A probe platform and method for detecting salbutamol based on magnetic-assisted Prussian blue nanozyme-DAB substrate

Abstract

The present invention discloses a probe platform and method for detecting salbutamol based on a magnetic-assisted Prussian blue nanozyme-DAB substrate. The probe platform for detecting salbutamol based on a magnetic-assisted Prussian blue nanozyme-DAB substrate comprises a magnetic-assisted Prussian blue nanozyme and a diaminobenzidine substrate, wherein the magnetic-assisted Prussian blue nanozyme is a core-shell complex of a Fe3O4 core and a Prussian blue shell. The present invention develops a probe platform for detecting salbutamol based on a magnetic-assisted Prussian blue nanozyme-DAB substrate. On the one hand, the signal intensity of the magnetic-assisted Prussian blue nanozyme can be amplified by more than 2 times by combining with the oxidation of the diaminobenzidine substrate; on the other hand, the analyte in the sample can be enriched and concentrated based on the magnetic-assisted Prussian blue nanozyme, and the analytical sensitivity can be increased by more than 10 times.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a probe platform and method for detecting salbutamol based on magnetic-assisted Prussian blue nanozyme-DAB substrate. Background Art

[0002] Salbutamol is a drug used clinically to treat bronchial asthma. However, due to its ability to reduce fat accumulation and increase carcass lean meat percentage, it is often illegally used as a veterinary drug additive. Studies have shown that long-term consumption of meat products containing salbutamol can easily lead to adverse reactions such as dizziness, nausea, palpitations and muscle tremors. Therefore, many countries, including China, have explicitly banned its use in livestock production. Therefore, it is necessary to strengthen the detection and monitoring of salbutamol.

[0003] At present, a variety of methods have been developed to detect salbutamol, mainly including instrumental analysis and immunoassay. Instrumental methods such as liquid chromatography tandem mass spectrometry (LC-MS / MS), high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), electrochemical sensors and enzyme-linked immunosorbent assay (ELISA) etc. These methods have the advantages of high specificity and high accuracy, but because the instruments are expensive and inconvenient to carry, they require professional operation, time-consuming and laborious, which limits their application in rapid on-site screening. In contrast, immunochromatographic analysis is widely used in on-site mass detection due to its advantages such as low cost, short time and no need for professional operation. However, the traditional colloidal gold immunochromatographic method has the disadvantages of low sensitivity, high false positive and false negative rates, and large interference from background signals, especially for samples with large sample matrix effects such as pig urine, which is a great challenge for the detection of trace analytes. Therefore, improving the detection sensitivity of the immunochromatographic method, improving the accuracy of the results, and reducing matrix interference are of great significance for extending its scope of application.

[0004] For the detection of small molecules, a competitive strategy is generally adopted. Sensitivity is mainly determined by two factors: signal intensity and the competition intensity between the analyte and the coated antigen in the reaction system. Reducing the amount of antibody / probe used can promote fierce competition, resulting in a more sensitive dose response relationship between analyte concentration and signal intensity. But on the other hand, the reduction of antibodies will lead to a weakening of signal intensity, which will not only make it difficult to judge the results, but may even lead to false positives. Therefore, how to reduce the amount of antibodies while maintaining the same signal intensity is a key problem to be solved to improve sensitivity. Summary of the invention

[0005] In order to make up for the defects of the existing technology and solve the problems of low sensitivity, high false positive and false negative rates, and large interference from background signals in the traditional colloidal gold immunochromatography method for detecting salbutamol, the present invention provides a probe platform and method for detecting salbutamol based on magnetic-assisted Prussian blue nanozyme-DAB substrate.

[0006] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0007] A probe platform for detecting salbutamol based on magnetically assisted Prussian blue nanozyme-DAB substrate, the probe platform comprising a magnetically assisted Prussian blue nanozyme coupled with a salbutamol antibody as a probe and diaminobenzidine (DAB) as a substrate, the magnetically assisted Prussian blue nanozyme being a core-shell complex of an Fe3O4 core and a Prussian blue shell.

[0008] Furthermore, the average particle size of the magnetically assisted Prussian blue nanozyme is 300 to 320 nm.

[0009] Furthermore, the synthesis method of the magnetic-assisted Prussian blue nanozyme coupled with salbutamol antibody is as follows:

[0010] (1) Synthesis of magnetic nanoparticles (Fe3O4): dissolving ferric chloride hexahydrate and trisodium citrate in a solvent and stirring; adding sodium acetate and continuing to stir to obtain a mixed solution; then transferring the mixed solution to a high-pressure reactor for high-temperature reaction, cooling and collecting a brown-black product after the reaction is completed, washing several times, and drying to obtain a dark brown powder;

[0011] (2) Synthesis of magnetically assisted Prussian blue nanozyme (Fe3O4@PB): The dark brown powder from step (1) was added to a solution containing potassium ferrocyanide and ferric chloride hexahydrate at the same concentration, and the reaction was stirred at room temperature. The color of the solution gradually changed from dark brown to dark green. The dark green product was collected, washed several times, and redissolved in ultrapure water to obtain a magnetically assisted Prussian blue nanozyme solution.

[0012] (3) Synthesizing the probe (Fe3O4@PB-Ab): Add salbutamol antibody to the magnetically assisted Prussian blue nanozyme solution of step (2) and react under vortexing; then add bovine serum albumin, continue the reaction, collect the precipitate by magnetic absorption, wash it several times with boric acid buffer solution, and finally dissolve it in boric acid buffer solution to obtain the probe.

[0013] Furthermore, the molar ratio of the ferric chloride hexahydrate to trisodium citrate is 1:1 to 5:1.

[0014] Furthermore, the concentrations of the potassium ferrocyanide and ferric chloride hexahydrate are 1-5 mM.

[0015] Furthermore, the reaction time of the stirring reaction at room temperature is 1 to 5 hours.

[0016] Furthermore, the molar mass of the sodium acetate is 10-20 mM.

[0017] Furthermore, the dark brown powder of step (1) is added to a solution containing potassium ferrocyanide and ferric chloride hexahydrate at the same concentration, specifically 8 to 12 mg of the dark brown powder of step (1) is added to 12 to 18 mL of a solution containing potassium ferrocyanide and ferric chloride hexahydrate at the same concentration. Furthermore, the salbutamol antibody is a monoclonal antibody, a polyclonal antibody or a nanobody.

[0018] A method for detecting salbutamol based on magnetic-assisted Prussian blue nanozyme-DAB substrate comprises the following steps: placing a sample to be tested or a sample extract in a centrifuge tube, adding the magnetic-assisted Prussian blue nanozyme coupled with a salbutamol antibody, incubating at room temperature and then magnetically absorbing, then discarding the supernatant and re-dissolving with a phosphate buffer and transferring all of it to a microwell; inserting a test strip into the microwell for reaction and then discarding the sample pad, adding a diaminobenzidine substrate for oxidation reaction, and then interpreting and reading the results.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides a probe platform for detecting salbutamol based on magnetic-assisted Prussian blue nanozyme-DAB substrate. On the one hand, the signal intensity of the magnetic-assisted Prussian blue nanozyme can be amplified by more than 2 times by combining with diaminobenzidine substrate oxidation; on the other hand, the analyte in the sample can be enriched and concentrated based on the magnetic-assisted Prussian blue nanozyme, and the analytical sensitivity can be increased by more than 10 times. The probe platform of the present invention not only solves the problems of low sensitivity, high false positive and false negative rates, and large background signal interference of the traditional colloidal gold immunochromatography method for detecting salbutamol, but also achieves the same or stronger signal intensity while reducing the amount of antibody used.

[0021] The present invention is specifically a competitive immunochromatographic method based on magnetic Prussian blue nanozyme combined with magnetic separation enrichment strategy and nanozyme oxidation strategy. The advantages are:

[0022] (1) The marker is dark in color and has strong biocompatibility: Since the magnetic Fe3O4 nanoparticles are brown in color, the color of the marker is deepened after the Prussian blue is deposited, which can improve the accuracy of the result interpretation. In addition, the conventional covalent connection achieved by organic crosslinking agents such as EDC / NHS or glutaraldehyde in the prior art may reduce the activity of the antibody or even denature it. The method of the present invention only prepares the probe by simple electrostatic adsorption of magnetic-assisted Prussian blue nanozymes and antibodies, eliminating the complex crosslinking process (such as the EDC / NHS method). The electrostatic adsorption method is used to couple the antibody without the need for an external crosslinking agent, which can well protect the antibody activity.

[0023] (2) Small amount of antibody used and high sensitivity: The present invention uses magnetic-assisted Prussian blue nanozyme combined with diaminobenzidine substrate oxidation. After oxidation, the oxidation products are aggregated and accumulated on the detection line, thereby forming a clearer band on the detection line, which plays a role in amplifying the signal. Only a small amount of probe is needed to produce a strong color development effect, which can significantly reduce the amount of antibody used (0.05 μg per test strip), triggering a more intense competition between the free analyte and the fixed antigen for the limited antibody binding sites, thereby bringing about a more sensitive concentration-response relationship between the analyte and the signal intensity, improving sensitivity, and reducing false positive phenomena. Conventional detection methods, if the amount of probe used is small, will lead to weak color development of the negative sample detection line and prone to false positive phenomena. On the other hand, compared with the traditional immunochromatography method, which is placed in a small volume sample for reaction, the present invention utilizes the immunomagnetic separation characteristics of the magnetic-assisted Prussian blue nanozyme, places the probe in a larger reaction volume for reaction, and concentrates it through magnetic separation enrichment, so that the probability of binding between the probe and the analyte in the reaction system is greatly improved. Through these two reaction strategies, the sensitivity was improved, and the elimination values ​​of salbutamol in pork and pig urine were 0.3 and 0.5 μg / kg, respectively, which were 10-17 times lower than the traditional colloidal gold method. The detection limit (LOD) was 0.022 and 0.030 μg / kg, respectively, which were 7-10 times lower than the traditional colloidal gold method. The LOQ was 0.030 and 0.057 μg / kg, respectively, which were 8-12 times lower than the traditional colloidal gold method.

[0024] (3) (3) The probe preparation process is simple: The labeling material synthesized by the present invention has a simple synthesis process and a high yield. It only requires the magnetic nanoparticles and the Prussian blue raw material to react at room temperature without the need for complex organic reagents. Since the magnetic-assisted Prussian blue nanozyme is magnetic, no large centrifugal equipment is required for centrifugation when preparing the probe. Only a magnet is needed to separate the solution from the solid, which greatly saves the synthesis and labeling time and simplifies the operation steps. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a scanning electron microscope image of the magnetic nanoparticles in Example 1 of the present invention;

[0026] Figure 2 This is a scanning electron micrograph of the magnetically assisted Prussian blue nanozyme in Example 1 of the present invention;

[0027] Figure 3 This is a comparison diagram of the effects of the magnetic-assisted Prussian blue nanoenzyme immunochromatography method of the present invention before and after adding no substrate and adding 3,3',5,5'-tetramethylbenzidine (TMB) and diaminobenzidine (DAB) substrates;

[0028] Figure 4This is a schematic diagram of the detection principle of the diaminobenzidine combined with magnetic-assisted Prussian blue nanozyme immunochromatographic test strip of the present invention, in which: 1: sample matrix; 2: salbutamol; 3: magnet; 4: magnetic-assisted Prussian blue nanozyme; 5: goat anti-mouse IgG; 6: coated antigen; 7: absorbent paper; 8: PVC bottom plate; 9: nitrocellulose membrane; 10: sample pad;

[0029] Figure 5 The standard curve and cut-off value of the diaminobenzidine combined with magnetic-assisted Prussian blue nanoenzyme immunochromatography method for detecting salbutamol in pig urine and pork;

[0030] Figure 6 This is a graph showing the cross-reaction detection in pig urine and pork using the diaminobenzidine combined with magnetic-assisted Prussian blue nanoenzyme immunochromatography method of the present invention.

[0031] Figure 7 This is a graph showing the relationship between the raw material feeding amount and the particle size in Example 4 of the present invention;

[0032] Figure 8 This is a graph showing the relationship between the synthesis time and particle size of the magnetic-assisted Prussian blue nanozyme in Example 4 of the present invention;

[0033] Fig. 9 The effect of different probe dosages on the detection effect in Example 4 of the present invention;

[0034] Fig.10 The effect of different magnetic enrichment times on the magnetic enrichment effect in Example 4 of the present invention;

[0035] Fig.11 The effect of different oxidation times on the oxidation effect in Example 4 of the present invention;

[0036] Fig.12 To develop the standard curve and cut-off value for the detection of salbutamol in pig urine and pork by colloidal gold immunochromatography;

[0037] Fig.13 Provided the standard curve and cut-off value for the detection of salbutamol in pig urine and pork using magnetic nanoparticle immunochromatography. DETAILED DESCRIPTION

[0038] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0039] Unless otherwise specified, the meanings of the terms in this specification are the same as those generally understood by those skilled in the art. However, if there is any conflict, the definitions in this specification shall prevail.

[0040] As used herein, "includes," "comprising," "including," "containing," "having," or other variations are intended to encompass non-exclusive inclusions, and no distinction is made between these terms. The term "comprising" means that other steps and ingredients that do not affect the end result may be added. The term "comprising" also includes the terms "consisting of" and "consisting essentially of." The compositions and methods / processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein.

[0041] All numerical values ​​or expressions involving component amounts, process conditions, etc. used in the specification and claims should be understood to be modified by "about" in all cases. All ranges involving the same component or property include endpoints, which can be independently combined. Since these ranges are continuous, they include every numerical value between the minimum and maximum values. It should also be understood that any numerical range cited in this application is expected to include all sub-ranges within the range. The present invention provides a probe platform for detecting salbutamol based on magnetic-assisted Prussian blue nanozyme-DAB substrate. Specifically, the probe platform includes a magnetic-assisted Prussian blue nanozyme coupled with a salbutamol antibody as a probe and diaminobenzidine as a substrate, and the magnetic-assisted Prussian blue nanozyme is a core-shell complex of a Fe3O4 core and a Prussian blue shell. Further, the average particle size of the magnetic-assisted Prussian blue nanozyme is 300-320nm.

[0042] On the one hand, the magnetic-assisted Prussian blue nanozyme can be combined with diaminobenzidine substrate oxidation to achieve the purpose of signal amplification; on the other hand, the magnetic-assisted Prussian blue nanozyme can enrich and concentrate the analytes in the sample, further improving the detection sensitivity.

[0043] The method for synthesizing the magnetic-assisted Prussian blue nanozyme coupled with salbutamol antibody is as follows:

[0044] (1) Synthesis of Fe3O4: dissolving ferric chloride hexahydrate and trisodium citrate in a solvent and stirring; adding sodium acetate and continuing stirring to obtain a mixed solution; then transferring the mixed solution to a high-temperature reactor for high-temperature reaction, cooling and collecting a brown-black product after the reaction is completed, washing several times, and drying to obtain a dark brown powder; the molar ratio of the ferric chloride hexahydrate to the trisodium citrate is 1:1 to 5:1; the molar mass of the sodium acetate is 10 to 20 mM;

[0045] (2) Synthesis of magnetically assisted Prussian blue nanozyme: 8-12 mg of the dark brown powder of step (1) was added to 12-18 mL of a solution containing potassium ferrocyanide and ferric chloride hexahydrate at the same concentration, and the reaction was stirred at room temperature for 1-5 h. The color of the solution gradually changed from dark brown to dark green. The dark green product was collected, washed several times, and redissolved in ultrapure water to obtain a magnetically assisted Prussian blue nanozyme solution; wherein the concentrations of potassium ferrocyanide and ferric chloride hexahydrate were 1-5 mM;

[0046] (3) Probe synthesis: Add salbutamol antibody to the magnetically assisted Prussian blue nanozyme solution of step (2) and react under vortexing; then add bovine serum albumin, continue the reaction, collect the precipitate by magnetic absorption, wash it several times with boric acid buffer solution, and finally redissolve it in boric acid buffer solution to obtain the probe.

[0047] The method for synthesizing magnetic-assisted Prussian blue nanozyme of the present invention is mainly a core-shell growth method, which does not require adjusting the pH value, and directly throws the magnetic nanoparticles into the raw material solution for reaction in one step, thereby shortening the operation time and simplifying the operation steps.

[0048] A method for detecting salbutamol using a probe platform based on magnetically assisted Prussian blue nanozyme-DAB substrate comprises the following steps: placing a sample to be tested or a sample extract in a centrifuge tube, adding the magnetically assisted Prussian blue nanozyme coupled with a salbutamol antibody, incubating at room temperature and then magnetically absorbing, then discarding the supernatant and re-dissolving with a phosphate buffer and transferring all to a microwell; inserting a test strip into the microwell for reaction and discarding the sample pad, adding a diaminobenzidine substrate for oxidation reaction and then interpreting and reading the result. The above-mentioned antibody can be a monoclonal antibody, a polyclonal antibody or a nanobody.

[0049] The detection principle of the test strip is: based on the principle of competitive reaction, the sample solution and the probe are added to the enzyme-labeled microwell, and the mixed solution flows to the detection area under the action of capillary force. When the sample does not contain the analyte, the probe will be captured by the coated antigen on the test line to form a visible strip. When the sample contains the analyte, the analyte and the coated antigen compete with each other. As the concentration of the analyte in the sample increases, the amount of probe captured by the coated antigen becomes less and less, and the color of the test line will become lighter and lighter until the line disappears. In addition, regardless of whether there is an analyte, the probe will be captured by the sheep anti-mouse IgG on the quality control line and develop color, otherwise the test strip is invalid. When strips appear on both the test line and the quality control line at the same time, it is negative; when only the quality control line has a strip, it is positive; when there is no strip on the quality control line or only the test line has a strip, it is judged to be invalid.

[0050] The signal enhancement strategies in the existing technologies mainly focus on the synthesis of new nanomaterials, which often involve complicated and tedious material synthesis steps, and have problems such as low product stability and reproducibility.

[0051] As a highly active nanozyme, Prussian blue has the advantages of good biocompatibility, low cost, high specific surface area, easy synthesis and surface modification. The introduction of Prussian blue material for use in immunochromatography methods can not only provide strong signals, but also realize secondary signal amplification through enzyme-catalyzed substrates, thereby improving detection sensitivity. DAB, as a chromogenic substrate for enzyme-catalyzed reactions, is widely used in colorimetric reactions such as protein blotting and immunohistochemistry, and is the most sensitive chromogenic substrate for horseradish peroxidase. The signal enhancement strategy of the present invention is mainly derived from the enzyme activity of the magnetic-assisted Prussian blue nanozyme. The signal intensity of the magnetic-assisted Prussian blue nanozyme can be amplified by more than 2 times by combining it with diaminobenzidine for enzyme-catalyzed reactions (see Figure 3 The catalytic efficiency is high and the antibody activity is not affected. Under enzyme catalysis conditions, it is oxidized to produce a dark brown product, which is darker in color than the commonly used TMB substrate (such as Figure 3 The Prussian blue nanozyme and diaminobenzidine are used together in the immunochromatography method, and a stronger signal amplification effect can be achieved by superimposing the background color of the nanozyme and the color of the enzyme catalysis product. Figure 3 The T-line intensities converted into grayscale values ​​are 1877.426, 4908.832, and 7397.782 from left to right, respectively. The color intensity of the specific diaminobenzidine substrate used in the present invention is deep, and the colors of other substrates and the color after superposition of the magnetic-assisted Prussian blue nanozyme may have double colors (color system change), that is, colors other than the gray-green color of the magnetic Prussian blue itself will appear. When quantitative readings are taken, the results may be misinterpreted due to the appearance of two-color bands and two peaks; and the color is enhanced after superposition of the diaminobenzidine substrate, that is, the color system will not change.

[0052] The main method to eliminate matrix interference in the prior art is dilution, but dilution will lead to reduced sensitivity. The present invention mainly uses the superparamagnetism of magnetic-assisted Prussian blue nanozyme to enrich and replace the matrix to eliminate the influence of the matrix. On the one hand, it can enrich the analyte in the sample, and on the other hand, it can remove some proteins and fats inside the solution, increase the migration rate of the solution, and the complex color background will disappear. Compared with the colloidal gold probe, the selection of the magnetic-assisted Prussian blue probe of the present invention can solve the problems of sample matrix influence, low sensitivity (low cut-off value, see Figure 5 and Fig.12 comparison), high false negative and false positive rates (see Table 1 for comparison).

[0053] In summary, the probe platform for detecting salbutamol based on magnetic-assisted Prussian blue nanozyme-DAB substrate of the present invention combines magnetic immune enrichment and diaminobenzidine substrate oxidation enhanced color development dual channels to improve the detection sensitivity. On the one hand, the signal intensity of magnetic-assisted Prussian blue nanozyme can be amplified by more than 2 times by combining with diaminobenzidine substrate oxidation; on the other hand, based on magnetic-assisted Prussian blue nanozyme, the analyte in the sample can be enriched and concentrated, and the analytical sensitivity can be increased by more than 10 times.

[0054] The present invention is described in detail below with reference to the examples, which are only preferred embodiments of the present invention and are not intended to limit the present invention. Unless otherwise specified herein, the concentrations of solutions are all volume concentrations.

[0055] Example 1 Synthetic probe

[0056] The synthesis method of magnetically assisted Prussian blue nanozyme coupled with salbutamol antibody specifically comprises the following steps:

[0057] (1) Synthesis of magnetic nanoparticles (Fe3O4): Dissolve 2g of ferric chloride hexahydrate and 1g of trisodium citrate in 20mL of ethylene glycol and stir vigorously for 60min. Then add 1g of sodium acetate and continue stirring vigorously for 30min to obtain a mixed solution. Then transfer the mixed solution to a high-pressure reactor and place it in a high-temperature oven at 200°C to react for 12h. After cooling, pour out the mixture in the reactor, collect the brown-black product with a magnet, and then wash the product with anhydrous ethanol and water three times respectively, and then dry it in a vacuum drying oven at 80°C for 12h. Finally, collect the dark brown powder and store it at 4°C for later use. The scanning electron microscope image of the magnetic nanoparticles is as follows Figure 1 shown.

[0058] (2) Magnetic-assisted synthesis of Prussian blue (Fe3O4@PB): 10 mg of the above dark brown powder was added to 15 mL of a solution containing 3 mM potassium ferrocyanide and ferric chloride hexahydrate, and the reaction was stirred vigorously at room temperature. The color of the solution gradually changed from dark brown to dark green. Finally, the dark green product was collected with a magnet, washed with ultrapure water three times, and then redissolved in 25 mL of ultrapure water and stored at 4°C for later use. The scanning electron microscopy image of the synthesized magnetic-assisted Prussian blue nanozyme is shown in Figure 2. Figure 2 As shown, the average diameter of the particles is 300-320 nm, the shape is uniform and they have good water solubility and dispersibility.

[0059] (3) Probe (Fe3O4@PB-Ab) synthesis: 10 μg of salbutamol monoclonal antibody was added to 1 mL of magnetically assisted Prussian blue solution. After reacting for 30 min under vigorous vortexing, 30 μL of 20% bovine serum albumin (w / v) was added. The reaction was continued for 30 min, and the precipitate was collected with a magnet. The precipitate was washed twice with 0.02 M pH 8.0 boric acid buffer solution (containing 0.5% Tween-20, 0.5% BSA, 5% sucrose, 0.3% PVP, and 0.03% procline-300), and then reconstituted with the same solution to 400 μL and stored at 4°C for use.

[0060] Example 2 Assembly of a Salbutamol Test Strip

[0061] The test strip for detecting salbutamol includes a sample pad, a nitrocellulose membrane, absorbent paper, and a PVC base plate; wherein the sample pad is soaked with a 0.1M pH 7.4 phosphate buffer solution (containing 0.5% Tween-20, 0.3% PVP, 0.5% sucrose, 0.5% BSA, and 0.03% procline-300), placed in a 37°C oven for drying for 12 hours, and then cut into 15 mm strips for standby use; the coated antigen, salbutamol-ovalbumin complex (0.8 mg / mL) and goat anti-mouse IgG (0.9 mg / mL), are sprayed on the nitrocellulose membrane to form a test line / quality control line, with a distance of 7 mm between the two lines, and then placed in a 37°C oven for drying for 12 hours for standby use; the commercial absorbent paper is not treated and cut into 25 mm strips for standby use; the sample pad, nitrocellulose membrane, and absorbent paper are sequentially pasted on the PVC base plate, with each part overlapping by 2 mm, and cut into 3.5 mm wide strips for standby use, such as Figure 4 shown.

[0062] Embodiment 3 detection method of salbutamol

[0063] (3.1) Sample pretreatment: Swine urine samples were tested directly without treatment; pork samples were minced and homogenized after fat removal, and then 5 g of pork sample was weighed into a 50 mL centrifuge tube, 5 mL of 0.05 M pH 7.4 phosphate buffer solution was added, extracted for 3 min, and centrifuged at 6000 rpm for 10 min at room temperature to obtain the supernatant for testing.

[0064] (3.2) Detection step: 1 mL of sample or sample extract was placed in a centrifuge tube, 2 μL of Fe3O4@PB-Ab prepared in Example 1 was added, incubated at room temperature for 8 min, placed in a magnetic stand for 3 min, then the supernatant was discarded and re-dissolved with 150 μL 0.02 M phosphate buffer and transferred to the microwell. Then the test strip prepared in Example 2 was inserted into the microwell for 3 min, the sample pad was discarded, 2 μL of diaminobenzidine was added for oxidation reaction for 2 min, and the result was judged and read. Figure 4shown.

[0065] (3.3) Method evaluation:

[0066] (a) Determination of cut-off value: The salbutamol standard solution was added to the negative sample according to the set concentration, and the test strips assembled under the optimal conditions were used for testing. The cut-off value of the diaminobenzidine combined with magnetic-assisted Prussian blue nanoenzyme immunochromatography was obtained by visual observation. When the T line completely disappeared, the corresponding minimum concentration of salbutamol was taken as the cut-off value. The results are shown in Figure 5 As shown, the cut-off values ​​of diaminobenzidine combined with magnetic-assisted Prussian blue nanoenzyme immunochromatography for detecting salbutamol in pig urine and pork were 0.3 and 0.5 μg / kg, respectively.

[0067] (b) Determination of LOD and LOQ: Add a series of concentrations of salbutamol to pig urine and pork samples, treat the samples according to the above-mentioned pretreatment method and then test them. Use a reader to read the optical density values ​​of the test line and the quality control line and record them as T value and C value respectively. Calculate the T / C value and draw a standard curve using Origin9.0 software. Use the concentration of salbutamol as the horizontal axis and B / B0 as the vertical axis (B is the T / C value of the test strip with salbutamol added, and B0 is the T / C value of the test strip without drug added) to fit the standard curve. The corresponding LOD and LOQ are defined as the concentration obtained by adding 3 times or 10 times the standard deviation to the average value of the B / B0 values ​​of 20 negative samples. Figure 5 As shown, the LOD of salbutamol in pork and pig urine detected by diaminobenzidine combined with magnetic-assisted Prussian blue nanoenzyme immunochromatography were 0.022 and 0.030 μg / kg, and the LOQ were 0.030 and 0.057 μg / kg, respectively.

[0068] (c) Determination of cross-reaction: Eight structural analogs (detection concentration of 1000 μg / kg) were selected and simultaneously detected using diaminobenzidine combined with magnetic-assisted Prussian blue nanozyme immunochromatography. The test results are as follows: Figure 6 The test strip developed by this method does not cross-react with other structural analogs and shows good specific reaction to salbutamol.

[0069] (d) Evaluation of result accuracy: Swine urine and pork samples were spiked at 0.5 times the cut-off value, 1 times the cut-off value, and 2 times the cut-off value, respectively. 50 samples were tested at each concentration. The colloidal gold immunochromatography method and the diaminobenzidine combined with magnetic-assisted Prussian blue nanozyme immunochromatography method were used for detection. The test results are shown in Table 1. It can be seen from the table that the accuracy of the results of the diaminobenzidine combined with magnetic-assisted Prussian blue nanozyme immunochromatography method is higher than that of the traditional colloidal gold immunochromatography method.

[0070] Table 1 Comparison of the accuracy of the conventional colloidal gold immunochromatography and the diaminobenzidine combined with magnetic-assisted Prussian blue nanoenzyme immunochromatography of the present invention

[0071]

[0072]

[0073] Example 4

[0074] In order to obtain the best assay performance, the inventors optimized the concentrations of potassium ferrocyanide and ferric chloride hexahydrate, the reaction time of magnetic-assisted Prussian blue nanozyme synthesis, the amount of probe, the magnetic enrichment time, the oxidation reaction time, and determined the optimal system conditions.

[0075] Specifically,

[0076] (4.1) Optimization of the dosage of synthetic raw materials: 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, and 6 mM concentrations of potassium ferrocyanide and ferric chloride hexahydrate were selected for reaction, and after optimization, 3 mM concentration was selected.

[0077] like Figure 7 As shown in the figure, from the relationship diagram between different raw material dosages and particle sizes, it can be seen that the higher the dosage, the larger the particle size of the synthesized magnetically assisted Prussian blue nanozyme, which reaches the maximum when the dosage is 3mM, and the shell thickness no longer increases. Considering the comprehensive raw material cost, the dosage of 3mM is selected as the most preferred.

[0078] (4.2) Synthesis time optimization: 1h, 2h, 3h, 4h, and 5h were selected for reaction. After optimization, 2h was selected as the synthesis time.

[0079] like Figure 8 As shown in the figure, from the relationship diagram between reaction time and particle size, it can be seen that the longer the reaction time, the larger the particle size of the synthesized magnetically assisted Prussian blue nanozyme, which reaches the maximum when the reaction time is 2 hours, and the shell thickness no longer increases. Considering the comprehensive time cost, a synthesis time of 2 hours is selected as the most preferred.

[0080] (4.3) Optimization of probe dosage: 1 μL, 2 μL, 3 μL, and 4 μL were selected for reaction, and finally 2 μL was selected as the probe dosage.

[0081] Too much probe will affect the inhibition effect of the experiment, resulting in incomplete inhibition. Fig. 9 It can be seen that when the amount of probe increases, the color intensity of the negative sample becomes higher and higher. Due to the excess of probe, the inhibition effect of the positive sample becomes worse. Considering the comprehensive cost and experimental effect, 2μL probe is selected as the best choice.

[0082] (4.4) Optimization of magnetic enrichment time: 4 min, 6 min, 8 min, and 10 min were selected for optimization, and 8 min was finally selected as the magnetic enrichment time.

[0083] The magnetic enrichment time determines the time cost and experimental effect of the method. Increasing the magnetic enrichment time can increase the probability of the probe and the reactant combining. Fig.10 It can be seen that when the time is less than 8 minutes, the inhibition is incomplete, and after 8 minutes, it can be completely inhibited and the color on the detection line disappears. Therefore, considering the time cost and experimental effect, 8 minutes is selected as the most preferred magnetic enrichment time.

[0084] (4.5) Oxidation reaction time: 1 min, 2 min, 3 min, and 4 min were selected for optimization, and finally 2 min was selected as the oxidation reaction time.

[0085] The length of oxidation time after adding the substrate determines the time cost of the method. Fig.11 It can be seen that when the oxidation time exceeds 2 minutes, the color development is too dark or even blackened due to the excessive accumulation of oxidation products, which also leads to a worse inhibition effect. At 2 minutes, the color development effect and nuclear transplantation effect are most suitable and the time cost is the lowest. Therefore, 2 minutes is selected as the most preferred oxidation reaction time.

[0086] Comparative Example 1

[0087] This comparative example is a magnetic nanoparticle immunochromatography method for detecting salbutamol, and the specific steps are as follows:

[0088] (1) Synthesis of magnetic nanoparticles (Fe3O4): Dissolve 2g of ferric chloride hexahydrate and 1g of trisodium citrate in 20mL of ethylene glycol and stir vigorously for 60min. Then add 1g of sodium acetate and continue to stir vigorously for 30min to obtain a mixed solution. Then transfer the mixed solution to a high-pressure reactor and place it in a high-temperature oven at 200℃ for reaction for 12h. After cooling, pour out the mixture in the reactor, collect the brown-black product with a magnet, and then wash the product with anhydrous ethanol and water three times respectively, and then dry it in a vacuum drying oven at 80℃ for 12h. Finally, collect the dark brown powder and store it at 4℃ for later use.

[0089] (2) Probe (Fe3O4@Ab) synthesis: 2 mg of Fe3O4 was placed in a 1.5 mL centrifuge tube, and 300 μL of EDC solution (2 mg / mL) and 400 μL (2 mg / mL) NHS solution were added to fully shake and dissolve. The mixture was placed on an oscillator and shaken vigorously for 15 min, and the supernatant was discarded. 1 mL of ultrapure water was added for reconstitution, and 20 μg of salbutamol monoclonal antibody was added. After reacting under vigorous vortexing for 30 min, 30 μL of bovine serum albumin with a mass concentration (w / v) of 20% was added. After continuing the reaction for 30 min, the precipitate was collected with a magnet, washed twice with 0.02 M pH 8.0 boric acid buffer solution (containing 0.5% Tween-20, 0.5% BSA, 5% sucrose, 0.3% PVP, and 0.03% procline-300), and then reconstituted to 200 μL with the same solution and stored at 4°C for use.

[0090] (3) Sample pretreatment: Swine urine samples were tested directly without treatment; pork samples were minced and homogenized after fat was removed. Then, 5 g of pork sample was weighed into a 50 mL centrifuge tube, 5 mL of 0.05 M pH 7.4 phosphate buffer solution was added, extracted for 3 min, and centrifuged at 6000 rpm for 10 min at room temperature to obtain the supernatant for testing.

[0091] (4) Detection steps: Add 2 μL of magnetic nanoparticle immunoprobe to 1 mL of pig urine or pork sample, incubate at room temperature for 8 min for full reaction, place the centrifuge tube on a magnetic rack for 3 min, remove the reacted sample solution and re-dissolve it with 150 μL of phosphate buffer, transfer it to the microwell and vertically insert the test strip to react for 3 min, then take it out, discard the sample pad and observe it with the naked eye within 1 min or read it with a reader.

[0092] Comparative Example 1 The cut-off values ​​of salbutamol in pig urine and pork detected by magnetic nanoparticle immunochromatography were 0.5 and 0.8 μg / kg, LODs were 0.05 and 0.09 μg / kg, and LOQs were 0.12 and 0.19 μg / kg, respectively. Fig.13 The antibody dosage was 0.2 μg per test strip.

[0093] The above-mentioned embodiments merely express the implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the patent scope of the present invention. Any technical solution obtained in the form of equivalent replacement or equivalent transformation shall fall within the protection scope of the present invention.

Claims

1. A method for detecting salbutamol based on magnetic-assisted Prussian blue nanozyme-DAB substrate, characterized in that: It includes the following steps: (I) Synthetic probes The synthesis method of magnetically assisted Prussian blue nanozyme coupled with salbutamol antibody specifically comprises the following steps: (1) Synthesis of magnetic nanoparticles: 2 g of ferric chloride hexahydrate and 1 g of trisodium citrate were dissolved in 20 mL of ethylene glycol and stirred vigorously for 60 min; 1 g of sodium acetate was then added and stirred vigorously for 30 min to obtain a mixed solution; the mixed solution was then transferred to a high-pressure reactor and placed in a 200°C high-temperature oven for reaction for 12 h; after cooling, the mixture in the reactor was poured out, and the brown-black product was collected with a magnet, and then the product was washed with anhydrous ethanol and water for 3 times, respectively, and then dried in a vacuum drying oven at 80°C for 12 h. Finally, the dark brown powder was collected and stored at 4°C for later use; (2) Magnetic-assisted Prussian blue synthesis: 10 mg of the dark brown powder of step (1) was added to 15 mL of a solution containing 3 mM potassium ferrocyanide and ferric chloride hexahydrate, and the mixture was stirred vigorously at room temperature. The color of the solution gradually changed from dark brown to dark green. The dark green product was collected with a magnet, washed three times with ultrapure water, and then redissolved in 25 mL of ultrapure water and stored at 4°C for later use. The synthesized magnetic-assisted Prussian blue nanozyme particles had an average diameter of 300 to 320 nm. (3) Probe synthesis: Add 10 μg of salbutamol monoclonal antibody to 1 mL of the magnetically assisted Prussian blue solution prepared in step (2), react under vigorous vortexing for 30 min, then add 30 μL of 20% w / v bovine serum albumin, continue the reaction for 30 min, collect the precipitate with a magnet, wash twice with 0.02 M pH 8.0 boric acid buffer solution, and then reconstitute to 400 μL with the same solution and store at 4°C for later use; (II) Assembly of salbutamol test strips The test strip for detecting salbutamol includes a sample pad, a nitrocellulose membrane, absorbent paper, and a PVC base plate; wherein the sample pad is soaked with a 0.1M pH 7.4 phosphate buffer solution, placed in a 37°C oven for drying for 12 hours, and then cut into 15 mm pieces for standby use; 0.8 mg / mL of the coated antigen-salbutamol-ovalbumin complex and 0.9 mg / mL of goat anti-mouse IgG are sprayed on the nitrocellulose membrane to form a test line / quality control line, with a distance of 7 mm between the two lines, and then placed in a 37°C oven for drying for 12 hours for standby use; the commercial absorbent paper is not processed and cut into 25 mm pieces for standby use; the sample pad, nitrocellulose membrane, and absorbent paper are sequentially pasted on the PVC base plate, with each part overlapping by 2 mm, and cut into 3.5 mm wide strips for standby use; (III) Detection method of salbutamol (3.1) Sample pretreatment: Swine urine samples were tested directly without treatment; pork samples were minced and homogenized after fat removal, and then 5 g of pork sample was weighed into a 50 mL centrifuge tube, 5 mL of 0.05 M pH 7.4 phosphate buffer solution was added, extracted for 3 min, and centrifuged at 6000 rpm for 10 min at room temperature to obtain the supernatant for testing; (3.2) Detection steps: Place 1 mL of sample or sample extract in a centrifuge tube, add 2 μL of the probe prepared in step (i), incubate at room temperature for 8 min, place in a magnetic stand for 3 min, then discard the supernatant and reconstitute with 150 μL 0.02 M phosphate buffer and transfer all to the microwell; then insert the test strip prepared in step (ii) into the microwell for reaction for 3 min, discard the sample pad, add 2 μL of diaminobenzidine for oxidation reaction for 2 min, and then interpret and read the results.

Images