A test strip, kit and method for simultaneous detection of multiple mycotoxins in feed based on self-calibration SERS lateral chromatography

Abstract

This invention discloses a test strip, reagent kit, and method for simultaneous detection of multiple mycotoxins in feed based on self-calibrated SERS lateral flow chromatography, belonging to the field of biodetection technology. The test strip includes a backing plate, a sample pad, a conjugate pad, an NC membrane, and an absorbent pad. The NC membrane is adhered to the backing plate, and the absorbent pad and the conjugate pad are respectively tightly pressed against both ends of the NC membrane. The sample pad is tightly pressed against the end of the conjugate pad furthest from the NC membrane. From the end closest to the conjugate pad to the end furthest from the conjugate pad, the NC membrane has a control line and at least one detection line sequentially arranged. This test strip based on self-calibrated SERS lateral flow chromatography has high detection efficiency, with advantages such as fast detection speed, high sensitivity, strong specificity, and simple operation. It can achieve simultaneous detection of multiple mycotoxins and is suitable for rapid detection in feed production sites and grassroots supervision.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a test strip, reagent kit, and method for simultaneous detection of multiple mycotoxins in feed based on self-calibrated SERS lateral flow chromatography. Background Technology

[0002] Mycotoxins produced from moldy feed are a significant factor jeopardizing the healthy development of livestock farming. Aflatoxin B1, zearalenone, and vomitoxin are among the most common and highly toxic mycotoxins in feed. These toxins possess strong carcinogenic, teratogenic, and immunosuppressive effects, and their bioaccumulation through the food chain seriously threatens animal health and human food safety. Currently, the main methods for detecting mycotoxins in feed include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS / MS), enzyme-linked immunosorbent assay (ELISA), and traditional lateral flow chromatography. While HPLC and LC-MS / MS offer high detection accuracy, they require expensive instruments, specialized operators, and complex sample pretreatment, resulting in long detection cycles and failing to meet the needs for rapid on-site detection. ELISA is cumbersome and time-consuming, has limited quantitative accuracy, and is difficult to perform multiplex detection. Traditional lateral flow chromatography is simple to operate and fast, but it suffers from low sensitivity, inaccurate quantification, and is susceptible to matrix interference leading to signal fluctuations.

[0003] Surface-enhanced Raman scattering (SERS) technology boasts advantages such as high sensitivity, resistance to photobleaching, and multiplex identification. Combining SERS with side-flow chromatography to form SERS side-flow chromatography technology holds promise for overcoming the shortcomings of traditional detection methods. However, existing SERS side-flow chromatography detection devices are prone to signal fluctuations when detecting complex matrix samples such as feed. This is due to the uneven distribution of SERS probes caused by the irregular porous structure of the NC membrane; furthermore, instrument errors and operational differences significantly affect the accuracy and repeatability of quantitative detection. In addition, most existing technologies can only achieve single-mycotoxin detection, failing to meet the practical needs of simultaneous screening for multiple mycotoxins in feed. Therefore, developing a SERS side-flow chromatography technology capable of eliminating signal fluctuations and achieving multiplex quantitative detection is of great significance for improving the detection level of mycotoxins in feed and ensuring feed quality and safety. Summary of the Invention

[0004] The purpose of this invention is to provide a test strip, reagent kit, and method for simultaneous detection of multiple mycotoxins in feed based on self-calibrated SERS lateral flow chromatography, thereby solving the problems existing in the prior art. This test strip based on self-calibrated SERS lateral flow chromatography has high detection efficiency, fast detection speed, high sensitivity, strong specificity, and simple operation. It can achieve simultaneous detection of multiple mycotoxins and is suitable for rapid detection in feed production sites and grassroots supervision.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a test strip based on self-calibrated SERS lateral flow chromatography, comprising a back plate, a sample pad, a conjugate pad, an NC membrane, and an absorbent pad; the NC membrane is attached to the back plate, the absorbent pad and the conjugate pad are respectively tightly pressed against both ends of the NC membrane, and the sample pad is tightly pressed against the end of the conjugate pad away from the NC membrane; From the end closest to the conjugate pad to the end furthest from the conjugate pad, the NC membrane is provided with at least one detection line and one quality control line in sequence; The control line is coated with goat anti-mouse IgG polyclonal antibody; each test line is coated with a mycotoxin capture hapten and embedded with an Au@ZIF-8 internal standard probe. The binding pad is loaded with the mycotoxin SERS immune probes corresponding to each detection line. The Au@ZIF-8 internal standard probe is formed by modifying the surface of Au nanoparticles with 4-mercaptopyridine and then encapsulating ZIF-8. The mycotoxin SERS immunoprobe is obtained by modifying Au nanoparticles with different Raman reporter molecules, reducing them to form an Ag shell, and then coupling them with different mycotoxin-specific detection antibodies.

[0006] Furthermore, the detection lines are three, each coated with aflatoxin B1 capture hapten, zearalenone capture hapten, and vomitoxin capture hapten, respectively.

[0007] Furthermore, the Raman reporter molecule includes Nile Blue, 5,5'-dithiobis(2-nitrobenzoic acid), or 4-mercaptobenzoic acid.

[0008] Furthermore, the coupling process further includes a step of blocking non-specific binding sites using bovine serum albumin.

[0009] Furthermore, the method for constructing the Au@ZIF-8 internal standard probe includes the following steps: 4-Mercaptopyridine ethanol solution was mixed and incubated with Au nanoparticles to modify the surface of the Au nanoparticles through thiol bonds. Polyvinylpyrrolidone solution was then added for further modification, and the precipitate was collected by centrifugation. The precipitate was redispersed and mixed with 2-methylimidazole solution and zinc acetate solution. After centrifugation and purification, the Au@ZIF-8 internal standard probe was obtained.

[0010] The present invention also provides a method for preparing the above-mentioned test strip, comprising the following steps: The sample pad, the conjugate pad, the NC membrane, and the absorbent pad are sequentially attached to the backing plate. A quality control line and a detection line are drawn on the NC membrane using a spray film applicator, and the Au@ZIF-8 internal standard probe is sprayed onto each detection line. Different mycotoxin SERS immunoprobes are mixed and sprayed onto the conjugate pad. After drying, the test strip is cut to obtain the test strip.

[0011] The present invention also provides the application of the above-described test strip in the preparation of a mycotoxin detection kit.

[0012] The present invention also provides a mycotoxin detection kit, comprising the test strips described above.

[0013] The present invention also provides the application of the above-described test strips or mycotoxin detection kits in the detection of mycotoxins.

[0014] This invention also provides a method for simultaneous detection of multiple mycotoxins in feed, comprising the following steps: After crushing and grinding the feed sample to be tested, methanol-acetonitrile mixed solution and PBS extraction buffer containing 2% Triton X-100 were added for extraction to obtain the sample test solution; The sample liquid to be tested was dropped onto the sample pad of the test strip. After standing and reacting, the SERS signal intensity of the characteristic peak of each detection line and the characteristic peak of the internal standard signal was collected by a 785 nm laser Raman spectrometer. Calculate the ratio of the SERS signal intensity of the characteristic peak of each detection line to the SERS signal intensity of the characteristic peak of the internal standard signal, substitute it into the corresponding standard curve, and obtain the concentration of mycotoxins in the sample respectively; the standard curve is a linear curve of intensity ratio versus concentration logarithm plotted using mycotoxin standards of gradient concentrations.

[0015] The present invention discloses the following technical effects: This invention develops a test strip based on self-calibrated SERS lateral flow chromatography, comprising a backplate, a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, and an absorbent pad. The NC membrane has a control line (C line) and multiple detection lines (T lines). Each T line is pre-immobilized with a capture antigen-BSA corresponding to a mycotoxin and an Au@ZIF-8 internal standard probe. The conjugate pad is loaded with Au@Ag core-shell SERS probes labeled with specific detection antibodies and different Raman reporter molecules. During detection, the feed sample extract is driven sequentially through each component by capillary action, forming a "capture antigen-SERS probe" semi-sandwich immune complex. By collecting the intensity ratio of the characteristic peak of the SERS probe at the T line to the characteristic peak of the internal standard probe, simultaneous quantitative detection of multiple mycotoxins is achieved.

[0016] This invention corrects signal fluctuations using the Au@ZIF-8 internal standard probe, solving the problems of signal instability and low quantitative accuracy in traditional SERS lateral flow chromatography detection. It has the advantages of fast detection speed, high sensitivity, strong specificity, and simple operation, and is suitable for rapid detection in feed production sites and grassroots supervision.

[0017] The Au@Ag core-shell structure of this invention exhibits a significant SERS enhancement effect, with detection limits for the three mycotoxins as low as 0.078 ng / mL, 0.024 ng / mL, and 0.068 ng / mL, respectively, far superior to traditional colloidal gold side-flow chromatography. An immunorecognition system constructed using mycotoxin-specific antibodies effectively avoids interference from components such as protein and starch in the feed matrix, with a cross-reactivity rate of less than 12%. The sample pretreatment of this invention is simple, the detection process requires no specialized personnel, and the results can be read using a portable Raman spectrometer, making it particularly suitable for feed production enterprises, farms, and grassroots regulatory departments. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the structure of a SERS side-flow chromatography test strip; where 1: sample pad; 2: conjugate pad; 3: detection line a; 4: detection line b; 5: detection line c; 6: absorbent pad; 7: embedded internal standard probe; 8: control line; 9: nitrocellulose membrane (NC); 10: PVC backing. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1 This embodiment provides a test strip based on self-calibrated SERS side-flow chromatography, such as... Figure 1 As shown, the test strip includes a PVC backing plate, a sample pad, a conjugate pad, a nitrocellulose membrane (NC membrane), and an absorbent pad. The NC membrane is adhered to the PVC backing plate, and the absorbent pad and conjugate pad are tightly pressed against both ends of the NC membrane, respectively. The sample pad is tightly pressed against the end of the conjugate pad furthest from the NC membrane. From the end closest to the conjugate pad to the end furthest from the conjugate pad, the NC membrane has three detection lines (T lines) and one control line (C line). Each detection line is coated with a specific capture hapten of a mycotoxin and embeds an Au@ZIF-8 internal standard probe to correct signal fluctuations during the detection process. The conjugate pad is loaded with three different mycotoxin SERS immunoprobes, each probe targeting a specific mycotoxin and labeled with different Raman reporter molecules, enabling the detection and recognition of multiple mycotoxins.

[0026] The preparation method of this test strip is as follows: (1) Preparation of Au@Ag core-shell SERS probes: 60 nm Au nanoparticles were prepared and incubated with three Raman reporter molecules, Nile Blue (NBA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), or 4-mercaptobenzoic acid (4-MBA), for 1 h. Unbound Raman reporter molecules were removed by centrifugation. Ascorbic acid and silver nitrate solution were added and stirred for 30 min to form an Ag shell (8 nm thick). The pH of the solution was adjusted to 7.0 (pH can reach up to 8.5). Corresponding mycotoxin-specific detection antibodies were added and incubated for 1 h. Bovine serum albumin (BSA) was added to block non-specific binding sites for 1 h. After centrifugation and purification, three SERS immunoprobes—Au@Ag—were obtained. NBA @Ag probe, Au DTNB @Ag probe and Au 4-MBA @Ag probes were all adjusted to a concentration of 1 mg / mL. The mycotoxin-specific detection antibodies were aflatoxin B1 monoclonal antibody (LD-Ab0026), anti-zearalenone monoclonal antibody (LD-Ab0184), and anti-vomiting toxin monoclonal antibody (LD-Ab0180), all purchased from Shandong Landu Biotechnology Co., Ltd.

[0027] Three SERS immunoprobes utilize the local surface plasmon resonance effect of the Au@Ag core-shell structure to enhance the Raman signal. The characteristic peaks of the three Raman reporter molecules do not overlap, enabling the labeling and detection of multiple mycotoxins.

[0028] (2) Preparation of Au@ZIF-8 internal standard probe: 20 μL of 10 mM 4-mercaptopyridine (4MPY) ethanol solution was mixed with 30 mL of Au nanoparticle solution (containing 1.49 mg of Au nanoparticles) and incubated overnight to allow 4MPY to modify the surface of Au nanoparticles through thiol bonds. Then, 300 μL of 2.5% polyvinylpyrrolidone (PVP) solution was added for further modification (surface modification) for 1.5 h. The precipitate was collected by centrifugation. After redispersing the precipitate, 3 mL of 2 M 2-methylimidazole solution and 0.8 mL of 0.3 M zinc acetate solution were added, and the mixture was reacted overnight in a water bath at 50 °C. The Au@ZIF-8 internal standard probe was purified by centrifugation, and the concentration was adjusted to 150 μg / mL. This Au@ZIF-8 internal standard probe exhibits good chemical stability and a uniform Raman signal, with its characteristic peak (1203 cm⁻¹) being the largest. -1 It does not overlap with the characteristic peaks of the three Raman reporter molecules used for detection, and can be used as a stable internal standard reference.

[0029] (3) Test strip assembly: The sample pad, conjugate pad, NC membrane and absorbent pad are pasted sequentially on the PVC backing, with adjacent components overlapping by 2 mm; the C line (goat anti-mouse IgG, 1 mg / mL) and three T lines (aflatoxin B1 capture hapten, zearalenone capture hapten and vomitoxin capture hapten, respectively, with a concentration of 0.8 mg / mL) are drawn on the NC membrane using a spraying device; the Au@ZIF-8 internal standard probe is sprayed onto each T line; the three SERS immunoprobes are mixed in equal volumes and sprayed onto the conjugate pad, and dried at 37℃ for 2 h; finally, the assembled device is cut into 3.8 mm wide test strips and sealed in a packaging bag containing desiccant for storage. The aflatoxin B1 capture hapten was purchased from Shanghai Lanbo Biotechnology Co., Ltd., catalog number ZD6017; the zearalenone capture hapten was purchased from Beijing Qihang Liye Technology Co., Ltd., catalog number XBK-0356; and the vomitoxin capture hapten was purchased from Shanghai Lanbo Biotechnology Co., Ltd., catalog number ZD6024.

[0030] Example 2 A method for detecting multiple mycotoxins in feed based on the test strip of Example 1 includes the following steps: (1) Sample pretreatment: Take the feed sample, crush and dispense it. Weigh 3.0 g of the ground sample and place it in a 50 mL centrifuge tube. Add 20 mL of methanol-acetonitrile mixed solution (volume ratio 3:1). Then, add PBS extraction buffer (pH=7.4) containing 2% Triton X-100. Vortex for 10 min, centrifuge at 8000 rpm for 15 min, and take the supernatant as the sample test solution.

[0031] (2) Detection procedure: Aspirate 50 μL of the sample solution to be tested and drop it onto the sample pad. Let it stand at room temperature for 12 min. The sample solution is driven by capillary action to flow through the conjugate pad, NC membrane and absorbent pad in sequence.

[0032] (3) Signal acquisition: A 785 nm laser Raman spectrometer was used for detection, and data were collected at 593 cm⁻¹ for each T-line. -1 (NBA characteristic peak), 1335 cm -1 (DTNB characteristic peak), 1589 cm⁻¹ -1 The SERS signal intensity (characteristic peak of 4-MBA) and 1203 cm⁻¹ -1 The internal standard signal intensity (4MPY characteristic peak). Laser power was 15 mW (maximum 30 mW), signal acquisition time was 1 s (maximum 3 s), and the silicon wafer was 520 cm² before acquisition. -1 The characteristic peaks are calibrated.

[0033] (4) Quantitative analysis: Calculate the ratio of the intensity of each characteristic peak to the intensity of the internal standard peak (I 593 / I 1203 I 1335 / I 1203 I 1589 / I 1203 Substituting these values ​​into the corresponding standard curves, the concentrations of aflatoxin B1, zearalenone, and vomitoxin in the samples were obtained. The standard curves were pre-plotted linear curves of intensity ratio versus concentration logarithm using mycotoxin standards at gradient concentrations.

[0034] The above method was used to detect multiple mycotoxins in feed samples. The concentrations of the three mycotoxins in the samples were calculated. The spiked recovery rate was 85-108%, and the RSD was ≤12%, which met the detection requirements.

[0035] The standard curve is as follows: Aflatoxin B1: y =3.785 x +0.936 (R) 2 =0.952), where y For I 593 / I 1203 , x The logarithm of concentration; Zearalenone: y =1.428 x -0.253 (R) 2 =0.972), where y For I 1335 / I 1203 , x The logarithm of concentration; Vomiting toxins: y =2.134 x +0.654 (R) 2 =0.969), where y For I 1589 / I 1203 , x This is the logarithm of the concentration.

[0036] Example 3 Sensitivity test: Using the test strips from Example 1, and following the method of Example 2, gradient concentrations of mycotoxin standards were tested. The detection limit for aflatoxin B1 was determined to be 0.078 ng / mL, with a linear range of 50–250 ng / mL; the detection limit for zearalenone was 0.024 ng / mL, with a linear range of 5–250 ng / mL; and the detection limit for vomitoxin was 0.068 ng / mL, with a linear range of 5–250 ng / mL. All detection limits were lower than the limits specified in the national feed hygiene standards.

[0037] Example 4 Specificity test: Using the test strip from Example 1, different toxins, including aflatoxin B1, zearalenone, vomitoxin, ochratoxin A, T-2 toxin, HT-2 toxin, fumonisin B1, and fumonisin B2, were detected according to the method of Example 2. The test results showed no significant cross-reactivity, indicating that the test strip based on self-calibrated SERS lateral flow chromatography of the present invention has good specificity.

[0038] Example 5 After the test strip of Example 1 was sealed and stored at room temperature for 3 months, the same concentration of standard was tested before and after storage. The results showed that the RSD of the signal intensity ratio was 13.7%, indicating that the test strip based on self-calibrated SERS side-flow chromatography of the present invention has good stability.

[0039] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A test strip based on self-calibrating SERS lateral flow chromatography, characterized in that, It includes a backplate, a sample pad, a conjugate pad, an NC membrane, and an absorbent pad; the NC membrane is attached to the backplate, the absorbent pad and the conjugate pad are respectively tightly pressed against both ends of the NC membrane, and the sample pad is tightly pressed against the end of the conjugate pad away from the NC membrane; From the end closest to the conjugate pad to the end furthest from the conjugate pad, the NC membrane is provided with at least one detection line and one quality control line in sequence; The control line is coated with goat anti-mouse IgG polyclonal antibody; each test line is coated with a mycotoxin capture hapten and embedded with an Au@ZIF-8 internal standard probe. The binding pad is loaded with the mycotoxin SERS immune probes corresponding to each detection line. The Au@ZIF-8 internal standard probe is formed by modifying the surface of Au nanoparticles with 4-mercaptopyridine and then encapsulating ZIF-8. The mycotoxin SERS immunoprobe is obtained by modifying Au nanoparticles with different Raman reporter molecules, reducing them to form an Ag shell, and then coupling them with different mycotoxin-specific detection antibodies.

2. The test strip according to claim 1, characterized in that, The detection lines consist of three lines, each coated with aflatoxin B1 capture hapten, zearalenone capture hapten, and vomitoxin capture hapten, respectively.

3. The test strip according to claim 2, characterized in that, The Raman reporter molecules include Nile blue, 5,5'-dithiobis(2-nitrobenzoic acid), or 4-mercaptobenzoic acid.

4. The test strip according to claim 1, characterized in that, The coupling process also includes a step of blocking non-specific binding sites using bovine serum albumin.

5. The test strip according to claim 1, characterized in that, The method for constructing the Au@ZIF-8 internal standard probe includes the following steps: 4-Mercaptopyridine ethanol solution was mixed and incubated with Au nanoparticles to modify the surface of the Au nanoparticles through thiol bonds. Polyvinylpyrrolidone solution was then added for further modification, and the precipitate was collected by centrifugation. The precipitate was redispersed and mixed with 2-methylimidazole solution and zinc acetate solution. After centrifugation and purification, the Au@ZIF-8 internal standard probe was obtained.

6. A method for preparing a test strip as described in any one of claims 1-5, characterized in that, Includes the following steps: The sample pad, the conjugation pad, the NC membrane, and the absorbent pad are sequentially attached to the back plate. Control lines and test lines were drawn on the NC membrane using a spraying device, and the Au@ZIF-8 internal standard probe was sprayed onto each test line. Different mycotoxin SERS immunoprobes were mixed, sprayed onto the conjugate pad, dried, and then cut to obtain the test strip.

7. The use of a test strip as described in any one of claims 1-5 in the preparation of a mycotoxin detection kit.

8. A mycotoxin detection kit, characterized in that, Includes the test strip as described in any one of claims 1-5.

9. The use of a test strip as described in any one of claims 1-5 or a mycotoxin detection kit as described in claim 8 in the detection of mycotoxins.

10. A method for simultaneous detection of multiple mycotoxins in feed, characterized in that, Includes the following steps: After crushing and grinding the feed sample to be tested, methanol-acetonitrile mixed solution and PBS extraction buffer containing 2% Triton X-100 were added for extraction to obtain the sample test solution; The sample liquid to be tested is dropped onto the sample pad of the test strip according to any one of claims 1-5. After standing and reacting, the SERS signal intensity of the characteristic peak of each detection line and the characteristic peak of the internal standard signal is collected by a 785 nm laser Raman spectrometer. Calculate the ratio of the SERS signal intensity of the characteristic peak of each detection line to the SERS signal intensity of the characteristic peak of the internal standard signal, substitute it into the corresponding standard curve, and obtain the concentration of mycotoxins in the sample respectively; the standard curve is a linear curve of intensity ratio versus concentration logarithm plotted using mycotoxin standards of gradient concentrations.

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