CN115014890A

CN115014890A - Sample processing method and method for measuring nitrofuran metabolite

Info

Publication number: CN115014890A
Application number: CN202210542080.8A
Authority: CN
Inventors: 张毅; 顾玉蓉; 杨玮民; 李绍峰; 吕剑锋; 温嘉恒; 陈奕丽; 陈春源
Original assignee: Shenzhen Polytechnic
Current assignee: Shenzhen Polytechnic
Priority date: 2022-05-18
Filing date: 2022-05-18
Publication date: 2022-09-06
Anticipated expiration: 2042-05-18
Also published as: CN115014890B

Abstract

The application relates to the technical field of biochemical analysis, in particular to a sample treatment method and a method for measuring nitrofuran metabolites. The sample processing method of the present application comprises the steps of: mixing an initial sample for measuring nitrofuran metabolites with a derivatization reagent under an acidic condition for derivatization treatment to obtain a derivatized sample; mixing the derived sample with polyethylene glycol alkyl phenyl ether, and performing cloud point extraction treatment under an alkaline condition to obtain a micelle phase solution; diluting the micelle phase solution with a buffer solution, and then filtering to obtain a sample solution to be detected. The sample treatment method is simple in process, and can effectively separate the nitrofuran metabolites, so that a good sample solution to be detected is provided for measuring the content of the nitrofuran metabolites, and the process does not use toxic solvents, so that the method has the characteristics of economy, safety, high efficiency, simplicity, convenience, time saving and environmental friendliness, and has a good application prospect.

Description

Sample processing method and method for measuring nitrofuran metabolite

Technical Field

The application belongs to the technical field of biochemical analysis, and particularly relates to a sample processing method and a method for measuring nitrofuran metabolites.

Background

Nitrofurans (Nitrofurans) are a class of synthetic broad-spectrum antibiotics whose basic structure is 5-nitrofuran, including furazolidone (furazolidone), furaltadone (furaltadone), nitrofurantoin (nitrofurazone), nitrofurazone (nitrofurazone), and the like. Nitrofurans are commonly used for the treatment and prevention of digestive system infections in swine, fish and poultry caused by Salmonella and E.coli infections, these drugs have a short half-life and can be rapidly metabolized in animals, and protein-bound metabolites can produce stable residues. The metabolites corresponding to furazolidone, furaltadone, nitrofurantoin and furacilin are respectively: 3-Amino-2-Oxazolidinone (3-Amino-2-oxozolidinone, AOZ), 5-methylmorpholine-3-Amino-2-oxazolidone (5-morpholino-methyl-3-Amino-2-oxozolidinone, AMOZ), 1-Amino-2-hydantoin (1-Aminohydantoin hydrochloride, AHD), and Semicarbazide (SEM).

At present, cloud point extraction technology is widely applied to research such as analytical chemistry, food and trace metal element analysis, and the like, thereby gaining wide attention of researchers at home and abroad. The cloud point extraction condition is mild, and a plurality of heat-unstable organic macromolecular substances and volatile organic compounds can be treated. Cloud point extraction has been developed from aqueous two-phase extraction, which mainly uses two important functions of surfactants: solubilization and cloud point phenomena, in which a surfactant is easily dissolved in water to form a clear solution when the temperature is lowered, and the solubility is reduced when the temperature is raised to a certain extent, and turbidity, precipitation, and delamination occur in an aqueous solution, so that the extraction method using this phenomenon is called cloud point extraction (CPE method).

The method for measuring the residue of the nitrofuran metabolites comprises a high performance liquid chromatography, a liquid chromatography-tandem mass spectrometry method, an enzyme-linked immunosorbent assay and the like. These methods have long detection time and large instrument volume, and are not suitable for being brought to a field for detection. In addition, the immune colloidal gold rapid detection kit meets the rapid detection requirement, and has the advantages of short detection time, high sensitivity, convenient carrying and the like. The measurement of the nitrofuran metabolites mainly aims at fresh and aquatic products, samples to be measured need to be pretreated, and for food fishes and the like rich in lipid matrixes, n-hexane is used for removing fat. However, the existing sample treatment method has complex steps and poor separation effect, so that the measurement effect of the nitrofuran metabolites needs to be improved.

Disclosure of Invention

The application aims to provide a sample treatment method and a method for measuring nitrofuran metabolites, and aims to solve the technical problem of simply and effectively separating the nitrofuran metabolites from a sample so as to detect the nitrofuran metabolites.

In order to achieve the purpose of the application, the technical scheme adopted by the application is as follows:

in a first aspect, the present application provides a method of sample processing comprising the steps of:

mixing an initial sample for measuring nitrofuran metabolites with a derivatization reagent under an acidic condition for derivatization treatment to obtain a derivatized sample;

mixing the derived sample with polyethylene glycol alkyl phenyl ether, and performing cloud point extraction treatment under an alkaline condition to obtain a micelle phase solution;

and diluting the micelle phase solution with a buffer solution, and then filtering to obtain a sample solution to be detected.

In a second aspect, the present application provides a method for determining a nitrofuran metabolite, comprising the following steps:

according to the sample processing method, a sample solution to be detected is obtained;

and detecting the nitrofuran metabolites in the sample solution to be detected by adopting a colloidal immune gold test strip.

The method comprises the steps of mixing an initial sample for measuring the nitrofuran metabolites with a derivatization reagent under an acidic condition to obtain a derivatized sample, mixing the derivatized sample with polyethylene glycol alkyl phenyl ether under an alkaline condition to perform cloud point extraction treatment, separating the derivatized nitrofuran metabolites in a micelle phase solution better under the action of the polyethylene glycol alkyl phenyl ether, and finally diluting and filtering the micelle phase solution to obtain a sample solution to be measured suitable for rapidly measuring the nitrofuran metabolites. The sample treatment method is simple in process, and can effectively separate the nitrofuran metabolites, so that a good sample solution to be detected is provided for the determination of the content of the nitrofuran metabolites, and the process does not use toxic solvents, and has the characteristics of economy, safety, high efficiency, simplicity, convenience, time saving and environmental protection, so that the method has a good application prospect.

According to the method for determining the nitrofuran metabolites, provided by the second aspect of the application, a sample solution to be detected is obtained by using a sample processing method specific to the application, and then the derivatized nitrofuran metabolites in the sample solution to be detected are detected by using a colloidal immunogold test strip; the sample treatment method is simple in process, and can effectively separate the nitrofuran metabolites, so that a good sample solution to be detected is provided for the determination of the content of the nitrofuran metabolites, and the detection based on the immune colloidal gold test strip has the advantages of short detection time, high sensitivity, convenience in carrying and the like, so that the nitrofuran metabolites in samples such as fresh and aquatic products can be better determined.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a graph of experimental data for determining the optimal concentration of Tritonx-114 provided in the examples of the present application;

FIG. 2 is a diagram illustrating the judgment of the result of the reagent plate for rapid immunodolloid gold detection provided in the embodiment of the present application;

FIG. 3 is a graph of blank measurements for two extractants provided in the examples herein;

FIG. 4 is a graph of SEM measurements corresponding to two extractants provided in the examples herein;

FIG. 5 is a graph of AMoz assay results provided in an embodiment of the present application;

FIG. 6 is a graph of AOZ measurements provided in the examples of the present application.

Detailed Description

In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly apparent, the present application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.

In this application, the term "and/or" describes an association relationship of associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a alone, A and B together, and B alone. Wherein A and B can be singular or plural. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.

It should be understood that, in various embodiments of the present application, the sequence numbers of the above-mentioned processes do not mean the execution sequence, some or all of the steps may be executed in parallel or executed sequentially, and the execution sequence of each process should be determined by its function and inherent logic, and should not constitute any limitation to the implementation process of the embodiments of the present application.

The terminology used in the embodiments of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in the examples of this application and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

The weight of the related components mentioned in the description of the embodiments of the present application may not only refer to the specific content of each component, but also represent the proportional relationship of the weight among the components, and therefore, the content of the related components is scaled up or down within the scope disclosed in the description of the embodiments of the present application as long as it is scaled up or down according to the description of the embodiments of the present application. Specifically, the mass described in the specification of the embodiments of the present application may be a mass unit known in the chemical industry field such as μ g, mg, g, kg, etc.

The terms "first" and "second" are used for descriptive purposes only and are used for distinguishing purposes such as substances from one another and are not to be construed as indicating or implying relative importance or to implicitly indicate the number of technical features indicated. For example, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX, without departing from the scope of embodiments of the present application. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature.

In a first aspect, an embodiment of the present application provides a sample processing method, including the following steps:

s01: mixing an initial sample for measuring nitrofuran metabolites with a derivatization reagent under an acidic condition for derivatization treatment to obtain a derivatized sample;

s02: mixing the derived sample with polyethylene glycol alkyl phenyl ether, and performing cloud point extraction treatment under an alkaline condition to obtain a micelle phase solution;

s03: and diluting the micelle phase solution with a buffer solution, and filtering to obtain a sample solution to be detected.

The sample processing method provided by the embodiment of the application is a sample processing method for rapidly determining nitrofuran metabolites, and comprises the steps of firstly mixing an initial sample containing the nitrofuran metabolites with a derivatization reagent under an acidic condition to perform derivatization processing to obtain a derivatized sample, then mixing the derivatized sample with polyethylene glycol alkyl phenyl ether to perform cloud point extraction processing under an alkaline condition, enabling the derivatized nitrofuran metabolites to be better separated in a micellar phase solution under the action of the polyethylene glycol alkyl phenyl ether in the process, and finally diluting and filtering the micellar phase solution to obtain a sample solution to be detected suitable for rapidly determining the nitrofuran metabolites. The sample treatment method is simple in process, and can effectively separate the nitrofuran metabolites, so that a good sample solution to be detected is provided for the determination of the content of the nitrofuran metabolites, and the process does not use toxic solvents, and has the characteristics of economy, safety, high efficiency, simplicity, convenience, time saving and environmental protection, so that the method has a good application prospect.

Specifically, in step S01, the initial sample may be an original sample to be used for measuring the nitrofuran metabolites, such as fresh, aquatic product, etc. Specifically, the edible muscle part of an aquatic product sample such as fish may be homogenized by a homogenizer to obtain an initial sample. Of course, the initial sample may be other sample solutions containing nitrofuran metabolites.

In order to better improve the detection sensitivity of the nitrofuran metabolites, the initial sample and the derivatization reagent are mixed under an acidic condition for derivatization treatment, so that a derivatized sample is obtained, and the nitrofuran metabolites can be better determined by detecting the nitrofuran metabolite derivatives. Further, the derivatization reagent can be 2, 3-dichloro-5, 6-dicyan-p-benzoquinone (DDQ), and the 2, 3-dichloro-5, 6-dicyan-p-benzoquinone can react with the nitrofuran metabolites to be derivatized so as to better detect the nitrofuran metabolites. During the derivatization treatment, acidic conditions may be provided by a hydrochloric acid solution. For example, the treatment of derivatization is carried out by adjusting the acidity with a 0.2mol/L hydrochloric acid solution, specifically, by adjusting the acidity so that 4 kinds of nitrofuran metabolites are hydrolyzed under weakly acidic conditions.

Further, the initial sample and the derivatization reagent are mixed and subjected to derivatization treatment under an acidic condition at the temperature of 58-62 ℃ for 50-70 min. Along with the lengthening of the derivatization time, the derivatization efficiency is gradually increased, and the generation amount of the derivative product is relatively stable after the derivatization time exceeds a certain time; along with the increase of the derivatization temperature, the derivatization efficiency is gradually improved, and when the derivatization temperature reaches a certain degree, the generation amount of a derivatization product is relatively stable, so that the derivatization temperature is finally selected to be 58-62 ℃ for 50-70 min in consideration of the requirement of a reagent plate as a rapid detection method, and the derivatization effect is better under the condition; for example, the incubation can be performed for 50-70 min under the condition of a constant-temperature water bath at 58-62 ℃.

Specifically, in step S02, the structural formula of the polyethylene glycol alkyl phenyl ether is as follows:

wherein R is an alkyl group having 6 to 10 carbon atoms, and n is an integer of 5 to 10.

The polyethylene glycol alkyl phenyl ether is used as a nonionic surfactant and is an environment-friendly surfactant, the polyethylene glycol alkyl phenyl ether corresponds to a cloud point temperature, and can be mutually dissolved with water when the cloud point temperature is lower than the cloud point temperature, so that the polyethylene glycol alkyl phenyl ether can be used for cloud point extraction treatment; further, the polyethylene glycol alkyl phenyl ether is triton X-114 (polyethylene glycol tert-octyl phenyl ether), and the structural formula is as follows:

n＝7～8。

due to the lower cloud point temperature (23 ℃) of Triton X-114, below which TrionX-114 is miscible with water; above this temperature, the separation into an aqueous phase and a micellar phase occurs; triton x-114 has a good solubilizing effect on biomolecules, and thus can be used for separation of aqueous phase proteins and lipoproteins.

Furthermore, polyethylene glycol alkyl phenyl ether is used as a surfactant, the addition amount has a certain influence on the extraction efficiency of the nitrofuran metabolites, and in the embodiment of the application, 2.0g of the edible muscle part of the homogenized aquatic product sample is taken as an example, and 4ml of aqueous solution of Tritonx-114 with the mass concentration of 1%, 2%, 3% and 4% is respectively added. Results as shown in fig. 1, the extraction efficiency of the target analytes (AOZ, AMOZ, AHD, and SEM) showed a tendency to rise before fall with increasing concentration of triton x-114. This is because when the concentration of the polyethylene glycol alkylphenyl ether is too low, the surfactant-enriched phase is too small to separate the phases during extraction and stratification, and as the concentration increases, more and more micelles are formed, and the solubilization effect is stronger, which is more advantageous for extraction of the target analyte. However, after the concentration of the Tritonx-114 is higher than 3%, the extraction efficiency tends to be reduced, because the concentration of the surfactant is increased, the solution viscosity is increased, the diffusion capacity is weakened, the micelle is difficult to permeate into the matrix, the internal structure of the micelle is changed, and the extraction rate is reduced. Therefore, the optimal concentration of Tritonx-114 is about 3%, such as 2.8-3.2%, in the embodiment of the present application, 2.8-3.2% Tritonx-114 solution can be selected for the cloud point extraction treatment.

Furthermore, the temperature of the cloud point extraction treatment is 38-42 ℃ and the time is 5-15 min. The temperature of the cloud point extraction treatment can be higher than the cloud point temperature of the polyethylene glycol alkyl phenyl ether, so that the nitrofuran metabolites can be better separated in the micellar phase solution. Because the equilibrium temperature is lower than the cloud point temperature of the polyethylene glycol alkyl phenyl ether, the two phases are in a mutual soluble state, when the equilibrium temperature rises, the equilibrium temperature exists in a hydrated form in an aqueous solution, so that H bonds in polyethylene glycol alkyl phenyl ether micelles are broken and dehydrated, the volume of a polyethylene glycol alkyl phenyl ether enriched phase is reduced, the polyethylene glycol alkyl phenyl ether is easier to settle, and the better separation of two aqueous phases is facilitated.

Further, mixing the derivatized sample and polyethylene glycol alkyl phenyl ether under an alkaline condition, wherein the alkaline condition for performing cloud point extraction treatment is that the pH value is 7.0-7.5; in cloud point extraction, a target analyte and polyethylene glycol alkyl phenyl ether micelles are subjected to hydrophobic distribution as a main mode, and the pH value of a sample solution influences the effect of two-phase distribution. When the pH of the extract is too high, the polyethylene glycol alkyl phenyl ether-rich phase is transferred from the lower phase to the upper phase of the solution, so that the two phases are not convenient to separate. Therefore, the alkaline condition is selected to be pH value of 7-7.5.

Among them, Tritonx-14 has a cloud point temperature of 23 ℃ and is therefore 15 to 25 ℃ higher than the cloud point temperature when it is desired to achieve an equilibrium temperature efficiently. However, high temperatures can cause decomposition of the drug and the extraction rate can be reduced. In addition, extending the equilibration temperature for a longer period of time facilitates distribution of the target analyte in the cloud point, increasing the equilibration time increases the extraction yield, but too long equilibration times do not have a significant effect on the extraction yield, increases the sample processing time, and affects the experimental cycle. According to the method, the balance temperature, the balance time and the temperature response value range of the target analyte of the Triton X-114 are examined through experiments, and the optimal conditions of the Trion X-114 for cloud point extraction of the nitrofuran metabolites are finally obtained: 2.8-3.2% Trion X-114, pH 7-7.5, equilibrium temperature 35 deg.C, and equilibrium time 10 min.

Further, in the step of mixing the derivatized sample with polyethylene glycol alkyl phenyl ether, sodium chloride may be added. NaCl belongs to electrolyte, and the addition of NaCl can lead to the increase of the aggregation number of polyethylene glycol alkyl phenyl ether micelles, so that the ionic strength of the solution can be further increased, the density of a water phase is increased, the two-phase separation is accelerated, the cloud point temperature is reduced, and the solubilization of a target analyte solution is improved, so that the addition of NaCl is favorable for cloud point extraction.

Specifically, in step S03, the micelle phase solution is diluted with a buffer solution and then filtered to obtain a sample solution to be tested. The buffer solution is selected from a tris solution, such as 8-12 mmol/L tris solution, so that the micelle phase solution can be diluted better. Further, the filtration treatment included filtration through a 0.22 μm filter. And filtering to obtain a uniformly dispersed sample solution to be detected so as to facilitate the subsequent determination of the nitrofuran metabolites.

In conclusion, in the cloud point extraction, polyethylene glycol alkyl phenyl ether is used as a surfactant, the Critical Micelle Concentration (CMC) is achieved in a solution, the solution is fully vortexed and vibrated, the solution is heated to the cloud point in a water bath, the temperature of the solution is kept for a period of time, the water phase is removed after centrifugal separation, a micelle phase solution containing the derivatized nitrofuran metabolites is obtained, and the micelle phase is properly diluted, so that a sample solution to be detected which can be directly used for the determination of the colloidal immunogold test strip is obtained.

In a second aspect, the embodiments of the present application provide a method for determining a nitrofuran metabolite, including the following steps:

t01: according to the sample processing method, a sample solution to be detected is obtained;

t02: and (3) detecting the nitrofuran metabolites in the sample solution to be detected by adopting a colloidal immunogold test strip.

The method for determining the nitrofuran metabolites provided by the embodiment of the application uses the sample solution to be detected obtained by the specific sample processing method of the application, and then adopts the colloidal immunogold test strip to detect the nitrofuran metabolites in the sample solution to be detected; the sample treatment method is simple in process, and can effectively separate the nitrofuran metabolites, so that a good sample solution to be detected is provided for the determination of the content of the nitrofuran metabolites, and the detection based on the immune colloidal gold test strip has the advantages of short detection time, high sensitivity, convenience in carrying and the like, so that the nitrofuran metabolites in samples such as fresh and aquatic products can be better determined.

According to the embodiment of the application, by applying the principle of competitive inhibition immunochromatography to the colloidal gold test strip, nitrofuran metabolites in a sample are combined with a specific antibody marked by colloidal gold in the flowing process, so that the combination of the antibody and an antigen-BSA conjugate on an NC membrane detection line (T line) is inhibited, and the change of the color depth of the detection line is caused; no matter whether the sample contains the substance to be detected or not, the quality control line (C line) is developed, and the detection is effective. Specifically, the step of detecting the sample solution to be detected by using the colloidal immunogold test strip comprises the following steps: and (3) dripping 70-80 mu L of sample solution to be detected into a sample hole of the colloidal immune gold test strip, reacting for 8-12 min, and judging the result.

In the embodiment of the application, Triton X-114 is used as an extractant for extracting nitrofuran metabolites, conditions such as optimal concentration, pH value, equilibrium time, equilibrium temperature and the like in low cloud point extraction of Triton X-114 are explored, and a sample processing method is established by using the conditions of cloud point extraction. Meanwhile, based on the sample processing method, a measuring method of the nitrofuran metabolites is determined. According to the method, samples containing about 20ppb nitrofuran metabolites (AOZ/SEM/AMOZ) are subjected to hydrolytic derivatization, Triton X-114 is added to extract a target solution, a slow release solution is added after extraction is completed, the sample solution to be detected is obtained after filtration, and then the sample solution is titrated into a colloidal immune gold test strip for manual interpretation to determine the detection result of the target. Therefore, Triton X-114 can effectively extract the nitrofuran metabolites, and the traditional method for extracting the nitrofuran metabolites by using an organic extractant ethyl acetate is broken through, and the method disclosed by the application does not use a toxic solvent, and has the advantages of economy, safety, high efficiency, simplicity, time saving, environmental friendliness and the like.

The following description is given with reference to specific examples.

Example 1

1.1 Experimental materials and instruments

AOZ, AMOZ, AHD, SEM standards (purity > 97%, LGC Labar GmbH), nitrofuran metabolite standard derivatization reagent (DDQ) (purity > 98%, tianjin altar technologies, ltd), hydrochloric acid (analytical grade, sekkaido, huachengda chemical ltd), dipotassium hydrogen phosphate (analytical grade, west longa chemical ltd), triton x-114 (molecular biology grade, YEASWN), 2-nitrobenzaldehyde (analytical grade, jiangsu ningkang chemical ltd), TBS buffer (Solarbio), and water for laboratory use is ultrapure water. The main instruments and equipment used in the experiment are as follows in table 1:

TABLE 1

1.2 preparation of reagents

(1) AOZ and AMOZ standard stock solutions: 1.0 mg/ml. Accurately weighing 10mg of standard substance, dissolving with methanol, diluting to a constant volume of 10ml in a volumetric flask, and storing at 4 ℃.

(2) SEM standard stock solution: 1.0mg/ml, dissolving the standard substance with methanol, metering to a 10ml volumetric flask, and storing at 4 ℃.

(3) ADH standard stock solution: 1.0mg/ml, dissolving the standard substance with methanol, metering to a 10ml volumetric flask, and storing at 4 ℃.

(4)10mM Tris, 150mM NaCl, pH 7.4: 1L of the packaged TBS buffered saline (1xTBS) was taken and dissolved in 1000ml of purified water.

(5) Triton x-114 (15%, w/w): measuring 16.68ml or less of triton X-114, adding into 100ml of TBS solution, gradually diluting to 1%, 2%, 3%, 4%, 5% and refrigerating for later use.

(6)0.2mol/L hydrochloric acid solution: 0.6ml of concentrated hydrochloric acid was measured and diluted to 100ml with water.

(7)0.05mol/L o-nitrobenzaldehyde solution: 0.0378g was weighed out and dissolved in 5ml of methanol.

(8)1mol/L dipotassium hydrogen phosphate solution: 17.42g of dipotassium hydrogenphosphate are weighed out and dissolved in 100ml of water.

(9)10mmol/L Tris solution: weighing 1.211g, dissolving in 80ml water, adding appropriate hydrochloric acid to adjust pH to 8.0, and adding water to make volume to 1L.

1.3 sample treatment method

Homogenizing edible muscle part of fish and aquatic product sample with homogenizer, weighing 2.0g homogenized sample in 50mL centrifuge tube to obtain initial sample, adding 5mL 0.2mol/L hydrochloric acid solution and 0.15mL derivatization reagent (DDQ), and mixing for 3 min; and (3) incubating for 60min under the condition of constant-temperature water bath at 60 ℃ to obtain a derivatized sample.

Taking out the derivatized sample, adding 3-5 mL of 1.0mol/L dipotassium phosphate solution and 0.4mL of 1.0mol/L sodium hydroxide solution, and adjusting the pH value to 7.5; adding 4ml of 3% triton x-114, performing vortex oscillation for 2min, performing constant-temperature water bath at 40 ℃ for 5min to obtain turbid solution, then immersing the test tube in an ice bath for 5min, centrifuging for 5min at 4000r/min to promote the separation of a surfactant-rich phase, precipitating the surfactant phase at the bottom of a conical tube, collecting a bottom concentrate by using a micro-injector, placing the recovered liquid drop in a sample injection vial, then adding a proper amount of 10mmol/L of tris (hydroxymethyl) aminomethane solution, fully and uniformly mixing, and filtering by a 0.22 mu m filter membrane to obtain the solution of the sample to be detected.

Example 2

Detection of nitrofurans metabolites

2.1 detection with colloidal immunogold test strip

The test strip applies the principle of competitive inhibition immunochromatography, and the derivatized nitrofuran metabolites in a sample are combined with the specific antibody marked by colloidal gold in the flowing process, so that the combination of the antibody and the antigen-BSA conjugate on an NC membrane detection line (T line) is inhibited, and the change of the shade of the color of the detection line is caused; no matter whether the sample contains the substance to be detected or not, the quality control line (C line) will be developed, indicating that the detection is effective.

The operation steps of the colloidal immune gold test strip are as follows: the test paper strip and the sample to be detected are at room temperature. ② the test strip is taken out from the original packaging bag and is used as soon as possible within 60min after being opened. And thirdly, sucking about 75 mu L of sample solution to be detected by a micropipette and vertically dropping the sample solution into the sample hole (S hole). And fourthly, starting timing of liquid flow, reacting for 10min, judging the result according to the detection requirement of the colloidal immune gold test strip, and determining the result to be invalid at other times.

The results of colloidal immunogold are shown in FIG. 2: a is invalid: the absence of a line C indicates that the operating process is incorrect or that the test strip has failed; in this case, the instructions should be read carefully again and retested with a new test strip. b is negative: the color development of the T line is stronger than that of the C line or five obvious differences from the color development of the C line indicate that the sample does not contain the substance to be detected or is lower than the detection limit. c is positive: the T line is obviously weaker than the C line or does not develop color, and the concentration of the substance to be detected in the sample is equal to or higher than the detection limit.

2.2 SEM assay

Respectively making blank samples and accurately transferring 0.2ml of SEM standard solution with the concentration of 100ng/ml to a negative standard sample, placing the negative standard sample in a 50ml polyethylene centrifuge tube, performing sample treatment by taking the SEM standard solution as an initial sample according to the step of '1.3 sample treatment method' in example 1 to obtain a sample solution to be detected (meanwhile, extracting a target analyte by taking ethyl acetate as an extracting agent disclosed by No. 783 of Ministry of agriculture, centrifuging, discarding a solution of the non-target analyte, adding 1ml of 10mmol/L of tris solution to dilute the target, filtering with a 0.22 mu m filter membrane to obtain a sample solution as a comparison), dripping the sample solution on a colloidal immune gold test strip to observe the result, and performing blank experiments by using two extracting agents (Triton x-114 and comparative ethyl acetate of the application) to prove that the detection result of the SEM colloidal immune gold test strip is effective, as shown in figure 3, the results were detected by SEM on both extractants, as shown in fig. 4.

2.3 AMOZ assay

And (3) respectively making a blank sample and accurately transferring 0.2ml of 100ng/ml AMOZ standard solution, adding the blank sample and the AMOZ standard solution into a negative standard sample, placing the negative standard sample into a 50ml polyethylene centrifuge tube, performing sample treatment by taking the AMOZ standard solution as an initial sample according to the step of the 1.3 sample treatment method in the embodiment 1 to obtain a sample solution to be detected, and then dropwise adding the sample solution to a colloidal immunogold test strip to observe a result. A blank experiment conducted by the Tritonx-114 extractant proves that the AMOZ colloidal immune gold test strip detection result is effective (on the left side of the figure 5), and the Tritonx-114 extractant can detect the extraction of the labeled AMOZ target (on the right side of the figure 5).

2.4 AOZ assay

And (3) respectively making a blank sample and accurately transferring 0.2ml of 100ng/ml of AOZ standard solution, adding the AOZ standard solution into a negative standard sample, placing the negative standard sample into a 50ml polyethylene centrifuge tube, performing sample treatment by taking the AOZ standard solution as an initial sample according to the step of the 1.3 sample treatment method in the embodiment 1 to obtain a sample solution to be detected, and then dropwise adding the sample solution to the colloidal immunogold test strip to observe a result. A blank experiment carried out by using the Tritonx-114 extractant proves that the detection result of the AOZ colloidal immune gold test strip is effective (on the left side of a figure 6), and the Tritonx-114 extractant can detect the extraction of a labeled AOZ target (on the right side of the figure 6).

In summary, according to the AOZ, AMOZ and SEM immune colloidal gold rapid detection reagent plate, the color development difference is developed by T, C lines during detection, and the determination result is positive by manual judgment. According to the ethyl acetate organic solvent extraction method in the 785 bulletin of the Ministry of agriculture, the requirement of manual judgment through T, C line color development difference during detection by using AOZ, AMOZ and SEM immune colloidal gold rapid detection reagent plates is met, and the detection result is positive. The color development effect of the device is better, the detection time is short, the sensitivity is high, the carrying is convenient, and the device has the advantages of economy, safety, high efficiency, simplicity, time saving and environmental protection.

The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method of sample processing, comprising the steps of:

and diluting the micelle phase solution with a buffer solution, and filtering to obtain a sample solution to be detected.

2. The method of sample processing according to claim 1, wherein the polyethylene glycol alkylphenyl ether has the following structural formula:

3. The method of sample processing of claim 2 wherein the polyethylene glycol alkylphenyl ether is triton X-114, having the formula:

4. the sample processing method according to claim 1, wherein the alkaline condition is a condition of pH of 7.0 to 7.5; and/or the presence of a gas in the gas,

the temperature of the cloud point extraction treatment is 38-42 ℃, and the time is 5-15 min.

5. The method of claim 1, wherein the step of mixing the derivatized sample with polyethylene glycol alkyl phenyl ether further comprises adding sodium chloride.

6. The sample processing method according to any one of claims 1 to 5, wherein the temperature of the derivatization treatment is 58 to 62 ℃ and the time is 50 to 70 min; and/or the presence of a gas in the gas,

the filtration treatment included 0.22 μm membrane filtration.

7. The method of any of claims 1 to 5, wherein the derivatizing agent is selected from the group consisting of 2, 3-dichloro-5, 6-dicyan p-benzoquinone; and/or the presence of a gas in the gas,

the buffer solution is selected from a tris solution; and/or the presence of a gas in the atmosphere,

the initial sample is selected from the group consisting of edible muscle from aquatic samples.

8. A method for measuring nitrofuran metabolites is characterized by comprising the following steps:

the sample treatment method according to any one of claims 1 to 7, obtaining a sample solution to be tested;

9. The assay method of claim 8, wherein the step of detecting the test sample solution with a colloidal immunogold test strip comprises: and (3) dripping 70-80 mu L of the sample solution to be detected into the sample hole of the colloidal immune gold test strip, reacting for 8-12 min, and judging the result.

10. The assay of claim 8, wherein the nitrofurans metabolites comprise at least one of AOZ, AMOZ, AHD, and SEM.