CN112444622A - Method for detecting malachite green, kit based on magnetic separation pretreatment and application thereof - Google Patents
Method for detecting malachite green, kit based on magnetic separation pretreatment and application thereof Download PDFInfo
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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
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
- B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
- B03C1/00—Magnetic separation
- B03C1/005—Pretreatment specially adapted for magnetic separation
- B03C1/01—Pretreatment specially adapted for magnetic separation by addition of magnetic adjuvants
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
- B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
- B03C1/00—Magnetic separation
- B03C1/32—Magnetic separation acting on the medium containing the substance being separated, e.g. magneto-gravimetric-, magnetohydrostatic-, or magnetohydrodynamic separation
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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
- G01N33/5302—Apparatus specially adapted for immunological test procedures
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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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
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
Abstract
The application discloses a method for detecting malachite green, which comprises the following steps: a) providing an animal muscle tissue homogenate; b) adding a reagent 1, a reagent 2 and a magnetic material into the animal muscle tissue homogenate, and after mixing, applying an external magnetic field to realize solid-liquid separation of an animal tissue homogenate solution to obtain a supernatant; c) and detecting malachite green in the supernatant. The application also discloses a kit comprising the reagent 1, the reagent 2 and a magnetic material and application of the kit in a method for detecting malachite green. The application provides a method for extracting a mixed solution, performing magnetic separation, performing quick phase separation, purifying octadecyl bonded silica gel particles, and directly measuring malachite green in animal muscle tissues by naked eyes.
Description
Technical Field
The application relates to a method for detecting malachite green, a kit based on magnetic separation pretreatment and application thereof, and belongs to the field of analysis and detection.
Background
With the continuous improvement of the living standard of people, the consumption of animal-derived food is more and more. In order to meet the increasing consumption demand and improve the breeding output of animals, people add a lot of forbidden drugs in the breeding process of the animals so as to ensure the survival rate of the animals, such as malachite green. The medicines involved in the culture process can be accumulated and concentrated in the organism through a food chain and finally transferred to a human body, and potential harm is brought to the edible safety of people. Malachite green is a chemical product, not only has the characteristics of high toxicity and high residue, but also can cause teratogenesis, carcinogenesis and mutagenesis. From 2002, malachite green is listed in 'animal medicine forbidden for food and animals and compound list thereof' in China, cannot be detected in animal food, and is listed as a forbidden medicine for aquaculture in 'standard of nuisanceless food and fishery medicine use criteria' (NY 5071-2002) in the agricultural industry in China. However, because malachite green has low price and good effect, the application of malachite green in the breeding links of various animals such as aquatic products is still prohibited frequently. Although the analysis methods of malachite green in animal bodies include detection methods such as High Performance Liquid Chromatography (HPLC), high performance liquid chromatography triple quadrupole tandem mass spectrometry (HPLC-MS/MS), enzyme-linked immunosorbent assay (ELISA), colloidal gold immunochromatography rapid detection method and the like, the preparation method of the sample is complicated due to the high content of interfering substances such as fat, protein and the like in animal tissue samples: it needs to adopt various pretreatment technologies such as solvent extraction, centrifugal separation, solid phase extraction and purification to purify and enrich. Therefore, a simple, efficient and low-cost method for detecting malachite green is needed.
Disclosure of Invention
According to the first aspect of the application, the method for detecting malachite green is provided, the defects of multiple steps and long time consumption of the pretreatment process of the existing method are overcome, based on the principle that the protein in the animal muscle tissue homogenate can be denatured into clusters and the magnetic material is wrapped in the clusters by solvent treatment, and the solid-liquid separation can be realized under the action of an external magnetic field, the method for extracting the mixed solution, quickly separating the magnetic separation and directly detecting the malachite green in the animal muscle tissue by naked eyes is provided, and the method has the advantages of simple pretreatment, high efficiency, solvent saving, high sensitivity and low cost.
The method for detecting malachite green is characterized by comprising the following steps: a) providing an animal muscle tissue homogenate; b) adding a reagent 1, a reagent 2 and a magnetic material into the animal muscle tissue homogenate, mixing, and then applying an external magnetic field to realize solid-liquid separation of an animal tissue homogenate solution to obtain a supernatant; c) and detecting malachite green in the supernatant.
In a preferred embodiment, the reagent 1 is a buffer.
Preferably, the reagent 2 is acetonitrile.
Preferably, the buffer solution is prepared by a method comprising: dissolving p-toluenesulfonic acid, hydroxylamine hydrochloride and ammonium acetate in water, and adjusting the pH to 4-5 by using an alkaline solution to obtain the buffer solution, wherein in the buffer solution, the concentration of the p-toluenesulfonic acid is 5-15mg/ml, the concentration of the hydroxylamine hydrochloride is 0.1-0.3mg/ml, the concentration of the ammonium acetate salt is 0.2-0.6mg/ml, and the concentration of the alkaline solution is 3-5 mol/L.
Specifically, the preparation method of the buffer solution comprises the following steps: dissolving p-toluenesulfonic acid, hydroxylamine hydrochloride and ammonium acetate in water, and adjusting the pH to 4.5 by using a 4mol/L sodium hydroxide solution to obtain the buffer solution, wherein in the buffer solution, the concentration of the p-toluenesulfonic acid solution is 10.32mg/ml, the concentration of the hydroxylamine hydrochloride is 0.2mg/ml, and the concentration of the ammonium acetate is 0.4 mg/ml.
In the application, "magnetic material" means can be used for examining in the muscle tissue homogenate and extract the object in-process, when using the solvent including acetonitrile and buffer, can examine the muscle tissue homogenate in the protein denaturation group of uniting, and magnetic material wraps up and forms the sample solid part that examines of magnetism in the protein denaturation group of matter, realizes the magnetic substance of solid-liquid separation under the effect of external magnetic field. In the present application, the magnetic propertiesThe material includes but is not limited to 3-5 parts of CaCl by weight23-5 parts of neutral alumina and 1-2 parts of Fe3O4。
Specifically, the magnetic material comprises 4 parts by weight of CaCl24 parts of neutral alumina and 1 part of Fe3O4. Wherein the neutral alumina can remove fat from the homogenate by adsorption.
Preferably, the external magnetic field includes, but is not limited to, a magnetic field generated by a ferromagnetic body or a magnetic field generated by an electric current.
As a preferred embodiment, in the step c), a dichlorodicyanobenzoquinone solution is further added to the supernatant. In malachite green gets into the organism, can produce the leuco malachite green that has stronger harm, in this application, through adding dichloro dinitrile group benzoquinone solution, can become malachite green with the leuco malachite green oxidation in the supernatant to improve detection accuracy.
As a preferred embodiment, in the step c), octadecyl silica gel particles are further added into the supernatant to further remove proteins, fats and other impurities in the supernatant by adsorption.
As a preferred embodiment, in step c), the method further comprises concentrating the supernatant to remove the solvent, adding a double solution to dissolve the concentrate, and then performing qualitative analysis with a colloidal gold immunochromatographic strip.
Preferably, the preparation method of the double solution comprises the following steps: dissolving sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate dodecahydrate and tween-20 in water, and adjusting the pH to 7-8 by using an acidic solution to obtain the compound solution, wherein in the compound solution, the concentration of the sodium chloride is 9-10g/L, the concentration of the potassium chloride is 0.2-0.3g/L, the concentration of the potassium dihydrogen phosphate is 0.2-0.3g/L, the concentration of the disodium hydrogen phosphate dodecahydrate is 2-3g/L, and the concentration of the tween-20 is 0.3-0.7 ml/L.
Specifically, sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate dodecahydrate and tween-20 are dissolved in water, and the pH is adjusted to 7.4 by using an acidic solution to obtain the compound solution, wherein in the compound solution, the concentration of the sodium chloride is 8g/L, the concentration of the potassium chloride is 0.2g/L, the concentration of the potassium dihydrogen phosphate is 0.27g/L, the concentration of the disodium hydrogen phosphate dodecahydrate is 2.87g/L, and the concentration of the tween-20 is 0.5 ml/L.
According to a second aspect of the present application, there is provided a kit, characterized by comprising: according to the first aspect of the present application, there is provided a reagent 1, a reagent 2 and a magnetic material.
In a preferred embodiment, the kit further comprises a dichlorodicyanobenzoquinone solution.
As a preferred embodiment, the kit further comprises octadecyl bonded silica gel particles.
As a preferred embodiment, the kit further comprises the reconstituted solution according to the first aspect of the present application and a colloidal gold immunochromatographic strip.
The colloidal gold immunochromatographic test strip is provided with a cellulose nitrate membrane, the cellulose nitrate membrane is coated with a detection line and a quality control line, the detection line contains malachite green antigen, and the quality control line contains an antibody capable of being specifically combined with the malachite green antigen;
if the color development ratio of the detection line is deeper than the quality control line or the same depth, the detection line is negative, and the concentration of malachite green in the matrix is lower than the detection limit;
if the color development of the detection line is lighter than the quality control line or the detection limit is green, the detection line is positive, and the concentration of malachite green in the matrix is higher than the detection limit;
if the quality control line is not developed, judging that the operation is improper or the colloidal gold immunochromatographic test strip is invalid. According to a third aspect of the present application, there is also provided the use of a kit according to the second aspect of the present application in a method for detecting malachite green.
The beneficial effects that this application can produce include:
1) the method is based on the principle that protein in a muscle tissue homogenate matrix can be denatured into clusters and ferroferric oxide magnetic particles are wrapped in the matrix by solvent treatment (such as acetonitrile and acetate buffer solution), and solid-liquid separation can be realized under the action of an external magnetic field.
2) The application provides a reagent kit for pretreating malachite green samples in animal muscle tissues based on magnetic separation, reagents used for pretreating the samples are low in price and easy to obtain, a solid-phase extraction column is not needed for purifying the samples, and the cost is low; the kit integrates required reagents into a pretreatment kit, and is convenient to carry and store.
Drawings
FIG. 1 shows the results of a positive surimi assay in one embodiment of the present application.
Detailed Description
The present application will be described in detail with reference to examples, but the present application is not limited to these examples.
Unless otherwise indicated, the materials in the examples of the present application were purchased commercially and tested using instrumentation under conditions and parameters recommended by the manufacturer.
In the examples, the colloidal gold immune layer test strip was prepared based on a malachite green monoclonal antibody, and purchased from south-developing-Japanese New Biotechnology, Inc. of Hangzhou.
Example 1
(1) And reagent preparation:
buffer solution: weighing 5.16 g of p-toluenesulfonic acid, 0.1 g of hydroxylamine hydrochloride and 0.2g of ammonium acetate, adding water for dissolving, diluting to 500mL, and adjusting the pH to 4.5 by using 4mol/L sodium hydroxide.
Fe3O4Magnetic material: 1g of CaCl21g of neutral alumina, 250mg of Fe3O4。
Compounding the solution: 8.00g of sodium chloride, 0.20g of potassium chloride, 0.27g of potassium dihydrogen phosphate and 2.87g of disodium hydrogen phosphate dodecahydrate are weighed out and dissolved in 900mL of water, 0.5mL of Tween-20 is added, the mixture is diluted to 1L with water, the mixture is mixed evenly, and the pH value is adjusted to 7.4 by hydrochloric acid.
(2) And (3) preparation: animal dissection, muscle tissue homogenization, and low-temperature preservation in a refrigerator at-20 ℃;
(3) and (3) extracting: weighing 5 g of minced muscle sample into a centrifugal test tube with a cover, sequentially adding 1ml of buffer solution and 5ml of acetonitrile, fully soaking, oscillating, and adding 2.25 g of Fe3O4Vibrating and mixing the magnetic material mixture, quickly separating the mixture under the action of an external magnet, and filtering the supernatant with a 0.22 mu m organic needle type filter membrane to be purified;
(4) and purifying: transferring 2.5mL of sample extract supernatant into a centrifuge tube, shaking and mixing with 100. mu.L of 0.001mol/L dichlorodicyanobenzoquinone solution, adding 300mg of octadecyl bonded silica gel particles (C)18Granules), nitrogen-blown concentration, and adding 0.2mL of redissolution to dissolve residual impurities.
(5) Dripping 100 μm above compound solution into colloidal gold-labeled micropores, mixing, and dripping into colloidal gold immunochromatographic test strip for direct visual detection.
Example 2
Adding malachite green into the blank fish sample respectively to obtain malachite green standard samples with the concentrations of 1, 2 and 4 mug/kg respectively, extracting according to the method described in the embodiment 1, detecting after purifying, directly measuring by naked eyes, repeating for 6 times, and the results are shown in the table 1; wherein, the visual determination result of the 2 mug/kg malachite green standard sample is shown in figure 1, and the comparison with the blank quality control sample shows that the T line color of the malachite green standard sample is lighter than that of the blank quality control sample, and the result is positive.
TABLE 1
+Positive for
-Negative of
The result shows that the established method has good reliability and can be used for rapidly detecting the malachite green in the aquatic products.
Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.
Claims (10)
1. A method of detecting malachite green, comprising:
a) providing an animal muscle tissue homogenate;
b) adding a reagent 1, a reagent 2 and a magnetic material into the animal muscle tissue homogenate, mixing, and then applying an external magnetic field to realize solid-liquid separation of an animal tissue homogenate solution to obtain a supernatant;
c) and detecting malachite green in the supernatant.
2. The method of claim 1, wherein reagent 1 is a buffer.
3. The method of claim 1, wherein the reagent 2 is acetonitrile;
preferably, the magnetic material comprises 3-5 parts by weight of CaCl23-5 parts of neutral alumina and 1-2 parts of Fe3O4；
Preferably, the external magnetic field comprises a magnetic field generated by a ferromagnetic body or a magnetic field generated by an electric current;
preferably, in the step c), a dichlorodinitrile benzoquinone solution is added into the supernatant;
preferably, in the step c), octadecyl bonded silica gel particles are added into the supernatant;
preferably, in the step c), the method further comprises concentrating the supernatant to remove the solvent, adding a complex solution to dissolve the concentrate, and detecting with a colloidal gold immunochromatographic test strip.
4. The method of claim 2, wherein the buffer is formulated by a method comprising:
dissolving p-toluenesulfonic acid, hydroxylamine hydrochloride and ammonium acetate in water, and adjusting the pH value to 4-5 by using a sodium hydroxide solution to obtain the buffer solution, wherein the concentration of the p-toluenesulfonic acid in the buffer solution is 5-15mg/ml, the concentration of the hydroxylamine hydrochloride in the buffer solution is 0.1-0.3mg/ml, the concentration of the ammonium acetate in the buffer solution is 0.2-0.6mg/ml, and the concentration of the alkaline solution in the buffer solution is 3-5 mol/L.
5. The method according to claim 1, wherein the method for preparing the double solution comprises: dissolving sodium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate dodecahydrate and tween-20 in water, and adjusting the pH to 7-8 by using an acidic solution to obtain the compound solution, wherein in the compound solution, the concentration of the sodium chloride is 9-10g/L, the concentration of the potassium chloride is 0.2-0.3g/L, the concentration of the potassium dihydrogen phosphate is 0.2-0.3g/L, the concentration of the disodium hydrogen phosphate dodecahydrate is 2-3g/L, and the concentration of the tween-20 is 0.3-0.7 ml/L.
6. A kit, comprising: reagent 1, reagent 2 and a magnetic material according to any one of claims 1 to 5.
7. The kit of claim 6, further comprising a solution of dichlorodicyanobenzoquinone.
8. The kit of claim 6, further comprising octadecyl bonded silica gel particles.
9. The kit of claim 6, further comprising the reconstituted solution of claim 9 or 10 and a colloidal gold immunochromatographic strip.
10. Use of a kit according to any one of claims 6 to 9 in a method for detecting malachite green.
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| US20070254323A1 (en) * | 2006-04-21 | 2007-11-01 | Jun Wang | Malachite green derivatives for immunoassay reagents to detect malachite green |
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| CN107064496A (en) * | 2017-06-05 | 2017-08-18 | 苏州快捷康生物技术有限公司 | A kind of malachite green colloidal gold immunochromatographimethod rapid detection card and preparation method thereof |
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