CN104535759A - Clenbuterol hydrochloride bionic immune column and detection method thereof - Google Patents

Abstract

The invention discloses a clenbuterol hydrochloride bionic immune column and a detection method thereof. The bionic immune column includes a solid phase extraction column, a test layer, a sieve plate, a solid phase extraction small column adapter and a syringe; the detection steps are: Activation, sample loading, column washing, addition of enzyme markers, column washing, color development. Qualitative or semi-quantitative clenbuterol content in the sample according to the color depth of the test layer. The biomimetic immune column is simple in preparation, convenient in operation, low in cost, good in specificity, does not require professional training, and has clear and easy-to-distinguish detection results, is easy to popularize, and is suitable for on-site detection.

Description

Clenbuterol hydrochloride bionic immune column and its detection method

technical field

The invention belongs to the interdisciplinary fields of biology, materials, and biological detection, and in particular relates to a clenbuterol hydrochloride bionic immune column and a detection method thereof.

Background technique

Clenbuterol (CLB) is an adrenal nerve stimulant, which has been clinically used to prevent and treat bronchial asthma, chronic bronchitis and other diseases. CLB is a kind of "lean meat extract" with high bioavailability, which can change the metabolic pathways in animals, promote protein synthesis in muscles and bones, accelerate growth, improve carcass quality, and increase lean meat percentage. However, CLB is easy to accumulate in the animal body, and the residual amount in the lung, liver, kidney and other organs, muscle and fat tissue is relatively large, and the residual time is relatively long, and it can accumulate in the human body through the food chain, causing toxic and side effects on the body. The main manifestations are arrhythmia, muscle tremor, dizziness, fatigue, etc., which can be life-threatening in severe cases. Most countries such as my country and the European Union strictly prohibit the application of CLB in livestock and poultry breeding.

The detection methods of clenbuterol mainly include instrumental methods (high performance liquid chromatography, liquid chromatography-mass spectrometry, etc.) and immunoassay methods (ELISA, colloidal gold test strips, etc.). Instrumental analysis is an accurate and sensitive detection method, but it requires expensive instruments and professional operators, and is not suitable for the detection of a large number of samples. The immunoassay method is sensitive, fast, easy to operate, and has a large amount of single-test samples. It can directly detect liquid samples and has a good consistency with instrumental detection methods. However, in this method, the antibody preparation period is long, it is not easy to store, and experimental animals are required.

Contents of the invention

purpose of invention

Aiming at the above-mentioned deficiencies of the prior art, the present invention aims to propose a biomimetic immune column and a detection method thereof that are simple in operation, strong in specificity, low in cost, and can semi-quantify the content of clenbuterol hydrochloride in a sample through color change.

Technical solutions

The present invention is achieved through the following technical solutions:

The clenbuterol hydrochloride bionic immune column is characterized in that it includes a solid phase extraction column, a test layer, a sieve plate, a solid phase extraction column adapter and a syringe; the solid phase extraction column and the syringe are connected through the solid phase extraction column adapter , the sieve plate is divided into an upper sieve plate and a lower sieve plate, the inside of the solid phase extraction column is a test layer, the top of the test layer is equipped with an upper sieve plate, and the bottom is equipped with a lower sieve plate; the test layer is clenbuterol hydrochloride biomimetic antibody and a homogeneous mixture of quartz sand.

The volume of the solid-phase extraction column is 3 mL; the sieve plate is a polypropylene sieve plate, and the pore size of the sieve plate is 20 microns.

Quartz sand is 100 mesh.

The clenbuterol hydrochloride biomimetic antibody is a molecularly imprinted polymer prepared by molecular imprinting technology, which is covalent bond polymerization technology or non-covalent bond polymerization technology.

A detection method of clenbuterol hydrochloride biomimetic immune column as above, characterized in that: the method steps are as follows:

(1) Activation: Phosphate buffer saline (PBS) activation;

(2) Sample loading: inject clenbuterol hydrochloride solution or sample solution into the solid phase extraction column with a pipette, and use the air pressure of the syringe to press the sample solution to the test layer;

(3) Wash the column: wash the column with PBS solution containing Tween;

(4) Add enzyme-labeled probe;

(5) Wash the column: wash the column with PBS solution containing Tween;

(6) Color development: Add TMB single-component color developer to develop color, and finally judge the concentration of clenbuterol hydrochloride in the solution according to the color depth of the test layer. The lighter the color, the greater the content of clenbuterol hydrochloride in the sample.

The pH value of PBS used in step (1) activation is 7.4, and the volume used for activation is 1 mL.

The clenbuterol hydrochloride solution used for sample loading in step (2) is a methanol solution with a volume of 1 mL and an adsorption time of 3 minutes.

The PBS solution used for washing the column in step (3) contains 0.5% Tween 20 by volume, and the dosage is 1 mL.

The enzyme-labeled probe added in step (4) is clenbuterol hydrochloride horseradish peroxidase marker, the amount used is 1 mL of 2000-fold diluted PBS, and the adsorption time is 4 minutes.

The PBS used in step (5) is Tween 20 containing 0.5% by volume, and the dosage is 4 mL.

Step (6) The dosage of TMB single-component color developer is 400 μL, and the color development time is 4 minutes.

Advantages and effects

The present invention has following advantage and beneficial effect:

The clenbuterol hydrochloride biomimetic immune column prepared by the invention is simple to prepare, has short detection time, low detection cost, and can semi-quantify the content of clenbuterol hydrochloride in a sample through the color depth, which is of great significance for improving food safety.

Description of drawings

Fig. 1 is a schematic diagram of the clenbuterol hydrochloride biomimetic immune column of the present invention.

Figure 2 is a schematic diagram of the connected clenbuterol hydrochloride biomimetic immune column.

The reference picture is the color change diagram of the detection results of different clenbuterol hydrochloride concentrations of the bionic immune column of the present invention; the concentrations of clenbuterol hydrochloride in columns 1-4 are 0, 10, 100, 1000 μg/L respectively.

Explanation of reference signs:

1 is the solid phase extraction column; 2 is the uniform mixture of clenbuterol hydrochloride molecularly imprinted biomimetic antibody and quartz sand; 3 is the upper sieve plate of the solid phase extraction column; 4 is the lower sieve plate of the solid phase extraction column; 5 is the solid phase extraction column. Phase extraction small column adapter; 6 is a syringe; 7 is a piston handle.

Detailed ways

The present invention is described in detail below with reference to accompanying drawing:

The invention is a clenbuterol hydrochloride biomimetic immune column, which is characterized in that it comprises a solid phase extraction column 1, a test layer 2, a sieve plate, a solid phase extraction small column adapter 5 and a syringe 6; the solid phase extraction column 1 and The syringe 6 is connected through the solid-phase extraction column adapter 5, and a piston handle 7 is arranged above the syringe 6. The sieve plate is divided into an upper sieve plate 3 and a lower sieve plate 4. The inside of the solid-phase extraction column 1 is the test layer 2, and the test layer The upper sieve plate 3 is installed at the top of the 2, and the lower sieve plate 4 is installed at the bottom; the test layer 2 is a uniform mixture of clenbuterol hydrochloride biomimetic antibody and quartz sand.

The volume of the solid-phase extraction column 1 is 3 mL; the sieve plate is a polypropylene sieve plate, and the pore size of the sieve plate is 20 microns.

Quartz sand is 100 mesh.

The clenbuterol hydrochloride biomimetic antibody is a molecularly imprinted polymer prepared by molecular imprinting technology, which is covalent bond polymerization technology or non-covalent bond polymerization technology.

A detection method of clenbuterol hydrochloride biomimetic immune column as above, characterized in that: the method steps are as follows:

(1) Activation: Phosphate buffer saline (PBS) activation;

(2) Sample loading: inject clenbuterol hydrochloride solution or sample solution into the solid phase extraction column with a pipette, and use the air pressure of the syringe to press the sample solution to the test layer;

(3) Wash the column: wash the column with PBS solution containing Tween;

(4) Add enzyme-labeled probe;

(5) Wash the column: wash the column with PBS solution containing Tween;

(6) Color development: Add TMB single-component color developer to develop color, and finally judge the concentration of clenbuterol hydrochloride in the solution according to the color depth of the test layer. The lighter the color, the greater the content of clenbuterol hydrochloride in the sample.

The pH value of the PBS used in step (1) activation was 7.4, and the volume used for activation was 1 mL.

The clenbuterol hydrochloride solution used for sample loading in step (2) is a methanol solution with a volume of 1 mL and an adsorption time of 3 minutes.

The PBS solution used for washing the column in step (3) contains 0.5% Tween 20 by volume, and the dosage is 1 mL.

The enzyme-labeled probe added in step (4) is clenbuterol hydrochloride horseradish peroxidase marker, the amount used is 1 mL of 2000-fold diluted PBS, and the adsorption time is 4 minutes.

The PBS used in step (5) was Tween 20 containing 0.5% by volume, and the dosage was 4 mL; the dosage of TMB single-component color developer in step (6) was 400 μL, and the color development time was 4 minutes.

The present invention will be further described below in conjunction with specific embodiments.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Example 1

The composition of clenbuterol hydrochloride biomimetic immune column (as shown in Figure 2) includes solid phase extraction column 1, sieve plate (upper sieve plate 3, lower sieve plate 4), solid phase extraction cartridge adapter 5, syringe 6, Test layer 2, the test layer is a uniform mixture of clenbuterol hydrochloride biomimetic antibody and quartz sand.

The lower sieve plate 4, the test layer 2 and the upper sieve plate 3 are loaded into the solid phase extraction column 1 in sequence.

The volume of the solid-phase extraction column is 3mL, the sieve plate is a polypropylene sieve plate, and the pore size of the sieve plate is 20 microns.

The syringe is a medical 5mL syringe.

The quartz sand is 100 mesh quartz sand.

The solid-phase extraction cartridge adapter was purchased from Yuexu Technology (Shanghai) Co., Ltd.

Preparation of clenbuterol hydrochloride biomimetic antibody

The clenbuterol hydrochloride biomimetic antibody was prepared by the covalent method. The specific steps were as follows: take 1 mmol of CLB derivatives in a round bottom flask, then add 5 ml of methanol-ethyl acetate mixed solution (3:2, V:V), After all the solids are dissolved, add 2 mmol of crosslinking agent ethylene glycol dimethacrylate and 30 mg of initiator azobisisobutyronitrile. After all the solids are dissolved, blow nitrogen for 5 minutes to remove oxygen, then seal the round bottom flask and place it in Placed in a 70 ^o C water bath for 48 h. After the polymerization reaction, the polymer biomimetic antibody was ground into particles with a mortar, and then placed in a vacuum oven at 45 ^o C to dry overnight. 1 g of the dried polymer biomimetic antibody was ultrasonically eluted with 200 mL of a mixed solution of hydrochloric acid (3 mol/L) and methanol (10:1, V:V) for 72 h, and then washed with a mixed solution of methanol-glacial acetic acid (9 :1, V:V) Soxhlet extraction of clenbuterol hydrochloride molecules until no clenbuterol hydrochloride was detected. After elution, the polymer biomimetic antibody is dried in a vacuum oven for use.

Preparation of Clenbuterol Hydrochloride Derivatives

Dissolve 10 mmol of clenbuterol hydrochloride in 50 mL of dichloromethane, add 40 mmol of triethylamine to the solution, then dropwise add 20 mmol of methacryloyl chloride, and react overnight at room temperature. After the reaction, wash with saturated sodium bicarbonate solution, separate the organic phase, wash the water phase twice with dichloromethane, combine all the separated organic phases, dry with anhydrous sodium sulfate, and use a rotary evaporator to The organic solvent was removed under reduced pressure, and the residue was purified with petroleum ether-ethyl acetate (3:1, V:V) with a silica gel column (silica gel diameter 200-300 mesh) to obtain the product CLB derivative.

Test layer preparation

Mix 10 mg of clenbuterol hydrochloride biomimetic antibody with 0.2 g of quartz sand and load it into a solid phase extraction column.

Enzyme-labeled probe preparation

The clenbuterol hydrochloride-horseradish peroxidase enzyme-labeled probe was prepared by EDC method.

Determination of detection limit

(1) Activation: Take the prepared biomimetic immune column and activate the column with 1 mL of phosphate buffer with a pH value of 7.4.

(2) Sample loading: Use a pipette to inject 1 mL of clenbuterol hydrochloride methanol solution (0, 10, 100, 1000 μg/L) of different concentrations into the solid phase extraction column, and then connect it through the solid phase extraction cartridge adapter Syringe and solid phase extraction column, push the plunger handle of the syringe, fix the sample solution on the test layer by air pressure, and keep it for 3 minutes. After the time is up, the sample solution is discharged from the solid phase extraction cartridge.

(3) Wash the column: Wash the test layer with 1 mL of PBS containing 0.5% (volume percent) Tween 20.

(4) Add enzyme-labeled probe: Add 1mL horseradish peroxidase-labeled clenbuterol hydrochloride PBS solution (1:2000, volume ratio) to the test layer, and keep it for 4 minutes, and then discharge it through the syringe. phase extraction column.

(5) Wash the column: Wash the column with 1mL×4 (wash the column 4 times) with PBS containing 0.5% (volume percent) Tween 20.

(6) Color development: Add 400 μL TMB single-component color developer to the test layer, observe the color depth after 4 minutes, judge the result in 4-5 minutes, and the result will be invalid if it exceeds 7 minutes.

Judging the content of clenbuterol hydrochloride in the sample loading solvent according to the color depth of the test layer, the lighter the color of the test layer indicates the higher the concentration of clenbuterol hydrochloride in the sample loading solution. The result (see reference picture) is that when the standard clenbuterol hydrochloride concentration is 10 μg/L, the test layer is colored and negative; when the standard clenbuterol hydrochloride concentration is 100, 1000 μg/L, the test layer No color, positive, indicating that the clenbuterol hydrochloride bionic immunoassay column has a detection limit of 100 μg/L for clenbuterol hydrochloride.

specificity test

The prepared bionic immunoassay column was used to detect 100 μg/L and 1000 μg/L clenbuterol hydrochloride and structural analogues terbutaline, isoproterenol, and salbutamol, and the results are shown in Table 1.

Note: Positive result (+): No color in the visually tested layer; Negative result (-): Color in the test area.

The results of the specificity test showed that the clenbuterol hydrochloride biomimetic immunocolumn had good specificity.

Example 2

A clenbuterol hydrochloride biomimetic antibody was prepared by a non-covalent method, and other conditions were the same as in Example 1.

Claims (10)

1. Clenbuterol hydrochloride bionic immune column, characterized in that it includes a solid phase extraction column (1), a test layer (2), a sieve plate, a solid phase extraction cartridge adapter (5) and a syringe (6); The phase extraction column (1) is connected to the syringe (6) through the solid phase extraction column adapter (5), and the sieve plate is divided into an upper sieve plate (3) and a lower sieve plate (4). It is the test layer (2), the top of the test layer (2) is equipped with an upper sieve plate (3), and the bottom end is equipped with a lower sieve plate (4); the test layer (2) is clenbuterol hydrochloride biomimetic antibody and quartz sand homogeneous mixture. 2. The clenbuterol hydrochloride biomimetic immune column according to claim 1, characterized in that: the volume of the solid phase extraction column (1) is 3mL; the sieve plate is a polypropylene sieve plate, and the pore size of the sieve plate is 20 microns. 3. The clenbuterol hydrochloride biomimetic immune column according to claim 1, characterized in that: the quartz sand is 100 mesh. 4. The clenbuterol hydrochloride biomimetic immune column according to claim 1, characterized in that: the clenbuterol hydrochloride biomimetic antibody is a molecularly imprinted polymer prepared by molecular imprinting technology, and the molecular imprinting technology is a covalent bond Polymerization techniques or non-covalent polymerization techniques. 5. A detection method of clenbuterol biomimetic immune column as claimed in claim 1, is characterized in that: the method steps are as follows: (1) Activation: Phosphate buffer saline (PBS) activation; (2) Sample loading: inject clenbuterol hydrochloride solution or sample solution into the solid phase extraction column with a pipette, and use the air pressure of the syringe to press the sample solution to the test layer; (3) Wash the column: wash the column with PBS solution containing Tween; (4) Add enzyme-labeled probe; (5) Wash the column: wash the column with PBS solution containing Tween; (6) Color development: Add TMB single-component color developer to develop color, and finally judge the concentration of clenbuterol hydrochloride in the solution according to the color depth of the test layer. The lighter the color, the greater the content of clenbuterol hydrochloride in the sample. 6. The detection method of clenbuterol hydrochloride biomimetic immunocolumn according to claim 5, characterized in that: the pH value of the PBS used in step (1) activation is 7.4, and the volume used for activation is 1 mL. 7. The detection method of clenbuterol hydrochloride biomimetic immunocolumn according to claim 5, it is characterized in that: the clenbuterol hydrochloride solution used for sample loading in step (2) is a methanol solution, the volume is 1mL, and the adsorption time is 3 minutes. 8. The detection method of clenbuterol hydrochloride biomimetic immune column according to claim 5, characterized in that: the PBS solution used for washing the column in step (3) contains 0.5% Tween 20 by volume, and the dosage is 1 mL. 9. The detection method of clenbuterol hydrochloride biomimetic immunocolumn according to claim 5, characterized in that: the enzyme-labeled probe added in step (4) is clenbuterol hydrochloride horseradish peroxidase marker, Use 1 mL of 2000-fold diluted PBS, and the adsorption time is 4 minutes. 10. The detection method of clenbuterol hydrochloride biomimetic immune column according to claim 5, characterized in that: the PBS used in step (5) is Tween 20 containing 0.5% by volume, and the dosage is 4mL; step (6) The dosage of TMB single-component chromogen is 400 μL, and the color development time is 4 minutes.