A sers / colorimetric dual-mode test strip for detecting mycotoxins and a preparation method and application thereof

Abstract

The application discloses a SERS / colorimetric dual-mode test strip for detecting mycotoxin, and a preparation method and application thereof. The test strip part comprises, in sequence, a sample pad, a binding pad, a nitrocellulose membrane and a water absorption pad fixed on a bottom plate, the binding pad is coated with a monoclonal labeled immune probe, the nitrocellulose membrane is provided with a quality control line and a detection line, the quality control line is provided with an anti-IgG antibody, the detection line is provided with a mycotoxin-bovine serum albumin coupling product, a hydrogel part is used for being pasted on the detection line, the hydrogel in the hydrogel part comprises TMB and NaAC-HAC buffer substances, the test strip can rapidly and sensitively detect mycotoxin, the peroxidase activity and SERS activity of the probe are utilized, the hydrogel is pasted on the T line after the T line is completely colored, color signals are output, and SERS / colorimetric dual-mode detection of mycotoxin is realized.

Description

Technical Field

[0001] This invention belongs to the field of immunological detection technology, specifically relating to a test strip for SERS / colorimetric dual-mode detection of fungal toxins, its preparation method, and its application. Background Technology

[0002] Mycotoxins are toxic secondary metabolites produced by toxin-producing filamentous fungi under suitable temperature and humidity conditions. Widely found in food and the environment, they possess teratogenic, carcinogenic, mutagenic, and immunosuppressive properties, posing serious health risks to humans and animals. Mycotoxins can bind to cells, producing general cytotoxic effects and inducing various diseases such as in situ liver cancer and gastric cancer. Furthermore, mycotoxins are readily soluble in organic solvents, chemically stable, and heat-resistant, making them difficult to destroy by heating or cooking. Therefore, they have become a significant factor in food contamination, highlighting the importance of establishing simple, rapid, and sensitive detection methods.

[0003] Currently, methods for detecting mycotoxins mainly include traditional chromatographic methods such as thin-layer chromatography, high-performance liquid chromatography, liquid chromatography-mass spectrometry, electrochemical methods, spectroscopic methods such as colorimetry, fluorescence methods, and surface-enhanced Raman spectroscopy, and immunoassay methods such as enzyme-linked immunosorbent assay (ELISA). However, most of these methods require expensive laboratory equipment, skilled technicians, and time-consuming and cumbersome operations. These inconveniences hinder their widespread application in on-site detection. Therefore, a simple and easy-to-use detection method is still needed to meet the demand for rapid on-site detection.

[0004] Lateral flow immunoassay (LFIA) combines immunoassay and lateral flow chromatography, enabling rapid detection of target analytes in samples through the specific reaction of antigens and antibodies and chromatographic action. LFIA primarily consists of a nitrocellulose (NC) membrane, a sample pad, a conjugation pad, and an absorbent pad sequentially fixed onto a polyvinyl chloride (PVC) substrate. The NC membrane has an antigen or antibody specifically designed to recognize the target analyte fixed at the test (T) line, and an anti-IgG antibody fixed at the control (C) line. The sample solution containing the immunoprobe migrates along the test strip using capillary action. When the test solution reaches the T line, the complex formed by the immunoprobe and the target analyte specifically binds to the substance fixed at the T line. The remaining test solution continues to migrate to the C line and undergoes an immune reaction with the IgG antibody, ultimately allowing for qualitative or quantitative detection. It is considered one of the most promising point-of-care testing methods. However, traditional lateral flow immunoassay test strips have limited detection sensitivity, allowing only qualitative and semi-quantitative analysis of the target analyte. Meanwhile, due to the complex composition of actual samples, the matrix has a significant impact on the test results during the detection process, which can easily lead to false positives and false negatives, resulting in low accuracy and limiting its application in some areas.

[0005] The lateral flow immunochromatographic test strip (SERS-LFIA) based on surface enhanced Raman spectroscopy (SERS) combines the high sensitivity and specificity of SERS with the convenience, simplicity, and low cost of lateral flow immunochromatographic test strips. Summary of the Invention

[0006] To address the shortcomings of the existing technologies, this invention combines SERS technology with lateral flow immunochromatography and uses hydrogel as the colorimetric signal output carrier to construct a lateral flow immunochromatographic test strip for detecting fungitoxins based on SERS. This combines the high sensitivity and specificity of SERS with the convenience and low cost of lateral flow immunochromatographic test strips. At the same time, the use of hydrogel as the colorimetric signal output reduces background color interference, and the use of dual-mode result comparison improves accuracy, thereby achieving rapid and sensitive detection of fungitoxins.

[0007] The technical solution of the present invention is as follows:

[0008] One objective of this invention is to provide a SERS / colorimetric dual-mode test strip for detecting mycotoxins, comprising a test strip portion and a hydrogel portion, wherein: the test strip portion includes a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad fixed sequentially on a base plate; the conjugate pad is coated with a monoclonal-labeled immunoprobe; the nitrocellulose membrane has a control line and a detection line; the control line contains an anti-IgG antibody, and the detection line contains a mycotoxin-bovine serum albumin coupling agent; the hydrogel portion is used to adhere to the detection line, and the hydrogel in the hydrogel portion contains TMB and NaAC-HAC buffer substances.

[0009] Furthermore, the detection line and the control line are drawn on the nitrocellulose membrane using a gold sputtering instrument at a dosage of 1 μL / cm. The detection line is coated with a fungal toxin-bovine serum albumin coupler at a concentration of 0.5 mg / mL, and the control line is coated with an anti-IgG antibody at a concentration of 0.5 mg / mL. The distance between the detection line and the control line is 5 mm.

[0010] The second objective of this invention is to provide a method for preparing a test strip for SERS / colorimetric dual-mode detection of fungal toxins, comprising the following steps:

[0011] S1: Prepare monoclonal labeled immune probes and store them at 4°C for later use;

[0012] S2: Prepare a nitrocellulose membrane and draw the detection line and control line on the nitrocellulose membrane in sequence;

[0013] S3: Immerse the sample pad and conjugate pad in the pretreatment solution and dry them for later use;

[0014] S4: The prepared nitrocellulose membrane, conjugation pad, sample pad and absorbent pad are sequentially pasted onto the PVC base plate, with each part overlapping by 2mm. After assembly, the parts are cut to obtain the test strip.

[0015] S5: Dissolve agarose and glucose in TAE buffer solution, then add TMB solution and NaAC-HAC solution to obtain the hydrogel portion.

[0016] Furthermore, the preparation method of the immune probe in step S1 includes the following steps:

[0017] (1) Add silver nitrate AgNO3 solution, ascorbic acid AA solution, sodium hydroxide NaOH solution and Ag seed solution to hexadecyltrimethylammonium chloride CTAC solution, and Ag NPs are obtained after reaction;

[0018] (2) Add Raman signaling molecule DTNB to Ag NPs for incubation, centrifuge and reconstitute to obtain modified Ag NPs;

[0019] (3) Modified Ag NPs were added to a cetyltrimethylammonium chloride CTAC solution, followed by chloroplatinic acid H2PtCl6 solution and ascorbic acid AA solution. After the reaction, the mixture was centrifuged and resuspended to obtain Ag@Pt NRs.

[0020] (4) Add fungal toxin antibody to Ag@Pt NPs solution for incubation, block with 1% BSA, centrifuge and resuspend to obtain Ag@Pt NPs-mAb immune probe.

[0021] Further, step S3 includes: cutting the sample pad and conjugate pad to a width of 1.4 cm, immersing them in the pretreatment solution for 30 min, drying them in a 37°C forced-air oven for 1 h, and storing them in a desiccant bag for later use.

[0022] Further, the pretreatment solution is a PBS solution containing 1% BSA, 1% trehalose, 2.5% sucrose and 1% Tween-20.

[0023] Further, step S4 includes: first, attaching the nitrocellulose membrane to the PVC base plate; then, overlapping the conjugate pad with the NC membrane by 2mm and attaching it to the PVC base plate; then, overlapping the sample pad with the conjugate pad by 2mm and attaching it to the PVC base plate; finally, overlapping the absorbent pad with the nitrocellulose membrane by 2mm and attaching it to the PVC base plate. After assembly, the sample is cut into 4mm wide test strips and stored at 4℃ for later use.

[0024] Further, the step of preparing the hydrogel in step S5 is as follows: weigh 0.6g agarose and 0.1g glucose and dissolve them in 60mL TAE solution. After heating and dissolving, take 10mL and let it stand to cool. Weigh 24mg TMB and dissolve it in 10mL ethanol. Take 1mL TMB and 1mL NaAC-HAC and add them to the agarose that has been left to stand to cool. Wait for it to solidify and then cut it for later use.

[0025] The third objective of this invention is to provide a test strip for detecting mycotoxins using a dual-mode SERS / colorimetric method in traditional Chinese medicine, grains, and environmental water samples.

[0026] Further, the detection droplet is added to the sample pad, and after it is laid flat, the SERS signal spectrum on the detection line is collected. After it is fully displayed, the hydrogel part is pasted onto the detection line, reacted, and then peeled off. The color change is observed and the RGB value is identified by taking a picture. The RGB value and SERS intensity value are substituted into the standard curve of fungal toxin concentration and the difference between RGB and SERS signals to obtain the corresponding concentration.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] The synthesized Ag@Pt-mAb nanomaterials of this invention exhibit excellent SERS activity. When applied to lateral flow immunochromatography, they enable simple, sensitive, and rapid detection of fungal toxins, directly detecting and collecting SERS signals. Furthermore, the Ag@Pt-mAb probes possess good oxidase activity, effectively catalyzing colorless TMB to blue ox-TMB, transforming the Image J signal output solely from the probe's own color into a color change of the catalytic product, effectively expanding the color output signal range. In addition, using a hydrogel as the color signal output, the color signal is transferred to the colorless hydrogel, effectively avoiding interference from the background color of the test strip's detection and control lines. This allows for visualized RGB analysis and dual-mode SERS detection testing, with cross-comparison of the dual-mode results improving accuracy. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the detection principle in this invention;

[0030] Figure 2 These are transmission electron microscope images of AgNPs and Ag@Pt NPs in this invention;

[0031] Figure 3 This is the elemental analysis spectrum of Ag@Pt NPs in this invention;

[0032] Figure 4 These are photographs of the test strip detection lines after different concentrations of fungal toxins were added in this invention;

[0033] Figure 5 This is the SERS signal spectrum of the test strip detection line after different concentrations of mycotoxins were added in this invention;

[0034] Figure 6 This is a linear graph of the SERS signal intensity difference on the detection line of the test strips for different concentrations of mycotoxins in this invention, which is a fitted graph of the logarithm of the concentration.

[0035] Figure 7 These are photographs of the hydrogel used in this invention and comparison images of it before and after reaction when placed on the detection line;

[0036] Figure 8 This is a linear graph of the RGB signal intensity value and the logarithmic value of the concentration on the detection line of different test strips in this invention. Detailed Implementation

[0037] The present invention will be further described below through specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the protection scope of the present invention.

[0038] Reference Figure 1 The following is an embodiment of the SERS / colorimetric dual-mode test strip for detecting fungal toxins, its preparation method, and its application, but the content of the present invention is not limited thereto.

[0039] Example 1

[0040] Prepare an immunoprobe (Ag@Pt-mAb) with peroxidase and SERS activity.

[0041] (1) Preparation of Ag@Pt NPs

[0042] First, add 0.5 mL of 10 mg NO 3加入 Ag seeds were obtained by adding 0.5 mL of 1% trisodium citrate and 0.6 mL of NaBH4 to 19 mL of water and allowing the mixture to stand for 2.5 hours. Subsequently, 1 mL of AgNO3 solution, 1 mL of ascorbic acid (AA) solution, 0.1 mL of sodium hydroxide (NaOH) solution, and 0.1 mL of Ag seed solution were added to 30 mL of hexadecyltrimethylammonium chloride (CTAC) solution. After reacting for 4 hours, the mixture was centrifuged to obtain Ag NPs. The Ag NPs were then incubated with the Raman signaling molecule DTNB, centrifuged, and reconstituted to obtain the modified Ag NPs. 2.5 mL of modified Ag NPs were added to 20 mL of cetyltrimethylammonium chloride (CTAC) solution, followed by 2 mL of chloroplatinic acid (H₂PtCl₆) solution and ascorbic acid (AA) solution. After reacting for 2 h, the mixture was centrifuged at 10,000 rpm for 10 min and resuspended to obtain Ag@Pt NPs. The transmission electron microscopy (TEM) images of the centrifuged Ag NPs and the TEM images and elemental analysis spectra of Ag@Pt NPs were compared with those of Ag@Pt NPs. Figures 2-3 .

[0043] (2) Preparation of Ag@Pt-mAb

[0044] The pH of Ag@Pt NRs solution was adjusted to 8.5 by adding K2CO3 solution dropwise. Then, 10 μL of mycotoxin antibody was added to the Ag@Pt NPs solution and incubated for 2 h to label the surface of Ag@Pt NPs with antibody. After blocking with 1% BSA for 30 min, the Ag@Pt NPs were centrifuged and resuspended in storage solution (containing 1% BSA, 1% sucrose, 1% trehalose, and 1% Tween-20) and stored at 4℃ to obtain the Ag@Pt NPs-mAb immunoprobe.

[0045] Example 2

[0046] Preparation of test strips

[0047] (1) Treatment of nitrocellulose membrane (NC membrane)

[0048] The fungal toxin zearalenone (ZEN) and bovine serum albumin (BSA) coupling compound, namely ZEN-BSA coupling compound (0.5 mg / mL), diluted with phosphate-buffered saline (PBS), and goat anti-mouse secondary antibody IgG (0.5 mg / mL) were used as the detection line (T line) and control line (C line), respectively. The lines were drawn on the NC membrane using a gold sputtering apparatus at a rate of 1 μL / cm, with a spacing of 5 mm between the two lines. The membrane was then dried in a 37°C oven for 1 hour and stored for later use.

[0049] (2) Pretreatment of sample pads and conjugate pads

[0050] After soaking the sample pads and conjugate pads in the pretreatment solution for 30 minutes, they were dried in a 37°C oven for 1 hour and then stored in a desiccant bag for later use.

[0051] (3) Assemble the test strips

[0052] Attach the NC membrane to the PVC base plate, then overlap the conjugate pad with the NC membrane by 2mm and attach it to the PVC base plate. Next, overlap the sample pad with the conjugate pad by 2mm and attach it to the PVC base plate. Finally, overlap the absorbent pad with the NC membrane by 2mm and attach it to the PVC base plate. Cut the sample pad into 4mm wide test strips and store them at 4℃ for later use.

[0053] Example 3

[0054] Preparation of hydrogels

[0055] Weigh 0.6g agarose and 0.1g glucose and dissolve them in 60mL TAE buffer solution. After heating and dissolving, take 10mL and let it cool. Weigh 24mg 3,3',5,5'-tetramethylbenzidine (TMB) and dissolve it in 10mL ethanol. Take 1mL and add it to the cooled agarose. Wait for it to solidify and cut it to a length and width of 4mm for later use.

[0056] Example 4

[0057] Detection of fungal toxins

[0058] (1) Preparation of ZEN standard solution

[0059] ZEN standard stock solution (1 mg / mL) was prepared using methanol, and ZEN solution was diluted with purified water to a series of concentrations from 0.1 ng / mL to 1000 ng / mL for lateral flow immunochromatographic detection.

[0060] (2) Detection process

[0061] Reference Figures 6-8In actual testing, a detection solution containing buffer solution, probe molecules, and ZEN of different concentrations is added to the sample pad of the above-mentioned lateral flow immunochromatographic test strip. After lying flat for 10 minutes, the SERS signal spectrum on the T line is collected. The hydrogel is placed on the T line and reacted for 10 minutes before being taken out, photographed, and RGB identified to establish a standard curve of fungal toxin concentration, SERS signal difference, and RGB value.

[0062] (3) Result determination and analysis

[0063] In visual inspection, refer to Figure 4 According to the negative control results (0 ng / mL), the detection results of ZEN at 0.1-1 ng / mL showed that the black intensity of the band on the T line did not decrease, indicating a negative result. The detection results of ZEN at 10 and 100 ng / mL showed that the black intensity of the band on the T line decreased significantly, indicating a weak positive result. When the concentration of ZEN was higher than 1000 ng / mL, no band appeared on the T line, indicating a positive result.

[0064] like Figure 5 As shown, during SERS detection, the SERS signal spectrum on the T-line is collected and substituted into... Figure 6 The concentration of mycotoxins in the test solution was calculated from the corresponding standard curve shown. For colorimetric detection, the colorless hydrogel was placed on the T-line and reacted for 10 minutes before being peeled off. An image was taken and its RGB values ​​were identified and substituted into the standard curve. Figure 8 The concentration of mycotoxins in the test solution was calculated from the corresponding standard curve shown. Using a SERS / colorimetric dual-mode comparison improves detection accuracy.

[0065] (4) Actual sample testing

[0066] To evaluate the applicability and accuracy of this method, we tested actual corn samples. First, the corn samples were pretreated according to the following steps: the corn was chopped and weighed, then mixed with 30 mL of methanol-water (7:3 v / v) and sonicated for 20 min. The supernatant was filtered through a 0.22 μm filter membrane, and the resulting liquid was centrifuged at 4000 rpm for 10 min. The resulting liquid was diluted with 10 mM PBS (containing 1% Tween 20) to prepare the sample detection solution, and experimental analysis was performed according to the above detection steps. ZEN was not detected in the tested samples, and the absence of ZEN in the samples was confirmed by liquid chromatography-mass spectrometry (LC-MS / MS). To further verify the analytical performance of this method, we performed spiked recovery experiments on all samples. Three concentrations of ZEN standard solution—low (0.1 ng / mL), medium (1.0 ng / mL), and high (10.0 ng / mL)—were added to the samples, and each concentration level was measured in triplicate. The results of the spiked experiments are shown in Table 1. The recoveries of spiked samples ranged from 98.2% to 103.2%, indicating that the method is suitable for the detection of actual samples.

[0067] Table 1. Spike recovery results of actual samples

[0068]

[0069] The above describes the preparation and application of an immunochromatographic test strip for detecting mycotoxins based on SERS technology according to the present invention. As can be seen from the specific embodiments, this SERS-LFIA method for determining mycotoxins has the advantages of simple operation, speed, and high sensitivity, and has the potential for application in rapid on-site detection.

[0070] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A test strip for detecting fungal toxins using a dual-mode SERS / colorimetric method, characterized in that, It includes a test strip and a hydrogel component, wherein: The test strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad, which are fixed in sequence on a base plate. The conjugate pad is coated with an immunoprobe labeled with a fungal toxin monoclonal antibody. The immunoprobe is an Ag@Pt NPs-mAb with both SERS activity and peroxidase-like activity. The nitrocellulose membrane has a control line and a detection line. The control line contains an anti-IgG antibody, and the detection line contains a fungal toxin-bovine serum albumin coupling compound. The hydrogel portion is used to adhere the detection line, and the hydrogel in the hydrogel portion contains agarose, glucose, TMB and NaAC-HAC buffer. The method for preparing the immune probe includes the following steps: (1) Add silver nitrate AgNO3 solution, ascorbic acid AA solution, sodium hydroxide NaOH solution and Ag seed solution to hexadecyltrimethylammonium chloride CTAC solution, and Ag NPs are obtained after reaction; (2) Add Raman signaling molecule DTNB to Ag NPs for incubation, centrifuge and reconstitute to obtain modified Ag NPs; (3) Add the modified Ag NPs to a cetyltrimethylammonium chloride CTAC solution, then add chloroplatinic acid H2PtCl6 solution and ascorbic acid AA solution, and centrifuge and resuspend after reaction to obtain Ag@Pt NPs; (4) Add fungal toxin antibody to Ag@Pt NPs for incubation, block, centrifuge and resuspend to obtain Ag@Pt NPs-mAb immune probe.

2. The test strip for SERS / colorimetric dual-mode detection of fungal toxins according to claim 1, characterized in that, The detection line and control line are drawn on a nitrocellulose membrane using a gold sputtering apparatus at a concentration of 1 μL / cm. The detection line is coated with a fungal toxin-bovine serum albumin coupler at a concentration of 0.5 mg / mL, and the control line is coated with an anti-IgG antibody at a concentration of 0.5 mg / mL. The distance between the detection line and the control line is 5 mm.

3. A method for preparing a test strip for SERS / colorimetric dual-mode detection of fungal toxins according to claim 1 or 2, characterized in that: Includes the following steps: S1: Prepare monoclonal antibody-labeled immune probes and store them at 4 ℃ for later use; S2: Prepare a nitrocellulose membrane and draw the detection line and control line on the nitrocellulose membrane in sequence; S3: Immerse the sample pad and conjugate pad in the pretreatment solution and dry them for later use; S4: The prepared nitrocellulose membrane, conjugation pad, sample pad and absorbent pad are sequentially pasted onto the PVC base plate, with each part overlapping by 2 mm. After assembly, the parts are cut to obtain the test strip. S5: Dissolve agarose and glucose in TAE buffer solution, then add TMB solution and NaAC-HAC solution to obtain the hydrogel portion.

4. The method for preparing the SERS / colorimetric dual-mode test strip for detecting mycotoxins according to claim 3, characterized in that, The preparation method of the immune probe in step S1 includes the following steps: The Ag@Pt NPs immunoprobe was obtained by incubating with fungal toxin antibodies, blocking with 1% BSA, centrifuging and resuspending.

5. The method for preparing the SERS / colorimetric dual-mode test strip for detecting mycotoxins according to claim 3, characterized in that, Step S3 includes: cutting the sample pad and conjugate pad to 1.4 cm wide, immersing them in the pretreatment solution for 30 min, drying them in a 37 ℃ forced-air oven for 1 h, and storing them in a desiccant bag for later use.

6. The method for preparing the SERS / colorimetric dual-mode test strip for detecting mycotoxins according to claim 5, characterized in that, The pretreatment solution is a PBS solution containing 1% BSA, 1% trehalose, 2.5% sucrose and 1% Tween-20.

7. The method for preparing the SERS / colorimetric dual-mode test strip for detecting mycotoxins according to claim 3, characterized in that, Step S4 includes: first, attaching the nitrocellulose membrane to the PVC base plate; then, overlapping the conjugate pad with the nitrocellulose membrane by 2 mm and attaching it to the PVC base plate; then, overlapping the sample pad with the conjugate pad by 2 mm and attaching it to the PVC base plate; finally, overlapping the absorbent pad with the nitrocellulose membrane by 2 mm and attaching it to the PVC base plate. After assembly, the sample is cut into 4 mm wide test strips and stored at 4 ℃ for later use.

8. The method for preparing the SERS / colorimetric dual-mode test strip for detecting mycotoxins according to claim 3, characterized in that, The steps for preparing the hydrogel in step S5 are as follows: Weigh 0.6 g agarose and 0.1 g glucose and dissolve them in 60 mL TAE solution. After heating and dissolving, take 10 mL of the solution and let it cool. Weigh 24 mg TMB and dissolve it in 10 mL ethanol. Take 1 mL TMB and 1 mL NaAC-HAC and add them to the cooled agarose solution. Wait for it to solidify and then cut it for later use.

9. The application of the SERS / colorimetric dual-mode test strip for detecting mycotoxins according to any one of claims 1 to 2 in the detection of mycotoxins in traditional Chinese medicine materials, grains and environmental water samples.

10. The application of the SERS / colorimetric dual-mode test strip for detecting mycotoxins according to claim 9, characterized in that, Add the detection droplet to the sample pad, lay it flat, collect the SERS signal spectrum on the detection line, and after it is fully displayed, stick the hydrogel part to the detection line, react and peel it off, observe the color change and take a picture to identify its RGB value. Substitute the RGB value and SERS intensity value into the standard curve of fungal toxin concentration and the difference between RGB and SERS signals to obtain the corresponding concentration.

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