CN110887964A - Sensitive probe, method for detecting tetracycline and application - Google Patents
Sensitive probe, method for detecting tetracycline and application Download PDFInfo
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- CN110887964A CN110887964A CN201911081355.7A CN201911081355A CN110887964A CN 110887964 A CN110887964 A CN 110887964A CN 201911081355 A CN201911081355 A CN 201911081355A CN 110887964 A CN110887964 A CN 110887964A
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- tetracycline
- molybdenum disulfide
- sensitive probe
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- monoclonal antibody
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9446—Antibacterials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses a sensitive probe, a method for detecting tetracycline and application of the sensitive probe, and the method comprises a signal carrier and a tetracycline monoclonal antibody, wherein the signal carrier is a molybdenum disulfide nanosheet, and the particle size of the molybdenum disulfide nanosheet is 450-550 nm, 200-300 nm and 40-60 nm. The two-dimensional molybdenum disulfide nanosheet is used as a signal carrier to construct a probe in immunoassay, the control of a very limited amount of antibodies is the key for improving the sensitivity, the specific surface area of molybdenum disulfide is large, functional groups are easy to modify on the surface, the adsorption capacity is strong, the biocompatibility is good, a small amount of antibodies are used for marking molybdenum disulfide as a detection probe, the high-sensitivity target object detection is realized, the detection limit of naked eyes can be as low as 0.023ng/mL, compared with the sensitivity of a traditional gold-labeled test strip, the sensitivity is at least improved by 100 times, and the method can be successfully applied to tetracycline detection in milk, honey and beef samples.
Description
Technical Field
The invention belongs to the field of biological detection, relates to a sensitive probe, a tetracycline detection method and application, and particularly relates to a sensitive probe which takes molybdenum disulfide nanosheets as a signal carrier and marks monoclonal antibodies, and a test strip and the sensitive probe can be used for rapidly detecting tetracycline antibiotics together and application.
Background
At present, the classical methods for qualitatively and quantitatively detecting tetracycline at home and abroad include liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), gas chromatography-mass spectrometry (GC-MS) and High Performance Liquid Chromatography (HPLC), although the methods have high accuracy, the instrument price is high, the pretreatment process is complex, the analysis speed is slow, the professional requirement is high, and the method is not suitable for large-batch samples or on-site rapid detection. In addition, there are also commonly used enzyme-linked immunosorbent assays (ELISA), chemiluminescence assays. The enzyme-linked immunosorbent assay can process a plurality of samples at one time, has high sensitivity, needs higher professional knowledge of operators, and is not beneficial to popularization and use. The chemiluminescence method is easy to realize automation, but has high environmental requirement and is not beneficial to field use.
Compared with the prior art, the immunochromatography method has the advantages of good stability, simple operation, no need of other instruments and equipment, high detection speed, visual and reliable result and suitability for on-site rapid detection. However, the low sensitivity of conventional gold colloidal-based test strips still prevents their widespread use, particularly in trace analysis.
Disclosure of Invention
Aiming at the defects and shortcomings in the prior art, the invention provides a sensitive probe, a tetracycline detection method and application for solving the technical problem of low sensitivity of immunochromatography detection.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
a sensitive probe comprises a signal carrier and a tetracycline monoclonal antibody, wherein the signal carrier is a molybdenum disulfide nanosheet, and the particle size of the molybdenum disulfide nanosheet is 450-550 nm, 200-300 nm and 40-60 nm.
Specifically, the preparation method of the sensitive probe comprises the steps of taking a molybdenum disulfide nanosheet as a signal carrier and adding a tetracycline monoclonal antibody to obtain the probe.
Further, the method for preparing the sensitive probe comprises the following steps:
(1) preparing molybdenum disulfide nanosheets: carrying out ultrasonic treatment on a mixed aqueous solution containing molybdenum disulfide and sodium cholate, and carrying out classification treatment by adopting a differential zone belt centrifugation method to obtain the molybdenum disulfide-sodium cholate composite material;
(2) preparing a sensitive probe: and (2) adding the tetracycline monoclonal antibody into the molybdenum disulfide nanosheet solution obtained in the step (1), mixing, reacting and centrifuging to obtain the tetracycline nano-antibody.
Further, in the step (1), the concentration ratio of the molybdenum disulfide to the sodium cholate is 1 (0.2-0.4).
Further, in the step (2), the concentration ratio of the molybdenum disulfide nanosheet to the tetracycline monoclonal antibody is 1: 0.001-0.003, the molybdenum disulfide nanosheet and the tetracycline monoclonal antibody are mixed and vibrated for 30-60 minutes, then 100 mu L of 10% bovine serum albumin is added for reaction for 30 minutes, and the reaction is sealed for 2-2.5 hours.
A method for detecting tetracycline comprises immersing a test strip into a test strip containing a sensitive probe of the invention for detection.
Furthermore, the test strip comprises a lining plate, a nitrocellulose membrane is attached to the lining plate, one end of the nitrocellulose membrane covers the water absorption pad, the other end of the nitrocellulose membrane sequentially covers the sample pad and the combination pad, and a detection line and a quality control line are transversely arranged on the non-covered surface of the nitrocellulose membrane; the detection line is sprayed with a tetracycline-bovine serum albumin conjugate, and the quality control line is sprayed with goat anti-mouse immunoglobulin.
Further, the preparation method of the tetracycline-bovine serum albumin conjugate sprayed on the detection line comprises the following steps: spraying the 1mg/mL tetracycline-bovine serum albumin conjugate solution on a detection line at a scribing rate of 1 mu L/cm to obtain a detection line;
the preparation method of the quality control line sprayed with the goat anti-mouse immunoglobulin comprises the following steps: the goat anti-mouse monoclonal antibody coating solution of 1mg/mL is sprayed on the nitrocellulose membrane at the speed of 1 muL/cm to form a quality control line, and the distance between the detection line and the quality control line is 5 mm.
The sensitive probe is used for detecting tetracycline in milk, honey or beef.
The method for detecting the tetracycline is used for detecting the tetracycline in milk, honey or beef.
Compared with the prior art, its advantage lies in with positive effect:
(1) the method is characterized in that a two-dimensional molybdenum disulfide nanosheet is used as a signal carrier to construct a probe in immunoassay for the first time, and the control of a very limited amount of antibodies is the key for improving the sensitivity, so that a cheap, sensitive, portable and quick-reading analysis system is developed for detecting tetracycline antibiotics by actual samples of milk, honey and beef;
(2) the prepared molybdenum disulfide has large specific surface area, easy modification of functional groups on the surface, strong adsorption capacity and good biocompatibility, a small amount of antibody is used for marking the molybdenum disulfide as a detection probe, high-sensitivity target object detection is realized, the naked eye detection limit can be as low as 0.023ng/mL, compared with the sensitivity of a traditional gold-labeled test strip, the sensitivity is at least improved by 100 times, the specificity is good, the whole process is finished within 11 minutes, and the method can be successfully applied to milk, honey and beef sample detection, which shows that the food matrix has almost no influence on the test strip. The novel two-dimensional material-based nanoprobe shows great application potential in the aspect of detecting antibiotic residues.
Drawings
FIG. 1 is a transmission electron microscope representation of a molybdenum disulfide nanosheet signal carrier prepared in accordance with the present invention;
FIG. 2 is a schematic diagram of a hierarchical molybdenum disulfide nanosheet signal carrier prepared according to the present invention;
FIG. 3 is an assembly diagram of an immunochromatographic test strip for rapid detection of tetracycline in accordance with the present invention;
FIG. 4 is a graph showing the results of 1) in example 3;
FIG. 5 is a graph showing the results of 2) in example 3;
FIG. 6 is a graph showing the results of example 4;
the following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
Detailed Description
In order to solve the problem of low sensitivity of immunochromatography detection, the invention constructs a novel two-dimensional signal carrier, and reduces the dosage of antibodies while ensuring the strength of a signal substance, thereby improving the sensitivity of an analysis system, and having important significance and application value for monitoring tetracycline antibiotics in animal-derived food. In order to obtain the best measurement performance, the inventor selects the nano material which is most suitable as the signal carrier, researches the best preparation method of the signal carrier coupling antibody and determines the best analysis condition. The finally prepared test strip is used for detecting tetracycline (TET), is particularly applied to the detection of tetracycline in milk, honey and beef, and has the characteristics of reliability, sensitivity, stability, portability, quickness and simplicity in operation.
As a novel signal carrier, the molybdenum disulfide nanosheet not only has proper size, good uniformity, dispersibility and stability, but also has unique biocompatibility and strong adsorption capacity, so that the molybdenum disulfide nanosheet is endowed with ultrahigh biomolecule loading capacity. Compared with materials such as carbon nanotubes, manganese dioxide nanoflowers, metal organic frameworks and the like, the synthesis is not limited by strict conditions, the chemical properties are stable, and the combination method with the antibody is simple. The invention synthesizes molybdenum disulfide nanosheets by utilizing an ultrasonic-assisted liquid phase stripping method, nanosheets with different sizes can be distinguished by a differential zone centrifugation method (DZC), larger particles can be settled faster than smaller particles, and thus, stripped MoS can be obtained2The nano sheets are distinguished according to the size, and a more uniform product is obtained.
The method for preparing the sensitive probe comprises the following steps:
(1) preparing molybdenum disulfide nanosheets: carrying out ultrasonic treatment on a mixed aqueous solution containing molybdenum disulfide and sodium cholate, and carrying out classification treatment by adopting a differential zone belt centrifugation method to obtain the molybdenum disulfide-sodium cholate composite material; the concentration ratio of the molybdenum disulfide to the sodium cholate is 1 (0.2-0.4);
(2) preparing a sensitive probe: and (2) adding the tetracycline monoclonal antibody into the molybdenum disulfide nanosheet solution obtained in the step (1), mixing, reacting and centrifuging to obtain the tetracycline nano-antibody. The concentration ratio of the molybdenum disulfide nanosheet to the tetracycline monoclonal antibody is 1 (0.001-0.003), the molybdenum disulfide nanosheet and the tetracycline monoclonal antibody are mixed and vibrated for 30-60 minutes, then 100 mu L of 10% bovine serum albumin is added for reaction for 30 minutes, and the reaction is sealed for 2-2.5 hours.
The working principle of the invention is as follows: immunoassays for non-protein small molecule compounds are based on the principle of competition, i.e., the analyte in the sample competes with the antigen immobilized on the detection line for binding to the signal-labeled antibody. The color intensity of the test area depends on the accumulation of signal material. During the test, as the concentration of tetracycline in the sample increases, more of the binding sites of the probe are occupied by tetracycline, resulting in a decrease in the number of probes that bind to the antigen on the test line, and the test line gradually fades until it completely disappears. Regardless of the tetracycline concentration in the sample, excess probe will bind to the goat anti-mouse immunoglobulin on the control line, so that the control line will always develop color. The probe constructed by the method meets the requirement of strong signal labeling of extremely limited antibodies, so that the probe needs less antibodies under the same signal material color development effect, and the key point of improving the sensitivity of competitive immunoassay is. When a weak positive sample is detected, the common gold-labeled antibody probe is not completely competed, and the rest can be combined with the antigen on the detection line, and a red negative result is shown and is expressed as a negative test result. The test strip provided by the invention has the advantages that few antibodies are easy to be fully combined by an analyte, no signal is displayed on a detection line, and a positive result is represented. This indicates that the sensitivity of the probe is far superior to that of the conventional probe strip.
Differential zone centrifugation: the density gradient is prepared by using a certain low-molecular solute in a centrifuge tube in advance, and the centrifugal operation is carried out after the feed liquid to be treated is added on the density gradient. The maximum density in the density gradient of the differential zone centrifugation is less than the density of a target product to be separated, in the centrifugation operation, various components in the feed liquid are settled in the density gradient at different speeds, and respective zones are formed according to the difference of the settlement coefficients of the components. After a certain time, different zones are respectively drawn from the centrifugal tube to obtain each purified component.
The immunochromatographic test strip is composed of five parts, wherein a nitrocellulose membrane, a sample pad, a combination pad and an absorption pad are sequentially attached to a lining plate, wherein tetracycline-bovine serum albumin conjugate (TET-BSA) and goat anti-mouse immunoglobulin (IgG) are coated on the nitrocellulose in a marking manner and are respectively used as a detection line (T) and a control line (C).
The experimental reagents used in the invention are all obtained from markets, no further treatment is carried out, and detection instruments and equipment and the like are all common instruments.
Example 1:
following the above technical scheme, the present embodiment provides a sensitive probe MoS2the-Abs and the preparation method comprise using molybdenum disulfide nanosheet as a signal carrier, and combining with tetracycline monoclonal antibody to form a sensitive probe, and specifically comprise the following steps:
(1) preparation of Signal Carrier
Will contain 2g of MoS2The powder and a 400mL mixed aqueous solution of 0.6g sodium cholate were sonicated at room temperature for 20h, the ultrasound generated cavitation bubbles, which helped the high energy jet, breaking up the bulk crystals, forming a greenish-black dispersion. MoS2The crystals are longitudinally stripped into 2D thin nanosheets which are stably dispersed in the surfactant solution.
MoS can be separated by differential zone centrifugation2And removing the active agent. Classified MoS2The sizes are respectively as follows: 450-550 nm (L-MoS)2)、200～300nm(M-MoS2) And 40 to 60nm (S-MoS)2) The transmission electron microscope results are shown in FIG. 1, wherein A is a block MoS2B is L-MoS2C is M-MoS2D is S-MoS2。
The specific procedure of differential zone centrifugation was as follows, 20mL of ultrasonically stripped grayish green MoS2The nanosheet solution was centrifuged at low speed for 30min and then 18mL of greenish black supernatant was collectedAnd (4) removing the large-size nanosheets. The supernatant was centrifuged at medium speed for 30min and the precipitate was collected for size screening. For more complete removal of the surfactant, the precipitate was resuspended by high speed centrifugation, ultrasonically dispersed in pure water and centrifuged at 12000rpm for 20min, and the precipitate was collected and dispersed in a defined volume of ultrapure water to prepare pure layered MoS2An aqueous solution. Just the L-MoS2For the samples, the low and medium speeds were 1500 and 3000rpm, respectively. For M-MoS2Samples, low and medium speed 3000 and 6000rpm respectively. For S-MoS2The samples, low and medium speed, were 6000 and 12000rpm, respectively. The operational flow is shown in FIG. 2.
(2) Signal carrier conjugated antibody
Add 2. mu.L of 1mg/mL tetracycline antibody to 1mL of MoS in step 12Shaking (1mg/mL) for 30min to bind tetracycline antibody to the carrier, and then adding 100. mu.L of 10% bovine serum albumin dropwise into MoS2Mixing the mixture uniformly, reacting for 30min, and standing at 4 ℃ for 2h to seal MoS2Redundant sites on the surface. The complex was then centrifuged at 10000rpm for 30min to remove excess BSA and washed three times with deionized water to collect purified MoS2Abs, i.e. 3 sensitive probes of different sizes. The precipitate obtained finally was dispersed in deionized water and stored at 4 ℃ to maintain stability and bioactivity for later use.
The tetracycline detecting probe used in examples 2-4 described below was prepared in example 1.
Example 2: preparation of immunochromatography test strip for rapidly detecting tetracycline antibiotics
The preparation method of the high-sensitivity immunochromatographic test strip for rapidly detecting tetracycline comprises the following steps:
the immunochromatographic test strip sensor consists of five parts, including a lining plate, a Nitrocellulose (NC) membrane, a combination pad, a sample pad and an absorption pad. First, the conjugate pad and the sample pad made of glass fiber were soaked in 2% bovine serum albumin to close the micropores, and dried at 37 ℃ overnight. Next, 1mg/mL of antigen (TET-BSA tetracycline-bovine serum albumin conjugate) and 1mg/mL of goat anti-mouse immunoglobulin were coated on the NC membrane as a detection (T) line and a control (C) line at a streaking rate of 1. mu.L/cm, respectively, with a distance of 5mm between the detection (T) line and the control (C) line. The NC film, conjugate pad, sample pad and absorbent pad were attached to the backing plate in sequence, overlapping 1-2mm, see fig. 3. The assembled strips were finally cut into test strips and then stored in a desiccator at room temperature until use.
Example 3: performance evaluation of immunochromatographic test strip for rapidly detecting tetracycline
The performance evaluation of the immunochromatographic test strip for rapidly detecting tetracycline as described above:
(1) sensitivity measurement
Dissolving tetracycline in deionized water, and continuously diluting to obtain test solutions with different concentrations in a range of 0.012-3 ng/mL, wherein the deionized water is a blank control. Immersing a sample pad of the test strip into a solution containing MoS2Visual results were observed within 11min after immunochromatography in 100. mu.L of test solution for Abs probe. For sensitivity evaluation, these standard solutions were tested by a common test strip and a newly developed test strip, respectively. When the T-line was visibly lighter than the negative control bar, the corresponding minimum tetracycline concentration was defined as the Visual Detection Limit (VDL), and when the T-line disappeared completely, the corresponding minimum concentration was considered as the threshold concentration.
The test results are shown in fig. 4, when the tetracycline content in the solution is low, a color band can be clearly observed on the test strip, and the intensity of the T line gradually decreases with the increase of the tetracycline concentration. For L-MoS2In other words, the color of the T line started to lighten and disappeared at 0.75ng/mL at a concentration of 0.023 ng/mL. The corresponding results were further confirmed by recording the light intensity of the strip at the T-line using a strip reader, which exhibited a good linear relationship (R) in the range of 0.012 to 0.75ng/mL20.974), the naked eye visual detection limit reaches 0.023 ng/mL. M-MoS2The line disappears at 1.5ng/mL, and the visual detection limit is 0.047 ng/mL; S-MoS2The line disappeared at 1.5ng/mL, and the visual detection limit was 0.094 ng/mL. The traditional gold-labeled test strip is used for detecting tetracycline, the naked eye visual detection limit is 3ng/mL, and the result shows that MoS2The constructed immunochromatography test strip has sensitivityThe method is far superior to the traditional gold marking method.
(2) Specific assay
Specificity was evaluated using Chlortetracycline (CTE), Oxytetracycline (OXY), ceftriaxone sodium (CTR), Azithromycin (AZM), florfenicol (FF), Streptomycin (STR), Neomycin (NEO), Ampicillin (AMP), Amoxicillin (AML), Erythromycin (EM) and Kanamycin (KM) as interfering substances, which were set at a final concentration of 100ng/mL, which was much greater than the final concentration of TET, 1.5 ng/mL. All experiments were performed in triplicate.
The results are shown in FIG. 5, except for tetracycline antibiotics, other antibiotics were not shown to disappear at the T-line. The excellent results are attributed to the antibody having a good specific recognition function for the target. The invention can detect tetracycline antibiotics with high specificity.
Example 4: application of immunochromatography test strip for rapidly detecting tetracycline
Rapid detection of L-MoS of tetracycline as described above2The application of the immunochromatographic test strip is that,
milk, honey and beef were used as actual samples. Tetracycline was added to the samples individually to achieve a continuous concentration of 3 to 0.012ng/mL (or ng/g). After mixing well, the spiked samples were kept at 4 ℃ overnight. For milk and beef samples, 1mL of spiked milk or 1g of spiked beef was placed in 2mL of 3% trichloroacetic acid and vortexed for 5 min. The mixture was then centrifuged at 10000rpm for 20min to remove protein, fat and other sample impurities. Finally, the collected supernatant was analyzed. For honey samples, 1g of the sample was mixed with 5mL PBS for 5min using a vortex mixer and centrifuged at 10000rpm for 20min and the collected supernatant was analyzed. The sample pad of the test strip was immersed in 100 μ L of test solution using PBS as a blank and the sample flowed through the strip by capillary action, allowing visualization within 15 min.
The detection result is shown in figure 6, the naked eye visual detection limit of the test paper strip in the milk, honey and beef detection solution is 0.023ng/mL, which is consistent with the detection limit of the standard tetracycline solution, and shows that the sample matrix has no or little influence on the analysis capability of the test paper strip test. Therefore, due to excellent sensitivity, specificity and applicability, the developed immunochromatographic test strip can well meet the requirement of rapidly screening tetracycline antibiotics in actual samples.
Claims (10)
1. The sensitive probe is characterized by comprising a signal carrier and a tetracycline monoclonal antibody, wherein the signal carrier is a molybdenum disulfide nanosheet, and the particle size of the molybdenum disulfide nanosheet is 450-550 nm, 200-300 nm and 40-60 nm.
2. The sensitive probe of claim 1, wherein the preparation method of the sensitive probe comprises the steps of taking a molybdenum disulfide nanosheet as a signal carrier and adding a tetracycline monoclonal antibody to obtain the probe.
3. The sensitive probe of claim 1 or 2, wherein the method for preparing the sensitive probe comprises:
(1) preparing molybdenum disulfide nanosheets: carrying out ultrasonic treatment on a mixed aqueous solution containing molybdenum disulfide and sodium cholate, and carrying out classification treatment by adopting a differential zone belt centrifugation method to obtain the molybdenum disulfide-sodium cholate composite material;
(2) preparing a sensitive probe: and (2) adding the tetracycline monoclonal antibody into the molybdenum disulfide nanosheet solution obtained in the step (1), mixing, reacting and centrifuging to obtain the tetracycline nano-antibody.
4. The sensitive probe according to claim 3, wherein in the step (1), the concentration ratio of the molybdenum disulfide to the sodium cholate is 1 (0.2-0.4).
5. The sensitive probe of claim 3, wherein in the step (2), the concentration ratio of the molybdenum disulfide nanosheet to the tetracycline monoclonal antibody is 1: 0.001-0.003, the molybdenum disulfide nanosheet and the tetracycline monoclonal antibody are mixed and vibrated for 30-60 minutes, then 100 μ L of 10% bovine serum albumin is added for reaction for 30 minutes, and the reaction is sealed for 2-2.5 hours.
6. A method for detecting tetracycline, which comprises immersing a test strip in a reagent containing a sensitive probe according to any one of claims 1 to 5.
7. The method for detecting tetracycline according to claim 6, wherein the test strip comprises a backing plate, a nitrocellulose membrane is attached to the backing plate, one end of the nitrocellulose membrane covers the absorbent pad, the other end of the nitrocellulose membrane sequentially covers the sample pad and the binding pad, and a detection line and a quality control line are transversely arranged on a non-covered surface of the nitrocellulose membrane; the detection line is sprayed with a tetracycline-bovine serum albumin conjugate, and the quality control line is sprayed with goat anti-mouse immunoglobulin.
8. The method of detecting tetracycline of claim 7, wherein the spraying of the tetracycline-bovine serum albumin conjugate onto the detection line is prepared by a method comprising: spraying the 1mg/mL tetracycline-bovine serum albumin conjugate solution on a detection line at a scribing rate of 1 mu L/cm to obtain a detection line;
the preparation method of the quality control line sprayed with the goat anti-mouse immunoglobulin comprises the following steps: the goat anti-mouse monoclonal antibody coating solution of 1mg/mL is sprayed on the nitrocellulose membrane at the speed of 1 muL/cm to form a quality control line, and the distance between the detection line and the quality control line is 5 mm.
9. A sensitive probe according to any of claims 1 to 5 for use in the detection of tetracycline in milk, honey or beef.
10. The method for detecting tetracycline according to any one of claims 6-8, which is used for detecting tetracycline in milk, honey or beef.
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| CN111551724A (en) * | 2020-04-03 | 2020-08-18 | 西北农林科技大学 | Fluorescent probe, method for detecting tetracycline and application |
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| CN114149592A (en) * | 2021-10-20 | 2022-03-08 | 江苏大学 | Composite ratiometric fluorescent probe and preparation method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111551724A (en) * | 2020-04-03 | 2020-08-18 | 西北农林科技大学 | Fluorescent probe, method for detecting tetracycline and application |
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| CN114149592A (en) * | 2021-10-20 | 2022-03-08 | 江苏大学 | Composite ratiometric fluorescent probe and preparation method and application thereof |
| CN114149592B (en) * | 2021-10-20 | 2023-01-17 | 江苏大学 | Composite ratiometric fluorescent probe and preparation method and application thereof |
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