Detection device for mycotoxin in buffalo milk
By introducing SERS technology and immunochromatographic analysis, and combining the electrostatic interaction between Au@4-MBA@Ag NPs and DON antibodies, the sensitivity and accuracy issues of DON detection in buffalo milk were resolved, achieving rapid detection with high sensitivity and low false positives.
Patent Information
- Application Number
- CN202520013508.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-03
- Publication Date
- 2026-01-06
- Estimated Expiration
- 2035-01-03
AI Technical Summary
Existing methods for detecting vomitoxin (DON) in buffalo milk suffer from low sensitivity, and rapid screening methods are easily affected by environmental factors, resulting in a high false positive rate and making it difficult to achieve large-scale on-site rapid detection.
A highly sensitive immunoprobe was prepared by combining colorimetric and Raman dual-signal SERS technology with immunochromatographic analysis. The electrostatic interaction between Au@4-MBA@Ag NPs surface molecules and DON antibody was utilized. The detection accuracy and sensitivity were improved by high-speed centrifugation and blocking.
This method enables rapid and accurate detection of DON in buffalo milk, improving detection sensitivity and reducing false positive rate, making it suitable for large-scale on-site testing.
Smart Images
Figure CN223770219U_ABST
Abstract
Description
Technical Field
[0001] This utility model relates to the technical field of buffalo milk detection, specifically to a buffalo milk fungal toxin detection device. Background Technology
[0002] Currently, methods for detecting dopamine toxicant (DON) mainly fall into two categories: instrumental analysis and rapid screening methods. While instrumental analysis offers advantages such as high sensitivity and precise quantitative analysis, it requires expensive equipment and significant time and expertise from professionals in the laboratory for complex procedures. Therefore, this method is unsuitable for large-scale, rapid on-site detection of DON. Rapid screening methods are widely used due to their short detection time, low cost, and lack of need for professional personnel. However, the results of rapid screening are susceptible to environmental factors, exhibiting high false positive rates and poor repeatability. Furthermore, the sensitivity of this method needs improvement when detecting low concentrations of DON. Summary of the Invention
[0003] The purpose of this invention is to provide a buffalo milk fungal toxin detection device to solve the above-mentioned defects caused by the prior art.
[0004] A buffalo milk mycotoxin detection device includes a detection stage, a detection tray, and an electron microscope sputtering apparatus. The detection tray is provided on the outer side of the detection stage. The detection tray stores the components used for detection and can be moved on the detection stage according to usage requirements to facilitate the storage and retrieval of the detection instruments. A configuration mechanism is provided directly above the detection stage. The configuration mechanism rotates and repositions multiple sets of glass beakers to facilitate the dispensing of the prepared detection solution.
[0005] Preferably, the configuration mechanism includes a dispensing plate, a groove, a positioning shaft, a storage tank, a glass beaker, and a bearing plate. The dispensing plate has a groove circumferentially formed on its outer side, and a glass beaker is connected to the outer side of the groove. The positioning shaft is connected to the top of the dispensing plate. The bearing plate is connected to the bottom of the dispensing plate, and a testing platform is connected to the bottom of the bearing plate. The glass beaker is connected to the outer side of the groove, and the storage tank is positioned directly above the positioning shaft.
[0006] Preferably, the dispensing plate is connected to the outside of the glass beaker through a groove annularly formed at its top.
[0007] Preferably, the testing tray includes a drawer, EP test tubes, silicon wafer disks, NC films, an electron microscope sputtering apparatus, and a microplate. A drawer is provided on one side of the testing tray, and multiple sets of EP test tubes are arranged inside the drawer. A silicon wafer disk is provided on one side of the EP test tubes, and multiple sets of NC films are arranged on one side of the silicon wafer disk. A microplate is provided on the other side of the NC films. The electron microscope sputtering apparatus is installed on the outside of the testing stage.
[0008] Preferably, the testing tray is connected to the testing table via a through-connected guide rod.
[0009] Preferably, the testing platform is connected to the bottom end of the liquid distribution plate via a bearing plate at the top.
[0010] Compared with the prior art, the present invention has the following advantages:
[0011] 1. In response to the current situation where the sensitivity of on-site screening products for vomitoxin in buffalo milk is not high, this project introduces SERS technology, characterized by high sensitivity, into LFIA and develops a colorimetric and Raman dual-signal mode SERS immunochromatographic analysis method for the rapid detection of DON in buffalo milk. This method retains the advantages of convenient and rapid immunochromatography, while significantly improving the detection sensitivity by utilizing SERS technology, and the colorimetric and Raman dual-signal mode helps to improve the detection accuracy.
[0012] 2. The effective adsorption and coupling of the antibody is achieved through electrostatic interaction between the carboxyl group (-COOH) of the 4-mercaptobenzoic acid (4-MBA) molecule on the surface of Au@4-MBA@Ag NPs and the amino group (-NH2) of the DON-specific monoclonal antibody. This process ensures the directional binding of the DON antibody to the nanoparticle surface, forming an immunoprobe. Subsequently, a 10% bovine serum albumin (BSA) solution is added for blocking, saturating the unbound sites on the nanoparticle surface, effectively reducing non-specific binding in subsequent detection, and improving the specificity and sensitivity of the probe. The immunoprobe is then purified by high-speed centrifugation, resuspended in an appropriate reconstitution solution, and stored at 4°C for later use to ensure its stability and activity. Attached Figure Description
[0013] Figure 1 This is a three-dimensional structural diagram of the present invention.
[0014] Figure 2 This is a schematic diagram of the testing platform structure from below in this utility model.
[0015] Figure 3 This is a schematic diagram of the internal structure of the drawer in this utility model.
[0016] Figure 4 This is a front view structural diagram of the testing platform in this utility model.
[0017] Figure 5 This is a schematic diagram of the detection process in this utility model.
[0018] in:
[0019] 1. Testing platform; 2. Dispensing tray; 3. Groove; 4. Positioning shaft; 5. Configuration mechanism; 6. Storage tank; 7. Glass beaker; 8. Testing tray; 9. Guide rod; 10. Bearing disc; 11. Drawer; 12. EP test tube; 13. Silicon wafer disc; 14. NC membrane; 15. Electron microscopy sputtering instrument; 16. Microplate. Detailed Implementation
[0020] To make the technical means, creative features, objectives and effects of this utility model easier to understand, the present utility model will be further described below in conjunction with specific embodiments.
[0021] like Figures 1 to 5 As shown, a buffalo milk mycotoxin detection device includes a detection stage 1, a detection tray 8, and an electron microscope sputtering device 15. The detection tray 8 is provided on the outer side of the detection stage 1. The detection tray 8 stores the detection components and can be moved on the detection stage 1 according to the usage requirements, which facilitates the storage and retrieval of the detection instruments. A configuration mechanism 5 is provided directly above the detection stage 1. The configuration mechanism 5 rotates and repositions multiple sets of glass beakers 7, thereby facilitating the dispensing of the prepared detection solution.
[0022] In this embodiment, the configuration mechanism 5 includes a dispensing plate 2, a groove 3, a positioning shaft 4, a storage tank 6, a glass beaker 7, and a bearing plate 10. The outer side of the dispensing plate 2 is provided with a groove 3, and the glass beaker 7 is connected to the outer side of the groove 3. The positioning shaft 4 is connected to the top of the dispensing plate 2. The bottom end of the dispensing plate 2 is connected to the bearing plate 10, and the bottom end of the bearing plate 10 is connected to the detection platform 1. The outer side of the groove 3 is connected to the glass beaker 7, and the storage tank 6 is provided directly above the positioning shaft 4.
[0023] In this embodiment, the dispensing plate 2 is connected to the outside of the glass beaker 7 through a groove 3 formed in an annular shape at the top. The dispensing plate 2 is used to position multiple sets of glass beakers 7 to prevent collisions between the glass beakers 7 during the configuration process.
[0024] In this embodiment, the testing tray 8 includes a drawer 11, EP test tubes 12, silicon wafer trays 13, NC films 14, an electron microscope sputtering device 15, and a microplate 16. A drawer 11 is provided on one side of the testing tray 8, and multiple sets of EP test tubes 12 are arranged inside the drawer 11. A silicon wafer tray 13 is provided on one side of the EP test tubes 12, and multiple sets of NC films 14 are arranged on one side of the silicon wafer tray 13. A microplate 16 is provided on the other side of the NC film 14. The electron microscope sputtering device 15 is installed on the outside of the testing stage 1. The materials are centrally stored using the drawer 11 inside the testing tray 8.
[0025] In this embodiment, the detection tray 8 is connected to the detection table 1 through a through-connected guide rod 9. The guide rod 9 allows for lateral displacement of the detection tray 8, thereby facilitating dynamic adjustment of the position of the detection tray 8.
[0026] In this embodiment, the detection platform 1 is connected to the bottom end of the dispensing plate 2 via a bearing plate 10 at the top. The bearing plate 10 positions the bottom end of the dispensing plate 2, facilitating the rotation and repositioning of the dispensing plate 2.
[0027] In practical applications, this buffalo milk fungal toxin detection device includes the following functions:
[0028] Step 1: Pour 70 mL of boiling water into multiple glass beakers 7 set at the top of the separatory plate 2, then add 5 mL of 1% sodium citrate, stir, and after the solution boils again, add 0.5 mL of 1% tetrachloroauric acid solution; after stirring for 60 s, add 2.5 mL of 0.1 M Tris solution, wait for 5 min, add 0.5 mL of 1% tetrachloroauric acid solution and repeat once, stop heating after the reaction is complete; after 15 min, stop stirring, let it cool naturally to room temperature, and finally obtain a wine-red AuNPs solution, which is stored at 4℃ in the dark for later use.
[0029] 2. Preparation of gold-core silver-shell nanoparticles: Synthesis of Au@4-MBANPs
[0030] Add 40 μL of 10 mM 4-MBA ethanol solution to a 10 mL AuNPs solution under stirring, and react at room temperature for 1 h to obtain 4-MBA modified gold nanoparticles (Au@4-MBANPs).
[0031] (2) Synthesis of Au@4-MBA@AgNPs
[0032] Aliquot 3 mL of Alu@4-MBANPs solution into three EP tubes (I-2), centrifuge at 10,000 rpm for 15 min, discard the supernatant, and resuspend twice with ultrapure water. Place the resuspended solution in a clean flask, heat and stir, and add 0.3 mL of 1% sodium citrate when the solution boils. When the solution boils again, add 60 μL of AgNO3 solution, and the solution gradually turns yellow. Continue boiling for 15 min, then stop heating, stir, and cool to room temperature.
[0033] A 2 mL Au@4-MBANPs solution was added to six clean glass beakers 7. 0, 20, 40, 60, 80, and 100 μL of 10 mL AgNO3 solution were added sequentially to each beaker to synthesize Au@4-MBA@AgNPs of different thicknesses. 10 μL of each sample was dropped onto a silicon wafer 13 and characterized using a Raman spectrometer with an excitation wavelength of 785 nm, a power of 30 mW, and an integration time of 2 s. Five points were randomly selected in the center region of the T-line of each sample for measurement, and the average value was taken as the final Raman signal intensity. Au@4-MBA@AgNPs with the highest Raman signal intensity were selected for subsequent experiments. 1 mL of Au@4-MBA@AgNPs was placed in a 1.5 mL EP tube 12, and an appropriate amount of 0.1 Mk2CO3 solution was added to adjust the solution to the optimal pH value. The mixture was mixed and allowed to stand for 5 min. Then, DON antibody was added to the solution, mixed, and incubated at room temperature for 1 h. 100 μL of 10% BSA solution was added, mixed, and blocked at room temperature for 1 h. After the reaction was complete, the mixture was centrifuged at 12000 rpm for 8 min, the supernatant was discarded, and the precipitate was resuspended in 200 μL of reconstitution solution and stored at 4 °C for later use.
[0034] Step 2: To achieve the best detection effect of the test strip, different sample diluents were used as negative samples and positive samples were prepared using different sample diluents for testing the test strip. The sample diluents included: PBST (10mM pH=7.4, containing 0.05% Tween-20), PBST (10mM pH=7.4, containing 0.01% Tween-20), PB (10mM pH=7.4), PB (10mM pH=7.4) plus 0.05% Tween-20, and PBS (10mM pH=7.4). 80 μL of each sample diluent (positive sample DON concentration is 0.01 μg / μL) was added to 20 μL of the immunoprobe and mixed thoroughly. The test strip was inserted into a 96-well microplate 16 containing the above mixture, and the color development of the test strip was observed to determine the optimal sample diluent. Take 1 mL of LAu@4-MBA@AgNPs and place it in six 1.5 mL EP tubes (12 in total). Add 0, 1, 2, 3, 4, and 5 μL of 0.1 M K2CO3 solution to each tube to prepare immunoprobes. Assemble test strips and perform Raman signal detection on negative and positive samples respectively. Determine the optimal labeling pH value based on the detection results. Take 6 mL of LAu@4-MBA@AgNPs solution and place it in six 1.5 mL EP tubes (12 in total). Add an appropriate amount of 0.1 M K2CO3 solution to each tube, mix well, and let stand for 5 min. Add 2, 4, 6, 8, and 10 μg of DON antibody to each tube to prepare immunoprobes. Assemble test strips and perform Raman signal detection on negative and positive samples respectively. Determine the optimal amount of labeled antibody based on the detection results.
[0035] Step 3: Dilute DON-BSA to 2 mg / mL with PBS, and dilute goat anti-mouse IgG polyclonal antibody to 2 mg / mL with 10 mMPB buffer (pH = 7.4). Then, using an electron microscope sputtering system 15, fix DON-BSA and goat anti-mouse IgG polyclonal antibody onto NC membrane 14 as T-lines and C-lines, respectively, and dry at 37°C for 3 hours. Attach NC membrane 14 to the center of a PVC base plate, with the C-line closer to the shorter side of the base plate. Place absorbent paper above the C-line, overlapping it with NC membrane 14 by 1–2 mm. Place the sample pad below the T-line, overlapping it with NC membrane 14 by 1–2 mm. Cut the test strips using a strip cutter, dry at 4°C, and store in the dark for later use.
[0036] Five NC membranes 14 were taken, and DON-BSA at concentrations of 0.1, 0.25, 0.5, 1.0, and 2.0 mg / mL were coated onto the NC membranes 14 using an electron microscope sputtering apparatus 15 to serve as T lines. Test strips were assembled, and the test strips were inserted into positive and negative samples for detection, respectively, and the color development of the test strips was observed. Raman signals were detected, and the Raman signal value of the strongest signal peak of 4-MBA (wavelength 1078 cm⁻¹) was analyzed to determine the concentration of antibody at the T line.
[0037] Five NC membranes 14 were used, and goat anti-mouse IgG polyclonal antibodies at concentrations of 0.5, 1.0, 1.5, and 2.0 mg / mL were immobilized on the membranes using an electron microscope sputtering apparatus 15 to serve as C-lines. Test strips were assembled, and the strips were inserted into positive and negative samples for testing, observing the color development. Raman signals were detected, and the Raman signal value at the strongest peak of 4-MBA (wavelength 1078 cm⁻¹) was analyzed to determine the concentration of the C-line antibody. The negative sample test strips were used to capture the T-line signal value every 2 minutes using a colloidal gold reader. The time corresponding to the stable T-line signal value was recorded as the optimal detection time, and the data were plotted and analyzed. DON solution was serially diluted to obtain DON dilutions of 10, 50, 100, 500, 1000, and 5000 μg / L. 80 μL of each dilution was placed in 16 wells of a 96-well microplate, and 20 μL of immunoprobe was added to each well to form a positive sample. Test strips from the same batch were inserted into the positive samples, with a negative control group included. After 16 minutes, the results were observed visually, and Raman signal values were measured. The Raman signal values of the 4-MBA characteristic peak were analyzed.
[0038] Step 4: Divide the 21 test strips into three groups: negative control, positive control, DON (zearalenone), ZEN (OTAA (ochratoxin A), FB1 (fumonisin B1), T-2 (T-2 toxin), and AFB1 (aflatoxin B1). Add three concentrations of the test sample (low (0.1 LOD), medium (LOD), and high (10 LOD)) to each of the 21 96-well plates, with three replicates for each concentration. Add 20 μL of the immunoprobe to each 96-well plate, mix well, and insert the test strip. After reacting for 16 min, observe the results visually and acquire Raman spectra. Analyze the signal value of the 4-MBA characteristic peak. Store the test strips in a light-proof container with desiccant at 4℃ and 25℃, respectively. Samples are taken monthly (1, 2, and 3 months), and the test strips stored at 4℃ and 25℃ are used to test negative and positive standards, respectively. Observe and record the T-line intensity to evaluate its stability.
[0039] Therefore, the above-disclosed embodiments are merely illustrative in all respects and are not the only ones. All modifications within the scope of this utility model or its equivalents are included in this utility model.
Claims
1. A device for detecting mycotoxins in buffalo milk, characterized by: Including detection platform (1), detection tray (8) and electron microscope gold spraying instrument (15), the outer side of the detection platform (1) is provided with detection tray (8), the detection tray (8) is used for accommodating the detection of zero component, and the detection tray (8) is moved on the detection platform (1) according to the use demand, so as to facilitate the storage and taking of the detection instrument, the upper side of the detection platform (1) is provided with configuration mechanism (5), the configuration mechanism (5) rotates and transposes a plurality of glass beakers (7), so as to facilitate the dispensing treatment of the configured detection solution.
2. The device for detecting mycotoxins in buffalo milk according to claim 1, characterized in that: The configuration mechanism (5) comprises a liquid distribution disc body (2), a groove (3), a positioning shaft (4), a liquid storage tank body (6), a glass beaker (7) and a bearing disc body (10), the outer side of the liquid distribution disc body (2) is annularly provided with a groove (3), the outer side of the groove (3) is connected with a glass beaker (7), the upper side of the liquid distribution disc body (2) is connected with a positioning shaft (4), the bottom end of the liquid distribution disc body (2) is connected with a bearing disc body (10), the bottom end of the bearing disc body (10) is connected with a detection platform (1), the outer side of the groove (3) is connected with a glass beaker (7), the upper side of the positioning shaft (4) is provided with a liquid storage tank body (6).
3. The device for detecting mycotoxins in buffalo milk according to claim 2, characterized in that: The liquid distribution disc body (2) is connected with the outer side of the glass beaker (7) through the groove (3) annularly arranged at the top end.
4. The device for detecting mycotoxins in buffalo milk according to claim 1, characterized in that: The detection tray (8) comprises a drawer (11), an EP test tube (12), a silicon wafer disc (13), an NC membrane body (14), an electron microscope gold spraying instrument (15) and a microwell plate (16), one side of the detection tray (8) is provided with a drawer (11), the inside of the drawer (11) is provided with a plurality of EP test tubes (12), one side of the EP test tube (12) is provided with a silicon wafer disc (13), one side of the silicon wafer disc (13) is provided with a plurality of NC membrane bodies (14), the other side of the NC membrane body (14) is provided with a microwell plate (16), and the electron microscope gold spraying instrument (15) is installed on the outer side of the detection platform (1).
5. The device for detecting mycotoxins in buffalo milk according to claim 4, characterized in that: The detection tray (8) is connected with the detection platform (1) through the guide rod (9) penetratingly connected.
6. The device for detecting mycotoxins in buffalo milk according to claim 1, characterized in that: The detection platform (1) is connected with the bottom end of the liquid distribution disc body (2) through the bearing disc body (10) arranged at the top end.