention;

[0050] Figure 7 It is the sensitivity and specificity of CeO2@RuNSs-CLICA prepared by the present invention;

[0051] Figure 8 It is the practical application of CeO2@RuNSs-CLICA prepared by the present invention;

[0052] The present invention is described in detail below with reference to the accompanying drawings and specific implementation methods. DETAILED DESCRIPTION

[0053] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described below are only part of the embodiments of the present invention, not all of the embodiments, and do not limit the present invention in any form. All technical solutions using this embodiment, including simple changes to this embodiment, belong to the scope of protection of the present invention.

[0054] In the present invention, nanocarrier CeO2@RuNSs and OTA-mAb are used to prepare a novel CL signal probe, which can detect OTA based on colorimetric ICA, and the CL substrate solution is added to the dried colorimetric ICA to further complete the CL signal detection.

[0055] The probe can efficiently catalyze the luminol-H2O2 system reaction by virtue of the synergistic effect of CeO2 and Ru, thus achieving the purpose of enhancing the luminescence efficiency. Finally, the probe was successfully used in the construction of CLICA and showed good potential in practical applications in wheat and coix seed.

[0056] To obtain the best detection performance, the inventors optimized the antigen coating concentration, antibody labeling amount, and probe addition amount to construct a colorimetric ICA. Based on this, the CL substrate solution was further optimized, including the antibody addition amount, luminol concentration, H2O2 concentration, and Tris-HCl concentration, and a highly sensitive visual CLICA was successfully prepared. This method has been successfully applied to the detection of OTA in wheat and coix seed, and its sensitivity, stability, and practicability have been verified.

[0057] The reaction principle of this test strip: Based on the competitive detection principle, the CeO2@RuNSs mAb probe is added to the sample to be tested to capture the OTA target, and the bound CeO2@RuNSs mAb-OTA immune complex moves to the detection area of the CLICA by capillary action.

[0058] For positive samples, no visible band appears on the test line for the CeO2@RuNSs-mAb probe (neither colorimetric nor CL signal). For negative samples, the CeO2@RuNSs-mAb probe can form a complex with OTA-BSA in the test line, and a black band is formed due to the visible color of CeO2@RuNSs (Mode 1: Detection is carried out based on colorimetric ICA under ambient light. Mode 2: By virtue of the efficient catalytic activity of CeO2@RuNSs (nanoparticles), the CL substrate solution is added to achieve CLICA analysis of OTA).

[0059] The CL signal probe of the present invention includes a carrier and a monoclonal antibody electrostatically adsorbed thereon; the carrier is CeO2@RuNSs, which is prepared from CeO2 and RuCl3·3H2O; the particle size of the carrier CeO2@RuNSs is 142 - 160 nm, and the particle size of CeO2 is 135 - 149 nm; the monoclonal antibody is OTA-mAb with a concentration of 1 mg / mL; after the carrier CeO2@RuNSs and OTA-mAb are electrostatically adsorbed, they are blocked with bovine serum albumin, and the CL probe is obtained after centrifugation.

[0060] The CLICA of the present invention includes attaching a nitrocellulose membrane to the corresponding position of a polyvinyl chloride bottom plate, pasting a blotting paper on the upper part of the nitrocellulose membrane, pasting a sample pad at the bottom end, and storing it sealed after cutting;

[0061] A CL signal probe, including a carrier and a monoclonal antibody electrostatically adsorbed thereon; the carrier is CeO2@RuNSs, which is prepared from CeO2 and RuCl3·3H2O; the particle size of the carrier CeO2@RuNSs is 151.8 ± 9.1 nm, and the particle size of CeO2 is 142.2 ± 7.1 nm; the monoclonal antibody is OTA-mAb with a concentration of 1 mg / mL;

[0062] Specifically, the preparation method of the CL signal probe is as follows:

[0063] Step 1: Dissolve PVP K30 and cerium(III) nitrate hexahydrate in ethylene glycol. Under vigorous stirring, add the HCl solution. After the solution is stirred until clear, transfer it to a polytetrafluoroethylene-lined autoclave and heat at 160 °C. Cool to room temperature, centrifuge and wash multiple times, and collect the gray precipitate, which is CeO2;

[0064] Specifically, the dosage of PVP K30 is 0.8 g, the cerium(III) nitrate hexahydrate is 2.0 g, the ethylene glycol is 60 mL, the HCl is 4 mL with a concentration of 1 M, and the heating time is 3 h;

[0065] Step 2: Dissolve the CeO2 obtained in Step 1 in ultrapure water. After ultrasonic treatment, slowly add the RuCl3·3H2O solution to the CeO2 solution. Stir at room temperature. The solution turns dark gray. Centrifuge and collect the precipitate, which is CeO2@RuNSs;

[0066] Specifically, the CeO2 solution is 30 mL with a concentration of 1 mg / mL, the ultrasonic time is 30 min, the stirring time is 12 h, the RuCl3·3H2O solution is 25 mL with a concentration of 0.2 mM;

[0067] Step 3: Dissolve the CeO2@RuNSs obtained in Step 2 in ultrapure water and incubate with OTA-mAb at room temperature, and block with bovine serum albumin. Centrifuge to obtain the CL signal probe;

[0068] Specifically, the CeO2@RuNSs solution is 1 mL with a concentration of 1 mg / mL, the OTA-mAb is 8 μL with a concentration of 1 mg / mL, the incubation time is 30 min, and the blocking time is 30 min.

[0069] The CL probe of the present invention is used for the application of detecting OTA in grains, and the grains include wheat and coix seed.

[0070] A CLICA for detecting OTA. After the test strip is incubated with the CL probe and the OTA detection sample, it is inserted into the CLICA for immunoreaction, dried and reserved. Then, a CL substrate solution is dropped on the dried test strip for CLICA signal detection;

[0071] Specifically, the CLICA includes attaching a nitrocellulose membrane to the corresponding position of a polyvinyl chloride bottom plate, pasting a blotting paper on the upper part of the nitrocellulose membrane, pasting a sample pad at the bottom end, cutting and storing it sealed;

[0072] The concentration of the OTA-BSA is 0.8 mg / mL, and the concentration of the goat anti-mouse secondary antibody is 0.5 mg / mL;

[0073] The preparation method of the nitrocellulose membrane: Both OTA-BSA and goat anti-mouse secondary antibody are sprayed on the nitrocellulose membrane at a scribing rate of 0.9 μL / cm, which are defined as the test line and the quality control line respectively, and dried overnight at 37 °C;

[0074] The overlapping widths of the absorbent paper and the sample pad with the nitrocellulose membrane are both 1.5 mm;

[0075] The preparation method of the sample pad: Put the glass fiber membrane into the blocking solution until it is completely soaked, and dry it overnight at 37 °C;

[0076] The cutting width is 3.2 mm;

[0077] Specifically, the CL substrate solution includes luminol, H2O2, and Tris-HCl;

[0078] The concentration of luminol is 0.06 M, the concentration of H2O2 is 0.25 M, and the concentration of Tris-HCl is 0.4 M;

[0079] The ratio of luminol, H2O2, and Tris-HCl in the CL substrate solution is 1:2:4;

[0080] Specifically, the CLICA signal detection includes inserting CeO2@RuNSs-CLICA into the detection solution. After the immunoreaction, the colorimetric result is obtained using a reader, and then placed in an oven to fix CeO2@RuNSs. Then, the CL substrate solution is dropped on the dried colorimetric test strip nitrocellulose membrane, the signal is collected using an iphone 13pro, and quantitative analysis is performed through Image J software;

[0081] The immunoreaction time is 15 min, the drying time is 3 h, and the volume of the CL substrate solution is 10 μL.

[0082] The experimental reagents used in the present invention are all commercially available and have not been further processed. The detection instruments and equipment, etc. are all common instruments, and the detection methods are all conventional detection methods.

[0083] Example 1: Synthesis and characterization of CeO2@RuNSs

[0084] Combined Figure 1 and 2 , this example gives the carrier CeO2@RuNSs and its preparation method. The carrier CeO2@RuNSs is prepared from CeO2 and RuCl3·3H2O; the particle size of the carrier CeO2@RuNSs is 151.8 ± 9.1 nm, and the particle size of CeO2 is 142.2 ± 7.1 nm;

[0085] The preparation method of the carrier CeO2@RuNSs includes:

[0086] Step 1: Dissolve PVP K30 and cerium(III) nitrate hexahydrate in ethylene glycol. Under vigorous stirring, add the HCl solution. After the solution is stirred until clear, transfer it to a polytetrafluoroethylene-lined autoclave and heat at 160 °C. Cool to room temperature, centrifuge and wash several times, and collect the gray precipitate, which is CeO2;

[0087] The dosage of PVP K30 is 0.8 g, the cerium(III) nitrate hexahydrate is 2.0 g, the ethylene glycol is 60 mL, the HCl is 4 mL with a concentration of 1 M, and the heating time is 3 h;

[0088] Step 2: Dissolve the CeO2 described in Step 1 in ultrapure water. After ultrasonic treatment, slowly add the RuCl3·3H2O solution to the CeO2 solution. Stir at room temperature, and the solution turns black-gray. Centrifuge to collect the precipitate, which is CeO2@RuNSs;

[0089] The CeO2 solution is 30 mL with a concentration of 1 mg / mL, the ultrasonic time is 30 min, the stirring time is 12 h, the RuCl3·3H2O solution is 25 mL, and the concentration is 0.2 mM;

[0090] A series of characterizations were carried out on CeO2 and CeO2@RuNSs in this example, and the results are analyzed as follows:

[0091] (1) Transmission electron microscopy: As shown in TEM images 2A and 2B, the synthesized CeO2 has excellent dispersibility and uniformity. By measuring 100 nanoparticles, the particle size of CeO2 is calculated to be 142.2 ± 7.1 nm. As shown in TEM images 3D and E, it is clearly observed that Ru is successfully loaded onto the surface of CeO2, and the particle size of CeO2@RuNSs is 151.8 ± 9.1 nm( Figure 2 F).

[0092] (2) X-ray energy dispersive spectroscopy (EDS): The EDS results( Figure 2 G) show that C, N, O, Ce, and Ru elements are present in the invented CeO2@RuNSs.

[0093] (3) XRD analysis: The XRD results( Figure 2 H) show that CeO2 shows clear peaks at 28.4° (111), 32.9° (200), 47.2° (220), 56.1° (311), 58.8° (222), and 69.1° (400) (PDF#97-018-2989), and CeO2@RuNSs shows an obvious peak at 38.4° (100) (PDF#97-005-2261), corresponding to Ru.

[0094] (4) XPS analysis: The survey spectrum of XPS ( Figure 2 I) The characteristic peaks of Ce 3d, O 1s, Ru 3p, N 1s and C 1s can be observed. The Ce 3d and Ru 3p are analyzed in detail.

[0095] The XPS spectrum of Ce 3d ( Figure 2 J) is fitted to ten peaks, including Ce 3d5 / 2 (ν0, 880.5 eV; ν, 882.3 eV; ν′, 885.3 eV; ν″, 889.1 eV; ν″′, 898.2 eV) and Ce 3d3 / 2 (u0, 900.3 eV; u, 901.0 eV; u′, 902.9 eV; u″, 907.4 eV; u″′, 916.6 eV). Among them, u′, u0, v′ and v0 come from Ce 3+ , u, u″, u″′, ν, ν″ and ν″′ come from Ce 4 + . These results clearly determine the existence of Ce 3+ and Ce 4+ oxidation states in CeO2@RuNSs.

[0096] The XPS spectrum of Ru 3p ( Figure 2 K) is fitted to four peaks. Among them, 462.5 eV and 484.7 eV correspond to Ru 3p3 / 2 and Ru 3p 1 / 2 respectively. The relatively weak energy bands at 465.4 eV and 486.8 eV belong to ruthenium oxide.

[0097] These results indicate the successful preparation of CeO2@RuNSs.

[0098] Example 2: Preparation method of CL probe

[0099] According to the above technical solution, the preparation method of the CL probe in this example is given, including the carrier CeO2@RuNSs, and adding OTA-mAb for electrostatic adsorption to obtain it. It includes the following steps:

[0100] Dissolve the invented CeO2@RuNSs in ultrapure water and incubate with OTA-mAb at room temperature, and block with bovine serum albumin, and centrifuge to obtain the CL signal probe;

[0101] Specifically, the CeO2@RuNSs solution is 1 mL, with a concentration of 1 mg / mL, OTA-mAb is 8 μL, with a concentration of 1 mg / mL, the incubation time is 30 min, and the blocking time is 30 min.

[0102] Example 3: Catalytic mechanism of CeO2@RuNSs and characterization of its binding ability with antibodies

[0103] CombinedFigure 3 and 4 In this example, the catalytic mechanism of CeO2@RuNSs and its binding ability with antibodies were characterized, and the results were analyzed as follows:

[0104] (1) UV-vis: As Figure 3 shown in A, the maximum emission wavelengths of the luminol-H2O2 system and the CeO2@RuNSs-luminol-H2O2 system are both 431 nm, without red shift or blue shift.

[0105] (2) CL intensity-time kinetic curve: Figure 3 As shown in B, compared with the luminol-H2O2 system, after adding CeO2@RuNSs, the CL intensity increased by nearly 59 times, and the duration was about 3600 seconds.

[0106] (3) ROS scavenging experiment: From Figure 3 C and Figure 4 analysis, it can be obtained that the maximum quenching rates of ascorbic acid, p-benzoquinone, tryptophan and tert-butanol are 99.3%, 99.6%, 5.2% and 5.5% respectively, indicating that O2 ·- plays a decisive role in the CeO2@RuNSs-luminol-H2O2 system.

[0107] (4) ESR: Figure 3 As shown in D, compared with DMPO-OH · and TEMP- 1 O2, the intensity of DMPO-O2 ·- is more obvious, indicating that the catalytic reaction of the CeO2@RuNSs-luminol-H2O2 system depends on O2 ·- , and a considerable amount of O2 ·- promotes the generation of OH · and 1 O2, which enhances the CL reaction as a secondary free radical.

[0108] These results indicate that CeO2@RuNSs has excellent catalytic activity. In addition, the invention gives its binding ability with OTA-mAb, and the results are analyzed as follows:

[0109] (5) UV-vis: From Figure 3 E, it can be seen that CeO2@RuNSs has a characteristic absorption peak at 320 nm derived from Ru, while after binding with OTA-mAb, a characteristic absorption peak appears at 280 nm.

[0110] (6) Zeta potential: Figure 3F shows that after the electrostatic adsorption of CeO2@RuNSs and OTA-mAb, the Zeta potential value changes from -31.6 mV to -25.1 mV.

[0111] (7) Antibody binding rate: See Figure 3 G. The binding rate of CeO2@RuNSs and the antibody is 91.1%, which is higher than that of AuNPs and the antibody (80.5%).

[0112] (8) Affinity constant: The affinity constant of CeO2@RuNSs-mAb is 2.99×10 7 M -1 .

[0113] (9) Dissociation constant: The dissociation constant of CeO2@RuNSs-mAb is 4.09×10 9 M.

[0114] These results can prove that after the effective coupling of CeO2@RuNSs and mAb, the immune activity of natural mAb is still maintained.

[0115] Example 4: Preparation and detection method of CLICA for highly sensitive visual detection of OTA in wheat and coix seed

[0116] Combined Figure 5 , this example gives the preparation method of CeO2@RuNSs-CLICA, including attaching the nitrocellulose membrane to the corresponding position of the polyvinyl chloride bottom plate, pasting the absorbent paper on the upper part of the nitrocellulose membrane, pasting the sample pad at the bottom end, cutting and storing it sealed;

[0117] Preparation method of nitrocellulose membrane: OTA-BSA and goat anti-mouse secondary antibody are both sprayed on the nitrocellulose membrane at a scribing rate of 0.9 μL / cm, which are defined as the detection line and the quality control line respectively, and dried overnight at 37°C;

[0118] The concentration of OTA-BSA is 0.8 mg / mL, and the concentration of goat anti-mouse secondary antibody is 0.5 mg / mL;

[0119] The overlapping width of the absorbent paper and the sample pad with the nitrocellulose membrane is 1.5 mm;

[0120] Preparation method of sample pad: Put the glass fiber membrane into the blocking solution until it is completely soaked, and dry it overnight at 37°C; the cutting width is 3.2 mm;

[0121] This example presents the CeO2@RuNSs-CLICA detection method, which includes inserting CeO2@RuNSs-CLICA into the detection solution. After the immunoreaction, the colorimetric result is obtained using a reader, and then it is placed in an oven to fix CeO2@RuNSs. Then, the CL substrate solution is dropped onto the dried colorimetric test strip, signals are collected using an iphone 13pro, and quantitative analysis is performed through Image J software;

[0122] The CL substrate solution includes luminol, H2O2, and Tris-HCl;

[0123] The concentration of luminol is 0.06 M, the concentration of H2O2 is 0.25 M, and the concentration of Tris-HCl is 0.4 M;

[0124] The ratio of luminol, H2O2, and Tris-HCl in the CL substrate solution is 1:2:4;

[0125] The immunoreaction time is 15 min, the drying time is 3 h, and the volume of the CL substrate solution is 10 μL.

[0126] Example 5: Parameter Optimization of the Invention of CeO2@RuNSs-CLICA

[0127] Combined with Figure 6 , this example presents the parameter optimization of the invention of CeO2@RuNSs-CLICA, including the addition amount of OTA-mAb, the concentration of luminol, the concentration of H2O2, and the concentration of Tris-HCl. The results are analyzed as follows:

[0128] (1) Addition amount of OTA-mAb: Figure 6 A and B show that when the amount of OTA-mAb is 8 μg, the CL signal intensity is about 24000, a bright blue light can be produced, and the best inhibition rate is exhibited.

[0129] (2) Concentration of luminol: See Figure 6 C. As the concentration of luminol increases from 0.04 to 0.10 mol / L, the CL intensity of the T line gradually increases, while the competitive inhibition rate shows a downward trend. Considering both the CL intensity and the inhibition rate, 0.06 mol / L of luminol is used in subsequent experiments.

[0130] (3) Concentration of H2O2: H2O2 is another important component in the CL substrate solution, which can generate ROS to excite luminol. When the concentration of H2O2 is too low, it is difficult to obtain an obvious CL signal, while when the concentration is too high, the background interference increases. When the concentration of H2O2 is 0.25 mol / L, an obvious blue light appears and the inhibition rate is the highest ( Figure 6 D).

[0131] (4) Tris-HCl concentration: Tris-HCl provides a suitable alkaline environment for the CL substrate to promote the reaction. When its concentration is 0.4 mol / L ( Figure 6 E), the CeO2@RuNSs-CLICA of the invention shows the optimal result.

[0132] Example 6: Sensitivity determination of CeO2@RuNSs-CLICA

[0133] Detection process: In a 96-well microplate, 200 μL of OTA with different concentrations (0 to 4 ng / mL) was incubated with CeO2@RuNSs-mAb for 3 min, and then the test strip was added for immunological reaction for 15 min, and the colorimetric result was read. To further obtain the CL detection signal, the CeO2@RuNSs-ICA used for colorimetric analysis before was dried in an oven, then the CL substrate solution was added, the signal was collected using an iphone 13pro, and quantitative analysis was performed using Image J software.

[0134] Detection results: The minimum concentration of OTA corresponding to the complete disappearance of the test line is defined as the disappearance line value; when the test line is visually observed to be significantly lighter than the blank control strip, the corresponding OTA concentration is defined as the visual limit of detection (vLOD); the OTA concentration at which the signal intensity of 50% competitive inhibition disappears is defined as the half-maximal inhibitory concentration (IC 50 ); the competitive inhibition rate IC 10 is defined as the limit of detection (LOD); the linear range is defined as the OTA concentration corresponding to 20% to 80% inhibition (IC 20 ~IC 80 ).

[0135] As Figure 7 shown in A-C, CeO2@RuNSs-CLICA has a good linear range in the range of 0.09 - 0.32 ng / mL, the disappearance line value is 1 ng / mL, and the vLOD is 0.25 ng / mL. In addition, the colorimetric CeO2@RuNSs-ICA and AuNPs-ICA also constructed good linear relationships in the ranges of 0.32 - 1.80 and 0.45 - 2.03 ng / mL respectively, and the corresponding disappearance line values and vLODs are 2 and 0.5 ng / mL respectively. At the same time, CeO2@RuNSs-CLICA (IC 50 is 0.17 ng / mL) is respectively compared with the colorimetric CeO2@RuNSs-ICA (IC 50 is 0.78 ng / mL) and AuNPs-ICA (IC 50It is 4.59 times and 4.76 times higher than that (0.81 ng / mL). In addition, the LOD of the invented CeO2@RuNSs-CLICA is 0.06 ng / mL, which is much lower than that of the traditional gold nanoparticle-based colorimetric ICA (0.23 ng / mL), showing excellent sensitivity.

[0136] Example 7: Specific determination of CeO2@RuNSs-CLICA

[0137] Detection process: In a 96-well microplate, 200 μL of vomitoxin, T2 toxin, patulin, deoxynivalenol, fumonisin B1, ochratoxin B, ochratoxin C (100 ng / mL) and ochratoxin A (2 ng / mL) were incubated with CeO2@RuNSs-mAb for 3 min respectively, and then the test strip was added for immunological reaction for 15 min, and the colorimetric result was read. To further obtain the CL detection signal, the CeO2@RuNSs-ICA used for colorimetric analysis before was dried in an oven, then the CL substrate solution was added, the signal was collected using an iphone 13 pro, and analyzed by Image J software.

[0138] Detection result: In the control group, the test line was significantly lower than the blank. If there was specific recognition, otherwise there was none.

[0139] Figure 7 D shows that the CL signal intensity of OTA was significantly weaker than that of other toxins (except OTB and OTC). It indicates that the invented CeO2@RuNSs-CLICA could not detect common fungi, but had specific recognition for the structural analogues OTB and OTC.

[0140] Figure 7 From Figures 7E and 7F, the cross-reactivity rates of CeO2@RuNSs-CLICA with OTB and OTC were 141.7% and 106.3% respectively.

[0141] The above results indicate that the invention has excellent sensitivity and good specificity.

[0142] Example 8: Practical application of CeO2@RuNSs-CLICA

[0143] Detection process: Weigh the ground sample (1 g), vortex with 40% methanol (5 mL) for 5 min for extraction. Then, centrifuge the mixture at 6000 rpm for 5 min. Finally, dilute the supernatant (30 times for wheat and 40 times for coix seed), and determine the recovery rate ((measured value (μg / kg) / spiked value (μg / kg)) × 100%).

[0144] To obtain the maximum extraction efficiency of OTA, the types and concentrations of extractants and the sample dilution factors were optimized in detail. In this work, OTA concentrations of 0 and 0.2 ng / mL were selected as the detection concentrations for optimization. Under the optimal extraction conditions, OTA concentrations of 3, 6, and 9 μg / kg, and 4, 8, and 12 μg / kg were selected respectively to study the actual detection ability of CeO2@RuNSs-CLICA in wheat and coix seeds.

[0145] Detection results: Using the blank group as a control and the detection line intensity and recovery rate as optimization indicators, the best extraction procedure was constructed.

[0146] Optimization of wheat extraction reagent: As the methanol content increased, the OTA recovery rate gradually increased and stabilized after 40% methanol ( Figure 8 A). Although the extraction effect of 40% acetonitrile was comparable ( Figure 8 B), considering the economic cost, 40% methanol was selected as the extraction reagent. The sample dilution factor directly affects the sensitivity of CeO2@RuNSs-CLICA in actual detection. When the wheat was diluted 30 times, the recovery rate met the requirements ( Figure 8 C), but as the dilution factor increased, the sensitivity showed a sharp downward trend.

[0147] Optimization of coix seed extraction reagent: Using the same optimization procedure, 40% methanol and a 40-fold dilution were finally selected as the optimal extraction parameters ( Figure 8 D-F).

[0148] Visual detection limit: As can be seen from Figure 8 G, the visual detection limits of the invented CeO2@RuNSs-CLICA in wheat and coix seeds were 3.6 μg / kg and 5 μg / kg respectively.

[0149] Spiked recovery experiment: As shown in Figure 8 H and I, the invented CeO2@RuNSs-CLICA provided detection results close to the theoretical values. The average recovery rates of wheat and coix seed samples were 92.0 - 114.9%, and the variations were 1.6 - 6.0%.

[0150] These results indicate that the invented CeO2@RuNSs-CLICA exhibits excellent detection ability for OTA in wheat and coix seeds.

[0151] The applicant declares that the present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement to the present invention, such as the equivalent substitution of the raw materials selected for the present invention, the addition of auxiliary components, and the selection of specific methods, all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A CeO2@Ru nanoparticle carrier, characterized in that: A spherical CeO2, wherein Ru is loaded outside the CeO2 sphere; The particle size of CeO2 spheres is 142.2 ± 7.1 nm, and the particle size of CeO2@Ru nanoparticle carrier is 151.8 ± 9.1 nm; The carrier is prepared from CeO2 and RuCl3 by a metal self-deposition method.

2. The CeO2@Ru nanoparticle carrier according to claim 1, characterized in that: The preparation of the CeO2@RuNSs carrier includes: CeO2 was dissolved in water. After ultrasonic treatment, RuCl3 solution was slowly added to the CeO2 solution and stirred at room temperature. The solution turned black-gray and the precipitate was collected by centrifugation to obtain CeO2@RuNSs. The concentration of the CeO2 solution is 1 mg / mL, the ultrasonic time is 30 min, the stirring time is 12 h, and the concentration of RuCl3 is 0.2 mM.

3. The CeO2@Ru nanoparticle carrier according to claim 1 or 2, characterized in that: PVP K30 and cerium nitrate were dissolved in ethylene glycol, and HCl solution was added under vigorous stirring; After the solution is stirred until clear, it is transferred to a polytetrafluoroethylene-lined autoclave, heated at 160°C, cooled to room temperature, centrifuged and washed several times, and the gray precipitate collected is CeO2; The amount of PVP K30 used is 0.8 g, cerium nitrate is 2.0 g, ethylene glycol is 60 mL, HCl is 4 mL, the concentration is 1 M, and the heating time is 3 h.

4. A chemiluminescent signal probe, characterized in that: The probe comprises a carrier and a monoclonal antibody electrostatically adsorbed thereon; The carrier is the CeO2@Ru nanoparticle carrier described in any one of claims 1-3; The monoclonal antibody is OTA-mAb, and the concentration is 1 mg / mL.

5. The chemiluminescent signal probe according to claim 4, characterized in that The preparation method is as follows: CeO2@Ru nanoparticles were dissolved in water and incubated with OTA-mAb at room temperature, blocked with bovine serum albumin, and centrifuged to obtain the CL signal probe. The CeO2@Ru nanoparticle solution is 1 mL and has a concentration of 1 mg / mL, the OTA-mAb is 8 μL and has a concentration of 1 mg / mL, the incubation time is 30 min, and the blocking time is 30 min.

6. Use of the chemiluminescent signal probe according to claim 4 or 5 in constructing a chemiluminescent test strip for detecting OTA in cereals.

7. The use according to claim 6, characterized in that: The cereals include wheat and / or coix seed.

8. A chemiluminescent test strip for detecting OTA in cereals, characterized in that: The chemiluminescent signal probe according to claim 4 or 5 is added to the sample to be tested, and the test strip is prepared and the CL substrate solution is prepared; The test strip comprises attaching a nitrocellulose membrane to a corresponding position of a polyvinyl chloride bottom plate, attaching absorbent paper to the upper part of the nitrocellulose membrane, attaching a sample pad to the bottom end, and sealing and storing after cutting; The nitrocellulose membrane preparation method comprises the following steps: OTA-BSA and goat anti-mouse secondary antibody are sprayed on the nitrocellulose membrane at a streaking rate of 0.9 μL / cm, which are defined as the detection line and the quality control line, respectively, and dried at 37° C. overnight; The concentration of OTA-BSA is 0.8 mg / mL, and the concentration of goat anti-mouse secondary antibody is 0.5 mg / mL; The overlap width of the absorbent paper and the sample pad with the nitrocellulose membrane is 1.5 mm; The sample pad preparation method is as follows: the glass fiber membrane is placed in a blocking solution until it is completely soaked, and then dried overnight at 37°C; The cutting width is 3.2 mm.

9. The chemiluminescent test strip for detecting OTA in cereals according to claim 8, characterized in that: The CL substrate solution includes luminol, H2O2 and Tris-HCl buffer, which are mixed evenly for use; The luminol concentration is 0.06M, the H2O2 concentration is 0.25M, and the Tris-HCl concentration is 0.4M; The volume ratio of luminol, H2O2 and Tris-HCl in the CL substrate solution is 1:2:

4.

10. The chemiluminescent test strip for detecting OTA in cereals according to claim 8, characterized in that: The detection method includes: The chemiluminescent test strip for detecting OTA in grains was inserted into the test solution, and after the immune reaction, the colorimetric result was obtained using a reader, and then placed in a drying oven to fix the CeO2@Ru nanoparticles; Then, CL substrate solution was added to the dried nitrocellulose membrane of the colorimetric test strip, the signal was collected, and quantitative analysis was performed using Image J software; The immune reaction time was 15 min, the drying time was 3 h, and the volume of the CL substrate solution was 10 μL.

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