CN110672399A - Sample pretreatment method suitable for immunological detection of drug residues in food - Google Patents
Sample pretreatment method suitable for immunological detection of drug residues in food Download PDFInfo
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Abstract
The invention provides a sample pretreatment method suitable for immunological detection of drug residues in food, which comprises the following steps: (1) treating the homogenized tissue sample with the extracting solution, and centrifuging the treated sample to collect supernatant; (2) mixing the supernatant collected in the step (1) with a polarity regulator to obtain a mixed solution; the mixed solution and the aqueous solution are not mutually soluble; (3) transferring the drug residue in the mixed solution in the step (2) into an aqueous solution or a buffer solution with pH of 5.0-9.0, and directly using the collected aqueous solution or the buffer solution with pH of 5.0-9.0 for immunological detection analysis. The target substance extracted by the method has no organic solvent interference, can be directly used for immunological method detection, simplifies the operation steps, improves the extraction efficiency and the detection efficiency, and does not influence the immunological detection result, so the method has important application value in the pretreatment application of the drug residue in food.
Description
Technical Field
The invention belongs to the field of food safety detection, and particularly relates to a sample pretreatment method suitable for immunological detection of drug residues in food.
Background
With the improvement of science and technology and the improvement of the living standard of people, the requirement on food safety is increasingly strict, so that the research on the effectiveness of the food safety detection technology is more urgent. In addition to improving the awareness of prevention, people must select a detection method which is convenient to popularize for supervision, and the development of immunological detection technology provides technical possibility for achieving the aim.
The immunological method is established on the basis of the specific reaction of antigen and antibody, so that the derived detection methods are various, almost all the immunological methods can be used for food safety detection, and the immunological methods with popularization potential in the food safety detection application mainly comprise the following methods: 1. enzyme-linked immunosorbent assay; 2. immune colloidal gold technology; 3. immunomagnetic bead method; 4. immunocapture PCR method; 5. immunosensor technology; 6. immunofluorescence techniques; 7. an immune chip.
No matter which immunological detection method is used, the pretreatment of a sample to extract a target object is an essential important link. However, in the conventional sample pretreatment method, the extracted target substance is polydispersed in an organic solvent, but the accuracy of the immunological detection method is affected by the organic solvent, so that a step of removing the organic solvent is required before the immunological detection, thereby reducing the detection efficiency of the target substance. Therefore, there is a need for further development of a sample pretreatment method directly applied to immunological detection of drug residues in foods.
Disclosure of Invention
In order to solve the problems in the prior art, the present invention aims to provide a sample pretreatment method adapted to immunological detection of drug residues in food, so that the sample is directly applied to the immunological detection without a post-treatment procedure after the pretreatment, and the detection efficiency of the drug residues in food is improved.
In order to achieve the purpose, the invention provides the following technical scheme:
a sample pretreatment method suitable for immunological detection of drug residues in food specifically comprises the following steps:
(1) treating the homogenized tissue sample with an extraction solution, and then collecting the supernatant by centrifugation; wherein the extracting solution is ethyl acetate or a mixture of ethyl acetate and one or more organic solvents;
(2) mixing the supernatant collected in the step (1) with a polarity regulator to obtain a mixed solution; the mixed solution and the aqueous solution are not mutually soluble;
(3) transferring the drug residue in the mixed solution in the step (2) into an aqueous solution or a buffer solution with pH of 5.0-9.0, discarding the mixed solution in the step (2), and directly using the collected aqueous solution or the buffer solution with pH of 5.0-9.0 for immunological detection analysis.
Further, the polarity regulator in step (2) is a non-polar organic solvent: n-hexane, cyclohexane, petroleum ether, chloroform, dichloromethane or n-pentane.
Further, the drug residue in the food product is soluble in ethyl acetate or a mixture of ethyl acetate and one or more organic solvents.
Further, the method for transferring the drug residue in the mixed solution of the step (2) to the aqueous solution or the buffer solution with the pH value of 5.0-9.0 in the step (3) comprises the following steps: pouring the mixed solution in the step (2) into a solid phase extraction column, enabling the mixed solution to completely flow through a column tube, and discarding an effluent liquid; adding water or buffer solution with pH of 5.0-9.0 as eluting solvent into the solid phase extraction column, eluting the drug residue adsorbed on the solid phase extraction column, collecting the eluate, and directly using for immunological detection and analysis.
Further, the method for transferring the drug residue in the mixed solution of the step (2) to the aqueous solution or the buffer solution with the pH value of 5.0-9.0 in the step (3) comprises the following steps: and (3) pouring the aqueous solution or the buffer solution with the pH value of 5.0-9.0 and the mixed solution in the step (2) into a separating funnel for fully and uniformly mixing, collecting the lower-layer aqueous solution or the buffer solution with the pH value of 5.0-9.0 after the solutions are layered, discarding the mixed solution in the step (2), and directly using the lower-layer solution for immunological detection and analysis.
Further, the mass percent of the ethyl acetate in the extracting solution is 20-100 wt%.
Has the advantages that: the invention provides a sample pretreatment method suitable for immunologically detecting drug residues in food, which comprises the steps of adding a nonpolar organic solvent into an extracting solution, thereby improving the hydrophobicity of the solvent and reducing the polarity of the solvent, further reducing the solubility of the drug residues to be separated in the solvent, mixing the solvent with an aqueous solution or a buffer solution with larger polarity, and increasing the solubility of the drug residues in the aqueous solution, so that the drug residues are transferred into the aqueous solution or the buffer solution and can be directly used for immunologically analyzing and detecting. The method for transferring the drug residue into the aqueous solution or the buffer solution can be used for extracting in an adsorption and elution mode by means of a solid-phase extraction column or directly mixing with a solvent and then extracting in an extraction mode, and the extraction mode is simple and convenient to operate. The drug residue obtained by the method is directly dispersed in aqueous solution or buffer solution, can be directly used as a substance to be detected for immunological analysis and detection, avoids the process of removing organic solvent after using organic solvent for elution, simplifies the operation steps and greatly improves the extraction efficiency. The pretreatment analysis of the actual sample shows that the target substance extracted by the pretreatment method does not interfere with the detection effect of the immune method, and the method has important application value in the pretreatment application of food drug residues.
Drawings
FIG. 1 is a graph showing the results of the test in example 1;
FIG. 2 is a graph showing the results of the test in example 2;
FIG. 3 is a graph showing the results of the test in example 3.
Detailed Description
The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The colloidal gold test strips used in the following examples were purchased from the market.
Example 1 Nitrofuran metabolite detection
1. Sample pretreatment
Weighing 6g of a homogeneous tissue sample in a 50mL centrifuge tube, sequentially adding 4mL of deionized water, 5mL of 1mol/L hydrochloric acid (3.1.14) and 0.2mL of 10mmol/L o-nitrobenzaldehyde solution, and fully oscillating for 3 min; incubating the centrifuge tube in water bath at 60 deg.C for 60 min; sequentially adding 5mL of 0.1mol/L dipotassium phosphate solution, 0.4mL of 1mol/L sodium hydroxide solution and 6mL of ethyl acetate, fully mixing for 3min, and centrifuging for 5min at 4000r/min at room temperature (20-25 ℃); transferring 3mL of centrifuged upper layer liquid into a 15mL centrifuge tube, adding 10mL of n-hexane solution, mixing for 4-5 times by reversing the upside down, and centrifuging for 1min at 4000 r/min. Connecting a negative pressure solid phase extraction device, connecting a 30mL syringe needle cylinder above the solid phase extraction column, pouring all the supernate into the 30mL syringe needle cylinder, starting an air pump, controlling the liquid flow rate to be about 1 drop/second, enabling all the liquid in the syringe to flow through the solid phase extraction column, removing the solution in the solid phase extraction column as clean as possible, and then closing the air pump. The pipette below the solid phase extraction column was replaced with a clean centrifuge tube and 1mL of aqueous solution was added to the solid phase extraction column. And opening the air pump, and controlling the flow rate of the liquid to be about 1 drop/second, so that the liquid in the centrifugal tube is the liquid to be detected after all the liquid in the solid-phase extraction column flows into the centrifugal tube.
2. Addition recovery experiment
Adding nitrofuran four metabolites with different contents into the negative samples respectively, wherein the adding contents of the nitrofuran four metabolites are 0 (blank control), 0.5, 1 and 2 mu g/kg respectively. And (3) pretreating the negative sample added with the target according to the pretreatment method in the step 1 to obtain the object to be detected.
The obtained substance to be detected was analyzed by colloidal gold test paper, and the detection result is shown in fig. 1.
Example 2 Chloramphenicol assay
1. Sample pretreatment
Weighing 5g of animal tissue sample, homogenizing in a homogenizer, placing the homogenized tissue sample in a centrifuge tube, adding 5mL of 20% sodium chloride aqueous solution, shaking and mixing uniformly, adding 10mL of ethyl acetate extractant, mixing fully for 2-5min, centrifuging at the room temperature of 20-25 ℃ at the rotation speed of 4000r/min for 10min, sucking 5mL of centrifuged supernatant, adding 15mL of petroleum ether, shaking for 1min to mix uniformly, adding 1mL of 0.01M PBS, shaking for 1min to mix uniformly, centrifuging or standing, collecting the layered lower-layer aqueous phase, and if necessary, extracting the organic solvent in the lower-layer liquid again by using 1 ~ 5mL of n-hexane or petroleum ether.
2. Addition recovery experiment
Different contents of chloramphenicol were added to the negative samples, respectively, at 0 (blank), 0.05, 0.1, 0.2. mu.g/kg. And (3) pretreating the negative sample added with the target according to the pretreatment method in the step 1 to obtain the object to be detected.
The obtained substance to be detected was analyzed by colloidal gold test paper, and the detection result is shown in fig. 2.
Example 3 Metronidazole detection
1. Sample pretreatment
Weighing 5g of animal tissue sample, homogenizing in a homogenizer, and placing the homogenized tissue sample in a centrifuge tube; then adding 10mL ethyl acetate, shaking and extracting for 3min, and centrifuging for 5min at 4000r/min at room temperature (20-25 ℃); 5mL of centrifuged upper layer liquid is removed; then adding 10mL of normal hexane and 5mL of petroleum ether, shaking for 2min to uniformly mix the mixture, connecting a negative pressure solid phase extraction device, connecting a 30mL syringe needle cylinder above the solid phase extraction column, pouring the mixed solution into the 30mL syringe needle cylinder, opening the negative pressure air extraction device to enable the liquid to completely flow through the solid phase extraction column, controlling the flow rate of the liquid to be about 1 drop/second, and closing the negative pressure air extraction device after removing the solution in the solid phase extraction column as much as possible. And (3) eluting the solid phase extraction column by using 1mL of aqueous solution as an eluent, collecting the eluent, and controlling the flow rate of the liquid to be about 1 drop/second.
2. Addition recovery experiment
And respectively adding metronidazole with different contents into the negative samples, wherein the addition contents of the metronidazole are respectively 0 (blank control), 0.5, 1.0 and 2.0 mu g/kg. And (3) pretreating the negative sample added with the target according to the pretreatment method in the step 1 to obtain the object to be detected.
The obtained substance to be detected was analyzed by colloidal gold test paper, and the detection result is shown in fig. 3.
The target object detected by the specific embodiment is a small molecule, so the colloidal gold test strip adopts a competition method, the color of the detection strip corresponding to the blank control sample is deepest according to the detection result, and the color of the corresponding detection strip gradually becomes lighter along with the increase of the concentration of the target object, so that the detection rule of the small molecule target object detected by the competition method is met. Therefore, the substance to be detected extracted by the pretreatment method does not interfere with a detection system, and can be directly applied to immunological detection and analysis. According to the embodiment of the invention, a series of standard substances with concentration gradients can be further detected by the colloidal gold test strip, then a standard curve for detecting the target substance is made for the strip at the detection line position through image analysis, and then the detection result of the actual sample is analyzed by using the standard curve, so that the actual detection result of the target substance in the sample is obtained.
Claims (6)
1. A sample pretreatment method suitable for immunological detection of drug residues in food is characterized by comprising the following steps:
(1) treating the homogenized tissue sample with an extraction solution, and then collecting the supernatant by centrifugation; wherein the extracting solution is ethyl acetate or a mixture of ethyl acetate and one or more organic solvents;
(2) mixing the supernatant collected in the step (1) with a polarity regulator to obtain a mixed solution; the mixed solution and the aqueous solution are not mutually soluble;
(3) transferring the drug residue in the mixed solution in the step (2) into an aqueous solution or a buffer solution with pH of 5.0-9.0, discarding the mixed solution in the step (2), and directly using the collected aqueous solution or the buffer solution with pH of 5.0-9.0 for immunological detection analysis.
2. The method for pretreating a sample suitable for immunologically detecting drug residues in food as claimed in claim 1, wherein the polarity-adjusting agent in step (2) is a non-polar organic solvent: n-hexane, cyclohexane, petroleum ether, chloroform, dichloromethane or n-pentane.
3. The method of claim 1, wherein the food drug residue is soluble in ethyl acetate or a mixture of ethyl acetate and one or more organic solvents.
4. The method for pretreating a sample suitable for immunologically detecting drug residues in food as claimed in claim 1, wherein the step (3) of transferring the drug residues in the mixed solution of step (2) into an aqueous solution or a buffer solution with pH of 5.0-9.0 comprises: pouring the mixed solution in the step (2) into a solid phase extraction column, enabling the mixed solution to completely flow through a column tube, and discarding an effluent liquid; adding water or buffer solution with pH of 5.0-9.0 as eluting solvent into the solid phase extraction column, eluting the drug residue adsorbed on the solid phase extraction column, collecting the eluate, and directly using for immunological detection and analysis.
5. The method for pretreating a sample suitable for immunologically detecting drug residues in food as claimed in claim 1, wherein the step (3) of transferring the drug residues in the mixed solution of step (2) into an aqueous solution or a buffer solution with pH of 5.0-9.0 comprises: and (3) pouring the aqueous solution or the buffer solution with the pH value of 5.0-9.0 and the mixed solution in the step (2) into a separating funnel for fully and uniformly mixing, collecting the lower-layer aqueous solution or the buffer solution with the pH value of 5.0-9.0 after the solutions are layered, discarding the mixed solution in the step (2), and directly using the lower-layer solution for immunological detection and analysis.
6. The method of claim 1, wherein the mass percentage of ethyl acetate in the extract is 20-100 wt%.
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