SERS / colorimetric dual-mode test strip for detecting mycotoxin as well as preparation method and application of test strip

Abstract

The invention discloses a test strip for detecting fungaltoxin in SERS / colorimetric dual modes as well as a preparation method and application of the test strip. Wherein the test strip part comprises a sample pad, a combination pad, a nitrocellulose membrane and a water absorption pad which are sequentially fixed on a bottom plate, and the combination pad is coated with a monoclonal labeled immune probe; a quality control line and a detection line are arranged on the nitrocellulose membrane; the quality control line is provided with an anti-IgG antibody, and the detection line is provided with a mycotoxin-bovine serum albumin conjugate; the hydrogel part is used for being adhered to the detection line, and hydrogel in the hydrogel part contains TMB (Tetramethylbenzidine) and a NaAC-HAC buffer substance; the test strip can rapidly and sensitively detect the mycotoxin, and by utilizing the peroxidase activity, SERS activity and other characteristics of the probe, hydrogel is pasted on a T line after the T line is completely developed, so that the T line outputs a color signal, and SERS / colorimetric dual-mode detection on the mycotoxin is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunological detection, and particularly relates to a test strip for SERS / colorimetric dual-mode detection of mycotoxins, a preparation method thereof, and an application thereof. Background Art

[0002] Mycotoxins are toxic secondary metabolites produced by toxigenic filamentous fungi under suitable temperature and humidity conditions. They are widely present in foods and the environment and have toxicities such as teratogenicity, carcinogenicity, mutagenicity, and immune function inhibition, which seriously affect the health of humans and animals. The binding of mycotoxins to cells can produce general cytotoxic effects and can induce various diseases such as in-situ liver cancer and gastric cancer. In addition, mycotoxins are soluble in organic solvents, have chemical stability and high temperature resistance, and are not easily destroyed by heating or cooking. Therefore, they have become one of the harmful factors of food contamination. Therefore, it is of great significance to establish a simple, rapid, and sensitive detection method.

[0003] Currently, the detection methods of mycotoxins mainly include traditional chromatographic methods such as thin-layer chromatography, high-performance liquid chromatography, liquid chromatography-mass spectrometry, electrochemistry methods, spectroscopic methods such as colorimetry, fluorescence, and surface-enhanced Raman spectroscopy, and immunoassay methods such as enzyme-linked immunosorbent assay. However, most of these methods require expensive experimental instruments, skilled technicians, and time-consuming and cumbersome operations. These inconveniences have hindered their wide application in on-site and instant detection. Therefore, there is still a need for a simple and easy-to-use detection method to meet the on-site rapid detection requirements.

[0004] Lateral Flow Immunoassay (LFIA) combines immunoassay and lateral flow chromatography techniques. Through the specific reaction of antigen-antibody and chromatography, it can rapidly detect the target substance in the sample. LFIA is mainly composed of a combination of a nitrocellulose (NC) membrane, a sample pad, a conjugate pad, and an absorbent pad fixed successively on a polyvinyl chloride (PVC) bottom plate. On the test (T) line of the NC membrane of LFIA, an antigen or antibody that can specifically recognize the target substance is fixed, and an anti-IgG antibody is sprayed and fixed on the quality control (C) line. The sample solution containing the immune probe migrates on the test strip by capillary action. When the test solution migrates to the T line, the complex formed by the immune probe and the target substance will specifically bind to the substance fixed on the T line, and the remaining test solution will continue to migrate to the C line to have an immune reaction with the IgG antibody. Finally, qualitative or quantitative detection is carried out, which is considered to be the most potential point-of-care testing method. However, the detection sensitivity of traditional lateral flow immunoassay test strips is limited, and only qualitative and semi-quantitative analysis of the target substance can be carried out. At the same time, due to the complex composition of the actual sample, during the detection process, the matrix has a great influence on the detection result, and false positives and false negatives are prone to occur, resulting in low accuracy and restricting its partial applications.

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

[0006] In order to solve the deficiencies existing in the above-mentioned prior art, the present invention combines SERS technology with lateral flow immunoassay technology, and uses hydrogel as a colorimetric signal output carrier to construct a lateral flow immunoassay test strip for detecting mycotoxins based on SERS. It combines the high sensitivity and specificity of SERS with the advantages of lateral flow immunoassay test strips such as convenience and low cost. At the same time, hydrogel is used as a colorimetric signal output to reduce background color interference, and the accuracy is improved by using dual-mode result comparison to achieve rapid and sensitive detection of mycotoxins.

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

[0008] One of the objectives of the present invention is to provide a test strip for SERS / colorimetric dual-mode detection of mycotoxins, which includes a test strip part and a hydrogel part, wherein: the test strip part includes a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad that are sequentially fixed on a bottom plate, and the conjugate pad is coated with a monoclonal-labeled immune probe; a quality control line and a detection line are provided on the nitrocellulose membrane; the quality control line is provided with an anti-IgG antibody, and the detection line is provided with a mycotoxin-bovine serum albumin conjugate; the hydrogel part is used to be pasted on the detection line, and the hydrogel in the hydrogel part contains TMB and NaAC-HAC buffer substances.

[0009] Further, the detection line and the quality control line are drawn on the nitrocellulose membrane by a gold spraying instrument at a dosage of 1 μL / cm. Among them, the concentration of the mycotoxin-bovine serum albumin conjugate coated on the detection line is 0.5 mg / mL; the concentration of the anti-IgG antibody coated on the quality control line is 0.5 mg / mL, and the distance between the detection line and the quality control line is 5 mm.

[0010] Another objective of the present invention is to provide a preparation method of a test strip for SERS / colorimetric dual-mode detection of mycotoxins, which includes the following steps:

[0011] S1: Prepare a monoclonal-labeled immune probe and store it at 4 °C for later use;

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

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

[0014] S4: Paste the treated nitrocellulose membrane, conjugate pad, sample pad, and absorbent pad on a PVC bottom plate in sequence, with each part overlapping by 2 mm. After assembly, cut it to obtain the test strip part;

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

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

[0017] (1) Add silver nitrate AgNO 3 solution, ascorbic acid AA solution, sodium hydroxide NaOH solution, and Ag seed solution to a cetyltrimethylammonium chloride CTAC solution, and react to obtain Ag NPs;

[0018] (2) Incubate Ag NPs with the Raman signal molecule DTNB, and after centrifugation, redissolve to obtain modified Ag NPs;

[0019] (3) Add the modified Ag NPs to the cetyltrimethylammonium chloride (CTAC) solution, then add chloroplatinic acid H 2 PtCl 6 solution and ascorbic acid (AA) solution. After the reaction, centrifuge and resuspend to obtain Ag@Pt NRs;

[0020] (4) Add the mycotoxin antibody to the Ag@Pt NPs solution for incubation. After blocking with 1% BSA, centrifuge and resuspend to obtain the Ag@Pt NPs-mAb immunoprobe.

[0021] Further, step S3 includes: Cut the sample pad and conjugate pad to a width of 1.4 cm, soak them in the pretreatment solution for 30 min, put them in a 37°C forced-air oven to dry for 1 h, and store them in a dry 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, attach the nitrocellulose membrane to the PVC bottom plate. Then, overlap the conjugate pad and the NC membrane by 2 mm and attach them to the PVC bottom plate. Next, overlap the sample pad and the conjugate pad by 2 mm and attach them to the PVC bottom plate. Finally, overlap the absorbent pad and the nitrocellulose membrane by 2 mm and attach them to the PVC bottom plate. After assembly, cut it, and store the cut test strip with a width of 4 mm at 4°C for later use.

[0024] Further, the steps for preparing the hydrogel in step S5 are: Weigh 0.6 g of agarose and 0.1 g of glucose, dissolve them in 60 mL of TAE solution, heat to dissolve, then suck out 10 mL and let it stand to cool. Weigh 24 mg of TMB and dissolve it in 10 mL of ethanol. Suck out 1 mL of TMB and 1 mL of NaAC-HAC and add them to the agarose that has stood to cool. Wait for it to form, and then cut it for later use.

[0025] The third object of the present invention is to provide an application of a SERS / colorimetric dual-mode detection test strip for mycotoxins in detecting mycotoxins in traditional Chinese medicines, cereal grains, and environmental water samples.

[0026] Further, add the detection liquid to the sample pad. After laying it flat, collect the SERS signal spectrum on the test line. After it is fully displayed, paste the hydrogel part on the test line, react, and then uncover it. Observe the color change and take a photo to identify its RGB value. Substitute the RGB value and the SERS intensity value into the standard curve of the mycotoxin concentration and the RGB and SERS signal differences 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 nanomaterial of the present invention has excellent SERS activity. When applied to the lateral flow immunoassay method, it can simply, sensitively, and rapidly detect mycotoxins, and can directly detect and collect the SERS signal results. At the same time, the Ag@Pt-mAb probe also has good oxidase activity, which can effectively catalyze colorless TMB into blue ox-TMB, converting the Image J signal output only by the color of the probe itself into the color change of the catalytic product, and can effectively expand the signal range of color output. In addition, using hydrogel as the color signal output, the color signal is transferred to the colorless hydrogel, effectively avoiding the interference of the background colors of the test line and the quality control line of the test strip itself. Visual RGB analysis and SERS detection dual-mode tests are realized, and the dual-mode results are used to compare with each other to improve the result accuracy. Description of the Drawings

[0029] Figure 1 is the detection principle diagram in the present invention;

[0030] Figure 2 is the transmission electron microscope image of AgNPs and Ag@Pt NPs in the present invention;

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

[0032] Figure 4 is the photo of the test line of the test strip after adding different concentrations of mycotoxins in the present invention;

[0033] Figure 5 is the SERS signal spectrum of the test line of the test strip after adding different concentrations of mycotoxins in the present invention;

[0034] Figure 6 is the fitting linear graph of the difference in SERS signal intensity and the logarithm of concentration on the test line of the test strip with different concentrations of mycotoxins in the present invention;

[0035] Figure 7 is the photo of the hydrogel and the comparison diagram before and after being placed on the test line in the present invention;

[0036] Figure 8 is the fitting linear graph of the RGB signal intensity value and the logarithm of concentration on the test line of different test strips in the present invention. Detailed Embodiments

[0037] The present invention will be further described below through the description of specific embodiments. However, 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. As long as they do not depart from the basic idea of the present invention, they are within the protection scope of the present invention.

[0038] Referring to Figure 1 , an embodiment of a test strip for SERS / colorimetric dual-mode detection of mycotoxins and its preparation method and application of the present invention are as follows. However, the content of the present invention is not limited thereto at all.

[0039] Example 1

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

[0041] (1) Prepare Ag@Pt NPs

[0042] First, add 0.5 mL of 10 mM AgNO 3加入 to 19 mL of water. Subsequently, add 0.5 mL of 1% trisodium citrate and 0.6 mL of NaBH4 in sequence and let it stand for reaction for 2.5 hours to obtain Ag seeds. Subsequently, add 1 mL of AgNO 3 solution, 1 mL of ascorbic acid (AA) solution, 0.1 mL of sodium hydroxide (NaOH) solution and 0.1 mL of Ag seed solution to 30 mL of cetyltrimethylammonium chloride (CTAC) solution. After reacting for 4 h, centrifuge to obtain Ag NPs. Incubate Raman signal molecule DTNB in Ag NPs, and after centrifugation, redissolve to obtain modified Ag NPs. Add 2.5 mL of the modified Ag NPs to 20 mL of cetyltrimethylammonium chloride (CTAC) solution, and then add 2 mL of chloroplatinic acid H 2 PtCl 6 solution and ascorbic acid (AA) solution. After reacting for 2 h, centrifuge at a speed of 10000 rpm for 10 min and resuspend to obtain Ag@Pt NPs; among them, the transmission electron microscope images of the centrifuged Ag NPs, the transmission electron microscope images of Ag@Pt NPs and the elemental analysis spectra are referred to Figures 2 - 3 .

[0043] (2) Prepare Ag@Pt-mAb

[0044] Drop K 2 CO 3The pH value of the solution was adjusted to 8.5, and then 10 μL of mycotoxin antibody was added to the Ag@Pt NPs solution and incubated for 2 h to label the antibody on the surface of Ag@Pt NPs. After blocking with 1% BSA for 30 min, it was centrifuged and resuspended in the preservation solution (containing 1% BSA, 1% sucrose, 1% trehalose, 1% Tween-20), and the obtained Ag@Pt NPs-mAb immunoprobe was stored at 4 °C.

[0045] Example 2

[0046] Preparation of test strip

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

[0048] The conjugate of mycotoxin zearalenone (ZEN) and bovine serum albumin (BSA) diluted with phosphate buffered saline (PBS), namely ZEN-BSA conjugate (0.5 mg / mL) and goat anti-mouse secondary antibody IgG (0.5 mg / mL) were used as the test line T line and the control line C line respectively, and were drawn on the NC membrane with a spraying gold instrument at a dosage of 1 μL / cm, and the distance between the two lines was 5 mm; it was placed in a forced air oven at 37 °C and dried for 1 h and then stored for later use.

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

[0050] The sample pad and conjugate pad were soaked in the pretreatment solution for 30 min, then placed in a forced air oven at 37 °C and dried for 1 h, and then stored in a dry bag for later use.

[0051] (3) Assembly of test strip

[0052] The NC membrane was attached to the PVC bottom plate, then the conjugate pad was overlapped with the NC membrane by 2 mm and attached to the PVC bottom plate, then the sample pad was overlapped with the conjugate pad by 2 mm and attached to the PVC bottom plate, and finally the absorbent pad was overlapped with the NC membrane by 2 mm and attached to the PVC bottom plate, and it was cut into test strips 4 mm wide and stored at 4 °C for later use.

[0053] Example 3

[0054] Preparation of hydrogel

[0055] Weigh 0.6 g of agarose and 0.1 g of glucose and dissolve them in 60 mL of TAE buffer solution. After heating and dissolving, 10 mL was taken and allowed to stand and cool. Weigh 24 mg of 3,3',5,5'-tetramethylbenzidine (TMB) and dissolve it in 10 mL of ethanol. 1 mL was taken and added to the agarose that had been allowed to stand and cool. Wait for it to form, and cut it into pieces with a length and width of 4 mm for later use.

[0056] Example 4

[0057] Detection of Mycotoxins

[0058] (1) Preparation of ZEN Standard Solution

[0059] Prepare the ZEN standard stock solution (1 mg / mL) with methanol, and dilute the ZEN solution with pure water to a series of concentrations from 0.1 ng / mL to 1000 ng / mL for lateral flow immunoassay detection.

[0060] (2) Detection Process

[0061] Refer to Figures 6 - 8 , in the actual detection, add the detection solution containing buffer solution, probe molecule, and different concentrations of ZEN to the sample pad of the above lateral flow immunoassay test strip. After lying flat for 10 min, collect the SERS signal spectrum on the T line. Place the hydrogel on the T line to react for 10 min, then take out and take a photo for RGB recognition, and establish the standard curve of mycotoxin concentration vs. SERS signal difference and RGB value.

[0062] (3) Result Judgment and Analysis

[0063] In visual detection with the naked eye, refer to Figure 4 , according to the negative control result (0 ng / mL), for the detection results of ZEN at 0.1 - 1 ng / mL, the black intensity of the strip on the T line does not weaken, which is negative; for the detection results of ZEN at 10, 100 ng / mL, the black intensity of the strip on the T line significantly weakens, which is weakly positive; when the concentration of ZEN is higher than 1000 ng / mL, no strip appears on the T line, which is positive.

[0064] As Figure 5 shown, in SERS detection, collect the SERS signal spectrum on the T line and substitute it into the corresponding standard curve shown in Figure 6 to calculate the concentration of mycotoxin in the detection solution. In colorimetric detection, place the colorless hydrogel on the T line to react for 10 min, then uncover it, take a photo for RGB recognition, and substitute it into the corresponding standard curve shown in Figure 8 to calculate the concentration of mycotoxin in the detection solution. Use the SERS / colorimetric dual-mode result comparison to improve the detection accuracy.

[0065] (4) Detection of Actual Samples

[0066] To evaluate the applicability and accuracy of this method, we detected actual corn samples. First, the corn samples were pretreated according to the following steps. First, the corn was chopped and weighed, and 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 obtained liquid was diluted with 10 mM PBS (containing 1% Tween 20) to prepare a sample detection solution, and experimental analysis was carried out according to the above detection steps. ZEN was not detected in the measured samples, and liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to confirm that ZEN was not contained in the samples. To further verify the analytical performance of this method, we carried out a standard addition recovery experiment on all samples. ZEN standard solutions with low (0.1 ng / mL), medium (1.0 ng / mL), and high (10.0 ng / mL) concentrations were added to the samples respectively, and each concentration level was determined in parallel 3 times. The results of the standard addition experiment are shown in Table 1. The recoveries of different samples were between 98.2% and 103.2%, indicating that this method is applicable to the detection of actual samples.

[0067] Table 1 Results of standard addition recovery of actual samples

[0068]

[0069] The above is the preparation and detection application of an immunochromatographic test strip for detecting mycotoxins based on SERS technology in the present invention. It can be seen from the specific implementation manner that this SERS-LFIA method for detecting mycotoxins in the present invention has the advantages of simple operation, rapidity, high sensitivity, etc., and has the application potential for on-site rapid detection.

[0070] The above embodiments only illustrate the principle and its efficacy of the present invention by way of example, and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A SERS / colorimetric dual-mode test strip for detecting mycotoxins, characterized in that: It includes a test strip part and a hydrogel part, wherein: The test strip part includes a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad fixed on the bottom plate in sequence, wherein the conjugation pad is coated with a monoclonal labeled immune probe; a quality control line and a detection line are arranged on the nitrocellulose membrane; the quality control line is provided with anti-IgG antibodies, and the detection line is provided with a mycotoxin-bovine serum protein coupling substance; The hydrogel part is used for sticking the detection line, and the hydrogel in the hydrogel part contains TMB and NaAC-HAC buffer substances.

2. The SERS / colorimetric dual-mode test strip for detecting mycotoxins according to claim 1, characterized in that: The test line and the quality control line are drawn on the nitrocellulose membrane by a gold spray instrument at a dosage of 1 μL / cm, wherein the concentration of the mycotoxin-bovine serum protein coupling substance coated on the test line is 0.5 mg / mL; the concentration of the anti-IgG antibody coated on the quality control line is 0.5 mg / mL, and the distance between the test line and the quality control line is 5 mm.

3. A method for preparing a SERS / colorimetric dual-mode test strip for detecting fungal toxins according to claim 1 or 2, characterized in that: The following steps are involved: S1: Prepare monoclonal labeled immunoprobe and store it at 4°C for future use; S2: prepare nitrocellulose membrane, and draw the detection line and quality control line on the nitrocellulose membrane in sequence; S3: Soak the sample pad and the conjugate pad in the pretreatment solution, and dry them for later use; S4: Paste the treated nitrocellulose membrane, conjugate pad, sample pad and absorbent pad on the PVC bottom plate in sequence, with each part overlapping by 2 mm. After the assembly is completed, cut the parts to obtain the test strip part; S5: dissolving agarose and glucose in TAE buffer solution, and then adding TMB solution and NaAC-HAC solution to obtain the hydrogel part.

4. The method for preparing the SERS / colorimetric dual-mode test strip for detecting fungal toxins according to claim 3, characterized in that: The method for preparing the immune probe in step S1 comprises the following steps: (1) adding silver nitrate AgNO3 solution, ascorbic acid AA solution, sodium hydroxide NaOH solution and Ag seed solution to hexadecyltrimethylammonium chloride (CTAC) solution to obtain Ag NPs after reaction; (2) Adding Raman signal molecule DTNB to Ag NPs for incubation, and then re-dissolving after centrifugation to obtain modified Ag NPs; (3) Adding the modified Ag NPs to the hexadecyltrimethylammonium chloride (CTAC) solution, then adding chloroplatinic acid (H2PtCl6) solution and ascorbic acid (AA) solution, centrifuging and resuspending after the reaction to obtain Ag@Pt NRs; (4) Add fungal toxin antibody to the Ag@Pt NPs solution for incubation, block with 1% BSA, and then centrifuge and resuspend to obtain the Ag@PtNPs-mAb immune probe.

5. The method for preparing the SERS / colorimetric dual-mode test strip for detecting fungal toxins according to claim 3, characterized in that: The step S3 comprises: cutting the sample pad and the conjugate pad into 1.4 cm width, soaking them in the pretreatment solution for 30 minutes, drying them in a 37° C. forced air oven for 1 hour, and storing them in a drying bag for later use.

6. The method for preparing the SERS / colorimetric dual-mode test strip for detecting fungal toxins 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 fungal toxins according to claim 3, characterized in that: The step S4 comprises: firstly attaching the nitrocellulose membrane to the PVC bottom plate, then overlapping the conjugate pad with the nitrocellulose membrane by 2 mm and attaching them to the PVC bottom plate, then overlapping the sample pad with the conjugate pad by 2 mm and attaching them to the PVC bottom plate, and finally overlapping the absorbent pad with the nitrocellulose membrane by 2 mm and attaching them to the PVC bottom plate, and after the assembly is completed, cutting them into 4 mm wide test strips and storing them at 4° C. for future use.

8. The method for preparing the SERS / colorimetric dual-mode test strip for detecting fungal toxins 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, heat and dissolve them, then absorb 10 mL and let it stand and cool, weigh 24 mg TMB and dissolve it in 10 mL ethanol, absorb 1 mL TMB and 1 mL NaAC-HAC, add them to the cooled agarose, wait for molding, and cut them for later use.

9. Use of the SERS / colorimetric dual-mode mycotoxin detection test strip according to any one of claims 1 to 2 in detecting mycotoxins in traditional Chinese medicines, cereals and food and environmental water samples.

10. The use of the SERS / colorimetric dual-mode test strip for detecting fungal toxins according to claim 9, characterized in that: The detection liquid is dropped into the sample pad, which is laid flat, and the SERS signal spectrum on the detection line is collected. After it is fully displayed, the hydrogel part is pasted on the detection line and then peeled off after the reaction. The color change is observed and photographed to identify its RGB value. The RGB value and SERS intensity value are substituted into the standard curve of the fungal toxin concentration and the RGB and SERS signal difference to obtain the corresponding concentration.

Images