A photothermal lateral flow assay device for simultaneous detection of multiple mycotoxins and application thereof

Abstract

The application discloses a photothermal lateral flow analysis device for simultaneously detecting multiple mycotoxins and application thereof, and the device comprises a test strip, n photothermal signal probes, an 808 nm plane laser light source and a thermal detection device, the n photothermal signal probes are prepared by respectively adsorbing n kinds of to-be-detected toxin antibodies on the surface of Fe3O4-PDA-Au complexes, and n is greater than or equal to 2; n kinds of to-be-detected toxin antigens are sequentially fixed on a detection area of the test strip, and a secondary antibody is fixed on a quality control area. The application steps are as follows: the n photothermal signal probes, a to-be-detected sample liquid and a buffer are mixed, a sample pad end of the test strip is inserted into the mixed liquid for chromatography, the whole detection area is irradiated by the 808 nm plane laser light, and the photothermal temperature of the detection area is collected by a thermal imaging or temperature measuring device; standard curves are respectively established according to the temperature and the concentration of the n to-be-detected toxins, and quantitative analysis of the to-be-detected toxins is carried out. The application can realize simultaneous photothermal qualitative and quantitative analysis of multiple mycotoxins.

Description

Technical Field

[0001] This invention relates to a photothermal lateral flow analysis device and its application for the simultaneous detection of multiple mycotoxins, belonging to the field of food analysis and detection technology. Background Technology

[0002] Because various fungi share similar ecological conditions for growth or toxin production, most fungi can produce multiple mycotoxins, and a single fungus can contaminate multiple agricultural products and foods. Coupled with dietary diversity, food is often contaminated with co-existing mycotoxins. Co-contamination of mycotoxins can lead to antagonistic, cumulative, or synergistic effects, posing a greater threat to humans. Therefore, there is an urgent need to develop high-throughput rapid detection methods for mycotoxins.

[0003] Sideflow chromatography is widely used for the detection of mycotoxins due to its fast detection performance, low cost, and good sensitivity. In the past 5-10 years, a signal amplification strategy based on current sideflow analysis technology has emerged. This strategy mainly relies on laser-excited plasma nanomaterials. When the excitation light matches the surface plasmon resonance of the nanoparticles, the particles absorb the light energy and release it as heat, achieving a signal amplification effect. Small changes in analyte concentration can be detected through temperature variations, with low background signal and high sensitivity. It has been successfully applied to the detection of various target analytes.

[0004] Compared to colorimetric and fluorescence lateral flow analysis techniques, photothermal lateral flow analysis has higher sensitivity and signal-to-noise ratio, and has received increasing attention in recent years. Zhang et al. developed an ultrasensitive photothermal lateral flow immunoassay method for the quantitative detection of ochratoxin A (OTA) based on multifunctional photothermal nanoparticles Fe3O4@Au (Talanta 2021, 222, 121478). Zhang et al. developed a photothermal lateral flow immunoassay method for the detection of deoxynivalenol (DON) using flower-like gold nanoparticles deposited as manganese dioxide nanocarriers as photothermal materials (Food Chemistry 2021, 341, 128231).

[0005] However, existing photothermal lateral flow analyses cannot achieve simultaneous detection of multiple analytes because they all use point lasers as light sources, which can only illuminate one detection site on the test strip at a time. Even if multiple analytes are placed in the detection area, each area can only be illuminated and its temperature measured sequentially. Multiple analytes can only be detected sequentially, and the total analysis time is the sum of the individual analysis times. Therefore, achieving simultaneous detection of multiple analytes in photothermal lateral flow analysis remains a challenge and bottleneck in the field. Summary of the Invention

[0006] Technical issues: Existing photothermal lateral flow analysis of mycotoxins has the following limitations: 1. The light source is a point laser, with a small spot area (5*8 mm). 2 First, each detection can only detect a single target object; second, data collection and analysis for a single target object takes 10-20 minutes, and the analysis time for multiple targets is the sum of the above times, so the detection efficiency needs to be improved.

[0007] Improving analytical throughput is a key issue in the development of point-of-care testing technologies and a current trend in analytical chemistry. Exploring and developing photothermal lateral flow analytical methods for the simultaneous detection and evaluation of polyfungal toxins, from a technical or instrumental perspective, has significant scientific and practical implications.

[0008] Technical solution:

[0009] On the one hand, this application provides a photothermal lateral flow analysis method for the simultaneous detection of multiple mycotoxins. The method utilizes a photothermal lateral flow analysis device to simultaneously detect multiple mycotoxins, and includes the following steps:

[0010] (1) Construct a photothermal lateral flow analysis device for simultaneous detection of multiple mycotoxins, specifically comprising four parts: preparation of test strips, preparation of n photothermal signal probes, construction of an 808 nm surface laser source, and thermal detection device. The test strip includes a base plate, on which a sample pad, an NC membrane, and an absorption pad are sequentially overlapped and pasted in a horizontal direction. The NC membrane includes a detection area and a quality control area, i.e., the C area. The detection area of ​​the test strip includes n independent T areas, each T area having a different toxin antigen to be tested fixed on it, denoted as T0. n In region C, n≥2, the corresponding secondary antibody is fixed in region C of the test strip; the photothermal signal probe is made of Fe3O4-PDA-Au composite material; the 808 nm surface laser source is formed by expanding an 808 nm point laser through a surface diverging laser beam expander, and the spot size can be adjusted to simultaneously cover all T regions. n district;

[0011] (2) Mix n kinds of photothermal signal probes, the sample solution to be tested, and the buffer solution to form a mixture. Insert the sample pad end of the test strip into the mixture for chromatography.

[0012] (3) n kinds of photothermal signal probes migrate through the detection area due to tomography and are detected by the corresponding T n Region capture; the detection region is irradiated with an 808 nm surface laser to simultaneously excite all T... n n photothermal signal probes in the area, simultaneously acquiring T using thermal imaging or temperature measurement equipment. n The photothermal temperatures of n photothermal signal probes in the region are analyzed qualitatively and / or quantitatively.

[0013] As an optional implementation method, the qualitative analysis refers to, based on Tn Whether the corresponding T zone in the region appears dark purple or whether there are changes in photothermal temperature determines whether the sample solution contains fungal toxins; the quantitative analysis refers to establishing standard curves based on photothermal temperature and the concentrations of n toxins to be tested, and performing quantitative analysis of n target substances.

[0014] As an optional implementation, in step (3), the detection area is T n The changes in light, heat, and temperature in the region are negatively correlated with the content of the corresponding fungal toxins being tested, specifically:

[0015] When the sample does not contain fungal toxins, T n The corresponding T region in the area appears dark purple and has a high light and heat temperature;

[0016] When the sample contains a certain fungal toxin, T n The corresponding T region is light purple or even colorless, and has a low light and heat temperature.

[0017] The quality control area, which serves as a reference for verifying the validity of the test strip results, always appears dark purple.

[0018] As an optional implementation method, the analytical method of this application is applicable to detecting whether the analyte contains a variety of fungal toxins, especially suitable for analytes of cereal origin. The cereals mentioned in this application include rice, wheat, millet, soybeans and other miscellaneous grains, which are a general term for cereal plants or food crops.

[0019] As an optional implementation, the fungal toxins include, but are not limited to, at least two of aflatoxin, vomitoxin, zearalenone, aspergillin, and fusarium toxins.

[0020] As an optional implementation, the method for preparing the photothermal lateral flow analysis device for simultaneous detection of multiple mycotoxins includes:

[0021] (1) Preparation of signal probes: Fe3O4-PDA-Au composite material was reacted with n kinds of toxin antibodies, i.e., primary antibody mAbs. n The Fe3O4-PDA-Au composite material, along with n types of toxin antibodies and bovine serum albumin (BSA), is then mixed to form a complex, thus preparing the Fe3O4-PDA-Au composite material, n types of toxin antibodies, and Fe3O4-PDA-Au-mAb of bovine serum albumin. n A photothermal signal probe was obtained, namely Fe3O4-PDA-Au-mAb. n It is stored in a sealed container in solution form until use.

[0022] (2) Constructing test strips: T nIn the test T zone of the test area, n kinds of test toxin antigens are fixed sequentially, and the secondary antibody against the primary antibody is added or sprayed onto the C zone. After drying, it is stored in a vacuum bag for later use.

[0023] As an optional implementation method, the specific method for preparing the Fe3O4-PDA-Au composite material is as follows: the prepared Fe3O4-PDA nanocrystal dispersion is diluted, the diluted Fe3O4-PDA solution is placed in a centrifuge tube, trisodium citrate solution is added, the mixture is mixed, HAuCl4 solution is added, ultrapure water is added, and then the reaction is carried out by shaking at room temperature to obtain the Fe3O4-PDA-Au composite material, which is then refrigerated for later use.

[0024] As an optional implementation, the photothermal signal probe is prepared as Fe3O4-PDA-Au-mAb. n The specific method is as follows: Fe3O4-PDA-Au composite material was placed in a centrifuge tube, and a weakly alkaline solution was added to adjust the pH of the system to 6-7.5. After shaking and mixing, the test toxin antibody was added separately. The mixture was shaken and reacted at room temperature for 30-60 min. Bovine serum albumin was then added for blocking, and the mixture was shaken and reacted at room temperature for 1-1.5 h. After centrifugation, the supernatant was removed, and the remaining substances were reconstituted in buffer solution to obtain Fe3O4-PDA-Au-mAb. n Store in the refrigerator for later use.

[0025] As an optional implementation, the buffer solution includes any one of phosphate buffer, borate buffer, and carbonate buffer.

[0026] As an optional implementation, the T n The test T region of the zone is sequentially fixed with n kinds of test toxin antigens, including the sequential dripping or spraying of the test toxin antigens into the T region, specifically: T n Add or spray 5-20 mM PBS solution containing 0.5-10 mg / mL of the antigen to be tested to each area.

[0027] As an optional implementation, the fixation of the secondary antibody in region C includes dripping or spraying a secondary antibody against the primary antibody source, i.e., anti-primary antibody, into region C. Specifically, this involves dripping or spraying a 5-20 mM PBS solution containing 0.05-10 mg / mL of secondary antibody into region C.

[0028] As an optional implementation, the running buffer is a solution containing sucrose, bovine serum albumin, Tween phosphate, or citrate.

[0029] On the other hand, the present invention provides a photothermal lateral flow analysis device for simultaneously detecting multiple mycotoxins, the photothermal lateral flow analysis device comprising four parts: a test strip, n kinds of photothermal signal probes, an 808 nm surface laser source, and a thermal detection device.

[0030] The test strip body includes a base plate, on which a sample pad, an NC membrane, and an absorbent pad are sequentially overlapped and pasted in a horizontal direction. The nitrocellulose membrane (NC membrane) is used to separate and detect the analyte from other substances in the sample. The sample pad is used for sample loading, and the absorbent pad is used to absorb excess liquid. The PVC base plate provides physical support for the test strip. The NC membrane includes a detection area, i.e., T... n The quality control area, also known as area C, has n≥2; where T n The region includes n independent T regions, with one T region set at intervals of 3~5 mm. Each T region is immobilized with a different toxin antigen to be tested, i.e., the molecule-protein conjugate to be tested. The C region is immobilized with a secondary antibody, which is a secondary antibody against the primary antibody, i.e., an anti-primary antibody.

[0031] The 808 nm surface laser source is formed by expanding an 808 nm point laser beam using a surface-diverging laser beam expander; the surface-diverging laser beam expander is used as an optical accessory for the 808 nm point laser, and can expand a 5*8 mm surface laser beam. 2 A small spot size can be magnified 1.5 to 30 times, and a surface laser can simultaneously irradiate n T-regions, where n ≥ 2. When the magnification factor is 2.5 times, a 5*8 mm area can be magnified. 2 The original light spot was enlarged to 12.5*20 mm. 2 The laser can simultaneously irradiate three T-zones;

[0032] The thermal detection device refers to thermal imaging or temperature measurement equipment.

[0033] As an optional implementation, the photothermal signal probe is an Fe3O4-PDA-Au composite material reacted with n kinds of target toxin antibodies, i.e., primary antibodies (mAbs). n The complex formed by the compounding of Fe3O4 with bovine serum albumin is called Fe3O4-PDA-Au-mAb. n The PDA is polydopamine. In this application, the signal probe is used independently of the photothermal test strip structure. After chromatographic migration, the photothermal signal probe flows through the detection area and is detected by the corresponding T... n Capture of the target molecule-protein conjugate in the region.

[0034] As an optional implementation, the thermal imaging or temperature measurement device includes, but is not limited to, any one of the following: mobile phone infrared thermal imaging analysis accessories, infrared thermal imagers, handheld infrared thermal imaging analyzers, or infrared thermal imaging temperature guns. The thermal imaging or temperature measurement device acquires photothermal imaging photos and outputs and displays them through a connected smart display terminal.

[0035] As an optional implementation, the smart display terminal includes a computer or a smartphone.

[0036] As an optional implementation, the primary antibody to be tested may be derived from sources including, but not limited to, mice, rats, and rabbits.

[0037] As an optional implementation, the secondary antibody includes, but is not limited to, sheep anti-mouse secondary antibody, rabbit anti-mouse secondary antibody, sheep anti-rabbit secondary antibody, and donkey anti-rabbit secondary antibody.

[0038] As an optional implementation, the T n The test T region of the zone is sequentially fixed with n kinds of test toxin antigens, including the sequential dripping or spraying of the test toxin antigens into the T region, specifically: T n Add or spray 5-20 mM PBS solution containing 0.5-10 mg / mL of the antigen to be tested to each area.

[0039] As an optional implementation, the fixation of the secondary antibody in region C includes dripping or spraying a secondary antibody against the primary antibody source, i.e., anti-primary antibody, into region C. Specifically, this involves dripping or spraying a 5-20 mM PBS solution containing 0.05-10 mg / mL of secondary antibody into region C.

[0040] As an optional implementation, the running buffer is a solution containing sucrose, bovine serum albumin, Tween phosphate, or citrate.

[0041] The detection principle of the photothermal lateral flow analyzer for detecting mycotoxins of the present invention is explained as follows, taking deoxynivalenol (DON), aflatoxin B1 (AFB1), and zearalenone (ZEN) as examples: When the sample does not contain any of the three mycotoxins, Fe3O4-PDA-Au-mAb n The conjugate is detected by the three corresponding detection regions (T) on the NC membrane. n The samples were captured sequentially in three T regions, all exhibiting a bright dark purple color. Simultaneous irradiation of the three T regions with an 808 nm surface laser resulted in a high photothermal temperature (∆T). When the sample contained one or more of the three mycotoxins, the mycotoxins in the sample reacted with some Fe3O4-PDA-Au-mAb during incubation with the photothermal signal probe. n Complex binding competitively inhibits T n The combination of the region and the photothermal signal probe leads to T n The region of Fe3O4-PDA-Au-mAb n Reduced capture results in a weaker purple hue or even colorlessness, and a lower photothermal temperature (∆T) under 808 nm surface laser irradiation. n The change in photothermal temperature in the region is inversely proportional to the content of mycotoxins in the sample. Excess Fe3O4-PDA-Au-mAb nThe complex binds to the goat anti-mouse secondary antibody on the control area (C area), always displaying a bright dark purple color, which serves as a reference for verifying the validity of the test strip results.

[0042] Beneficial effects:

[0043] The detection method provided by this invention can simultaneously perform photothermal quantitative detection of multiple mycotoxins, especially mycotoxins in cereals, within 20 minutes in a single chromatography step. It has higher sensitivity and expands the detection throughput without increasing the detection time compared with existing photothermal sideflow analysis methods. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of the structure of the device of the present invention;

[0045] Figure 2 This document presents the response of the test strip of the present invention to negative and positive samples, as well as the standard curve.

[0046] Figure 3 This describes the specific response of the test strip detection area and probes in this invention. Detailed Implementation

[0047] The present invention can be better understood from the following embodiments. However, the specific material ratios, process conditions, and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0048] The following embodiments of this application disclose a photothermal lateral flow analysis method for simultaneously detecting multiple fungal toxins, including the following steps:

[0049] (1) Preparation of photothermal signal probe: Fe3O4-PDA-Au composite material was compounded with n kinds of toxin antibodies to be tested, and then mixed with bovine serum albumin to prepare Fe3O4-PDA-Au-mAb. n That is, a photothermal signal probe;

[0050] (2) Assemble the test strip: In the detection area (T n n different toxin antigens to be tested were sequentially fixed in the quality control area (C area), and secondary antibodies were fixed in the quality control area (C area).

[0051] (3) Mix n kinds of photothermal signal probes, the sample solution to be tested, and the buffer solution, then insert the sample pad end of the test strip into the mixture for chromatography;

[0052] (4) Irradiate the detection area with an 808nm surface laser, collect the temperature with thermal imaging or temperature measurement equipment, establish standard curves based on the temperature and the concentration of n toxins to be tested, and perform quantitative analysis of n target substances.

[0053] This invention enables simultaneous photothermal quantitative analysis of multiple mycotoxins. Based on the number of T-regions, the magnification factor of the surface-diverging laser beam expander in the surface laser can be adjusted to regulate the surface laser irradiation area, ensuring the spot area meets the coverage requirements of n T-regions. The following examples use AFB1, ZEN, and DON as examples of mycotoxins to provide a thorough and detailed explanation of the preparation and application methods of the entire device.

[0054] Example 1:

[0055] The preparation of a photothermal lateral flow analyzer for the simultaneous detection of multiple mycotoxins and its detection of negative and positive samples are described in the following steps.

[0056] 1. Material preparation

[0057] 1.1 Preparation of Fe3O4-PDA nanocrystals

[0058] Under nitrogen protection, FeCl3·6H2O (1.622 g) and FeCl2·4H2O (0.994 g) were dissolved in 20 mL of deionized water, with 5 mL of ammonia (28% w / v%) added. The resulting solutions were stirred at room temperature for 10 min. Then, 4.4 g of sodium citrate was added to the mixture under constant mechanical stirring at 90 °C, producing Fe3O4 nanoparticles coated with citrate. The solution was yellowish-brown and collected using a permanent magnet. 6 mg of dopamine hydrochloride was added to 50 mL of PBS (pH 8.5) containing 5 mg of Fe3O4 nanoparticles. After shaking at room temperature for 4 h, Fe3O4-PDA nanoparticles were obtained by centrifugation and washed three times with water. Relatively purified Fe3O4-PDA nanoparticles were obtained and stored at 4 °C for later use.

[0059] 1.2 Preparation of Fe3O4-PDA-Au composite materials

[0060] Dilute the Fe3O4-PDA dispersion with deionized water to a volume fraction of 1%. Take the diluted Fe3O4-PDA solution into a centrifuge tube, add ultrapure water, trisodium citrate solution (0.3 mol / L), and HAuCl4 solution (m / v=1%). Then immediately place it on a vortex shaker and shake for 7 min at room temperature to obtain the composite nanomaterial Fe3O4-PDA-Au of Fe3O4-PDA nanocrystals and AuNPs. Store it in a refrigerator at 4℃ for later use.

[0061] 1.3 Preparation of photothermal signal probe Fe3O4-PDA-Au-mAb n

[0062] Take 1 mL of Fe3O4-PDA-Au into three centrifuge tubes respectively, add 0.2 mol / L K2CO3 solution to adjust the pH of the system to 6.5, 6.5 and 6 respectively, shake to mix well, then add 5 μL of 1 mg / mL DON-mAb, 3 μL of AFB1-mAb and 1 μL of ZEN-mAb respectively, mix well and react at room temperature (150 rpm) for 45 min. After the reaction, 100 μL of bovine serum albumin (m / m=10%) was added for blocking for 1 h (150 rpm). After centrifugation at 12000 r / min for 30 min, the supernatant was removed and then reconstituted in 100 μL of buffer (20 mmol / L Na3PO4, 5% BSA, 0.25% Tween-20, 10% sucrose) to obtain three photothermal probes of the target analytes: Fe3O4-PDA-Au-mAb1, Fe3O4-PDA-Au-mAb2, and Fe3O4-PDA-Au-mAb3 (mAb1, mAb2, and mAb3 correspond to DON-mAb, AFB1-mAb, and ZEN-mAb, respectively, and are denoted as T1, T2, and T3). The probes were stored at 4℃ for later use.

[0063] 1.4 Detection Area (T) n Preparation of solution in the zone

[0064] DON-BSA, AFB1-BSA, and ZEN-OVA were diluted with 10 mM PBS solution (pH 7.4) to 0.4 mg / mL, 0.6 mg / mL, and 0.2 mg / mL, respectively, corresponding to solutions in the T1, T2, and T3 regions.

[0065] 1.5 Preparation of solutions for the quality control zone (Zone C)

[0066] The goat anti-mouse secondary antibody was diluted to 0.2 mg / mL with 10 mM PBS solution (pH 7.4).

[0067] 2. Preparation of test strips

[0068] according to Figure 1 The membrane assembly method involves attaching the NC membrane to the center of a PVC base plate. The sample pad and absorbent pad overlap the left and right ends of the NC membrane, respectively, covering it by approximately 2 mm. The assembled card is then cut into 3 mm wide strips to obtain blank test strips. These are then used in... Figure 1 China T n Add 0.5 μL of T to zone C and zone C dropwise at 5 mm intervals. n Prepare zone A solution and 0.5 μL zone C solution. After spotting, place the test strips in an oven and dry at 37°C for 60 min, then store in a vacuum bag for later use.

[0069] 3. Drawing working curves

[0070] Prepare 10 mL of 1 mg / mL DON, AFB1, and ZEN standard solutions using acetonitrile. Dilute the DON standard solution with 10 mM PBS solution (pH 7.4) to concentrations of 1 ng / mL, 5 ng / mL, 50 ng / mL, 150 ng / mL, and 400 ng / mL.

[0071] Similarly, the AFB1 standard solution was diluted to concentrations of 0.5 ng / mL, 2.5 ng / mL, 10 ng / mL, 50 ng / mL, and 200 ng / mL as test solutions for later use.

[0072] Similarly, the ZEN standard solution was diluted to concentrations of 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, and 15 ng / mL as test solutions for later use.

[0073] Take 24 μL of each of the three test solutions, totaling 72 μL, and place them in centrifuge tubes. Then, take 3 μL of each of the three photothermal probes, totaling 9 μL, and add them to the centrifuge tubes. Add 9 μL of running buffer to the centrifuge tubes and mix for 10 min. Then, insert the test strip into the centrifuge tubes for chromatography. After chromatography, use an 808 nm surface laser (power 1.96 W / cm²) to perform the chromatography. 2 Irradiate for 5 minutes, and monitor the temperature changes of detection areas T1, T2, and T3 using infrared thermal imaging accessories.

[0074] The results show that... Figure 2 (A) Figure 2 (B), Negative test strip T n The area all showed a distinct dark purple color, indicating high light and heat temperature; the positive test strip T n The area appears light purple or even colorless, and has a low temperature due to light and heat.

[0075] The operating curves of DON, AFB1, and ZEN are as follows: Figure 2 As shown in (C), (D), and (E).

[0076] Example 2:

[0077] Specificity verification of a photothermal lateral flow analyzer for simultaneous detection of multiple mycotoxins for multiple mycotoxins includes the following steps:

[0078] 1. Preparation of test paper materials

[0079] Same as Example 1

[0080] 2. Preparation of test strips

[0081] Same as Example 1

[0082] 3. Sample pretreatment

[0083] Use 10 mM PBS solution (pH 7.4) as the negative test solution.

[0084] 4. Sample testing

[0085] Add 72 μL of negative test solution to each of three centrifuge tubes, followed by 9 μL of Fe3O4-PDA-Au-mAb1, 9 μL of Fe3O4-PDA-Au-mAb2, and 9 μL of Fe3O4-PDA-Au-mAb3 (labeled T1, T2, and T3, respectively). Then add 9 μL of running buffer to each tube, mix, and incubate for 10 min. Insert the test strips into the centrifuge tubes for chromatography. After chromatography, use an 808 nm laser (1.96 W / cm²) equipped with a planar diverging laser beam expander. 2 Irradiate for 5 minutes, and monitor the detection area T using infrared thermal imaging accessories. n Temperature changes, results show as follows Figure 3 .

[0086] like Figure 3 As shown, the detection areas of the Fe3O4-PDA-Au-mAb1 probe chromatography strip, except for the DON detection area, are all relatively light and have a low photothermal temperature; the detection areas of the Fe3O4-PDA-Au-mAb2 probe chromatography strip, except for the AFB1 detection area, are all relatively light and have a low photothermal temperature; the detection areas of the Fe3O4-PDA-Au-mAb3 probe chromatography strip, except for the ZEN detection area, are all relatively light and have a low photothermal temperature; the specific recognition results between the probes are good.

[0087] Example 3:

[0088] A photothermal lateral flow analyzer for the simultaneous detection of multiple mycotoxins is applied to actual sample analysis, comprising the following steps:

[0089] 1. Preparation of test paper materials

[0090] Same as Example 1

[0091] 2. Preparation of test strips

[0092] Same as Example 1

[0093] 3. Sample pretreatment

[0094] Take 5 g of pre-milled grain powder without mycotoxin detection, 0.5 g of NaCl, and mix with 20 mL of 84% acetonitrile aqueous solution. Sonicate for 20 min, centrifuge for 10 min, filter the supernatant through a 0.22 μm filter membrane and treat with nitrogen blowing. Redissolve the sample with PBS. Prepare 10 mL of 1 mg / mL standard solutions of deoxynivalenol (DON), aflatoxin B1 (AFB1), zearalenone (ZEN), ochratoxin A (OTA), and patulin (PAT) using acetonitrile. Dilute the DON standard solutions to concentrations of 4 ng / mL, 40 ng / mL, and 400 ng / mL using actual sample solutions.

[0095] Similarly, the AFB1 standard solution was diluted to concentrations of 1 ng / mL, 10 ng / mL, and 100 ng / mL as test solutions for later use.

[0096] Similarly, the ZEN standard solution was diluted to concentrations of 0.5 ng / mL, 5 ng / mL, and 15 ng / mL as test solutions for later use.

[0097] Similarly, the OTA and PAT standard solutions were diluted to 100 ng / mL and used as test solutions.

[0098] 4. Sample testing

[0099] Add 72 μL of the actual sample solution to each of the three centrifuge tubes, followed by 9 μL of Fe3O4-PDA-Au-mAb1, 9 μL of Fe3O4-PDA-Au-mAb2, and 9 μL of Fe3O4-PDA-Au-mAb3 (denoted as T1, T2, and T3, respectively). Then add 9 μL of running buffer to each tube, mix, and incubate for 10 min. Insert the test strip into the centrifuge tube for chromatography. After the test strip chromatography is complete, use an 808 nm laser (power 1.96 W / cm²) equipped with a planar diverging laser beam expander. 2 Irradiate for 5 minutes, and monitor the detection area T using infrared thermal imaging accessories. n The temperature change was used to substitute the temperature value into the working curve of the corresponding toxin obtained in Example 1 to obtain its detection concentration value, and further its recovery rate and relative standard deviation were obtained. The results are shown in Table 1.

[0100] For test samples containing 120 ng / mL of DON, AFB1, and ZEN, the corresponding detection areas on the test strips showed a lighter color and a lower photothermal temperature. For test samples containing OTA and PAT, the detection areas on the test strips showed a deeper dark purple color and a higher photothermal temperature. The photothermal side-flow analyzer demonstrated good accuracy in detecting actual samples. The detection limits for DON, AFB1, and ZEN were 0.62 ng / mL, 0.38 ng / mL, and 0.27 ng / mL, respectively.

[0101] Table 1. Accuracy test results of the photothermal sideflow analyzer for various mycotoxins applied to actual sample analysis.

[0102]

[0103] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A photothermal lateral flow analysis method for simultaneous detection of multiple mycotoxins, characterized in that, The method for simultaneously detecting multiple mycotoxins using a photothermal lateral flow analyzer includes the following steps: (1) Construct a photothermal lateral flow analysis device for the simultaneous detection of multiple mycotoxins, specifically comprising four parts: preparation of test strips, preparation of n photothermal signal probes, construction of an 808 nm surface laser source, and thermal detection devices. The test strip includes a base plate, on which a sample pad, an NC membrane, and an absorbent pad are sequentially overlapped in a horizontal direction. The NC membrane includes a detection area and a quality control area, i.e., a C area. The detection area of ​​the test strip includes n independent T areas, each T area having a different analyte antigen fixed on it, denoted as T0. n In region C, n≥2, the test strip has a corresponding secondary antibody fixed in region C; The photothermal signal probe is a Fe3O4-PDA-Au composite material, which is reacted with n kinds of toxin antibodies (i.e., primary antibody mAbs) to be tested. n The complex formed after compounding with bovine serum albumin (BSA) is Fe3O4-PDA-Au-mAb. n The PDA is polydopamine; the preparation method of the Fe3O4-PDA-Au composite material includes: diluting the Fe3O4-PDA nanocrystal dispersion, taking the diluted Fe3O4-PDA solution into a centrifuge tube, adding trisodium citrate solution, mixing well, adding HAuCl4 solution, adding ultrapure water, and then shaking the reaction at room temperature to obtain the Fe3O4-PDA-Au composite material, which is then refrigerated for later use; The 808 nm surface laser source is formed by expanding an 808 nm point laser using a surface-diverging laser beam expander. The surface-diverging laser beam expander serves as an optical accessory for the 808 nm point laser, enlarging a small spot size by 1.5 to 30 times. By adjusting the spot size, it can simultaneously cover all T... n district; The thermal detection device includes a thermal imaging or temperature measuring device, which includes, but is not limited to, any one of a mobile phone infrared thermal imaging analysis accessory, an infrared thermal imager, or an infrared thermal imaging temperature gun. The thermal imaging or temperature measuring device acquires photothermal imaging photos and outputs and displays them through a connected smart display terminal, which includes a computer or a smartphone. (2) Mix n kinds of photothermal signal probes, the sample solution to be tested, and the buffer solution to form a mixture, and insert the sample pad end of the test strip into the mixture for chromatography; (3) n photothermal signal probes migrate through the detection area due to tomography and are detected by the corresponding T n Region capture; the detection region is irradiated with an 808 nm surface laser to simultaneously excite all T... n n photothermal signal probes in the area, using thermal imaging or temperature measurement equipment to simultaneously collect T n The photothermal temperatures of n photothermal signal probes in the region are analyzed qualitatively and / or quantitatively.

2. The method according to claim 1, characterized in that, In step (3), the detection area is T n The changes in light, heat, and temperature in the region are negatively correlated with the content of the corresponding fungal toxins being tested, specifically: When the sample does not contain fungal toxins, T n The corresponding T region in the area appears dark purple and has a high light and heat temperature; When the sample contains a certain fungal toxin, T n The corresponding T region is light dark purple or colorless, and has a low light and heat temperature; The quality control area, which serves as a reference for verifying the validity of the test strip results, always appears dark purple.

3. The method according to claim 1, characterized in that, The fungal toxins include, but are not limited to, at least two of aflatoxin, vomitoxin, zearalenone, aflatoxin, and fusarium toxin.

4. The method according to claim 1, characterized in that, The photothermal signal probe Fe3O4-PDA-Au-mAb n The preparation method includes: placing the Fe3O4-PDA-Au composite material into a centrifuge tube, adjusting the pH of the system to 6-7.5 with a weak alkaline solution, shaking to mix, then adding the antibody to be tested, and reacting with shaking at room temperature for 30-60 min. Next, adding bovine serum albumin, and reacting with shaking at room temperature for 1-1.5 h, centrifuging to remove the supernatant, and then redissolving the remaining material in buffer solution to obtain Fe3O4-PDA-Au-mAb. n That is, n kinds of photothermal signal probes; the photothermal signal probes are stored in a sealed container in solution and refrigerated for later use.

5. The method according to claim 1, characterized in that, The detection zone of the test strip is respectively dripped or sprayed with 5-20 mM PBS solution containing 0.5-10 mg / mL of the target toxin antigen; the C zone is dripped or sprayed with 5-20 mM PBS solution containing 0.05-10 mg / mL of secondary antibody.

6. A photothermal lateral flow analysis device for detecting multiple fungal toxins, characterized in that, The device comprises four parts: a test strip, n kinds of photothermal signal probes, an 808 nm surface laser source, and a thermal detection device. The test strip includes a base plate, on which a sample pad, an NC membrane, and an absorbent pad are sequentially overlapped and pasted in a horizontal direction. The NC membrane includes a detection area, i.e., T. n The quality control area, also known as area C, has n≥2, where T n The region includes n independent T regions, each T region is immobilized with a different toxin antigen to be tested, i.e., the test molecule-protein conjugate, and the C region is immobilized with a secondary antibody; The 808 nm surface laser source is formed by expanding an 808 nm point laser using a surface-diverging laser beam expander. The surface-diverging laser beam expander serves as an optical accessory for the 808 nm point laser, enlarging a small spot size by 1.5 to 30 times. By adjusting the spot size, it can simultaneously cover all T... n district; The thermal detection device refers to a thermal imaging or temperature measurement device, which includes, but is not limited to, any one of a mobile phone infrared thermal imaging analysis accessory, an infrared thermal imager, a handheld infrared thermal imaging analyzer, or an infrared thermal imaging temperature gun. The thermal imaging or temperature measurement device acquires photothermal imaging photos and outputs and displays them through a connected smart display terminal, which includes a computer or a smartphone. The n photothermal signal probes are Fe3O4-PDA-Au composite materials, which are respectively reacted with n target toxin antibodies, i.e., primary antibody mAbs. n The complex formed after compounding with bovine serum albumin is Fe3O4-PDA-Au-mAb. n The PDA is polydopamine.

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