A β-adrenergic receptor agonist multi-residue detection immunosensor and its detection method

Abstract

The invention discloses an electrochemical immunosensor capable of detecting various beta-adrenergic receptor stimulants (short for beta-stimulants). The sensor comprises a base electrode, a graphene / chitosan complex membrane and multi-determinant antigens of beta-stimulants, wherein the graphenee / chitosan complex membrane is modified on the surface of the base electrode, and the multi-determinant antigens of the beta-stimulants are fixed on the surface of the graphene / chitosan complex membrane. The multi-determinant antigens fixed on the surface of the sensor are salbutamol-ractopamine-BSA or salbutamol-ractopamine-OVA. The invention also provides an electrochemical immunodetection method for various beta-stimulant residues. The electrochemical immunosensor can be used for detecting six beta-stimulants including clenbuterol, salbutamol, ractopamine, terbutaline, mabuterol and tulobutezol through carrying out cross immune reaction on the beta-stimulants based on wide-spectrum specific antibodies of the beta-stimulants by taking K3[Fe(CN)6] as a probe.

Description

technical field

[0001] The invention belongs to the technical field of food safety detection and analytical chemistry, and relates to an electrochemical immunosensor based on antibody cross-reaction and a detection method thereof, in particular to a β-adrenoceptor agonist (hereinafter referred to as β-agonist) Multi-residue detection immunoelectrochemical sensor and its detection method. Background technique

[0002] β-adrenergic receptor agonists, referred to as β-agonists, are the chemical residue hazards with the highest detection rate and the most serious hazards in animal-derived foods, the most famous of which is clenbuterol (CL) , commonly known as "clenbuterol". In the 1980s, researchers from the U.S. fatty amine company accidentally discovered that "lean meat powder" can enhance the decomposition of animal fat, promote protein synthesis, greatly shorten the time to market of meat products, and significantly improve carcass lean meat rate, feed return rate and econo...

Examples

Embodiment 1

[0036] Example 1 Preparation of multi-cluster antigen (SAL-RAC-BSA conjugate)

[0037] 1) Coupling the SAL hapten on BSA by the mixed anhydride method, and then purifying:

[0038] Dissolve 0.2 mmol salbutamol sulfate in 5-10 ml absolute ethanol, add 0.2 mmol glutaric anhydride, react at room temperature for 3-5 h, and centrifuge. Dissolve the solid in the lower layer in 5-10 ml of N,N-dimethylformamide, add 0.2 mmol of tri-n-butylamine and 0.2 mmol of isobutyl chloroformate, and continue the reaction for 1-3 h. The above reaction solution was slowly added to 5-10 ml of phosphate buffer solution containing 5-10 mg / ml BSA, and reacted overnight at room temperature. The above reaction solution is dialyzed 3 to 6 times with phosphate buffer solution, and freeze-dried to obtain the freeze-dried powder of the SAL-BSA conjugate. According to the UV-Vis absorption spectra of SAL, BSA and SAL-BSA conjugates ( figure 1 ) to calculate the available coupling ratio, BSA:SAL = 1:8.5;

...

Embodiment 2

[0041] Example 2 Preparation of broad-spectrum specific antibody (anti-SAL-RAC-BSA antibody)

[0042] Healthy white rabbits weighing about 2 kg were immunized with the SAL-RAC-BSA conjugate synthesized in Example 1 as an immunogen. For the first immunization, 0.25 mg of immunogen was mixed with an equal amount of Freund's complete adjuvant, fully emulsified, and injected subcutaneously at multiple points on the back. Two weeks later, the same dose of immunogen and the same amount of Freund's incomplete adjuvant were used for emulsification and booster immunization, and booster immunization was performed every two weeks, for a total of three times. Ten days after the last immunization, blood was collected from the jugular vein of the white rabbits, placed at 4°C for 30 minutes, and then purified by ammonium sulfate multi-stage precipitation to obtain the anti-SAL-RAC-BSA polyclonal antibody.

Embodiment 3

[0043] Example 3 Electrochemical immunosensor and its preparation

[0044] A glassy carbon electrode with a diameter of 3 mm was sequentially filled with Al with a diameter of 0.3 μm and 0.05 μm 2 o 3 Grinding with polishing powder, followed by ultrasonic cleaning with absolute ethanol-distilled water and distilled water for 5 min, and then rinsed with distilled water. 4 μl of graphene / chitosan dispersion was drop-coated on the electrode, and then 2 μl of 0.2 mg / ml albuterol-ractopamine-bovine serum albumin solution was drop-coated on the surface of the electrode and dried at room temperature. Finally, the electrodes were soaked in 5% BSA solution and incubated in a 37°C oven for 30 min to block the remaining active sites.

technical field

[0001] The invention belongs to the technical field of food safety detection and analytical chemistry, and relates to an electrochemical immunosensor based on antibody cross-reaction and a detection method thereof, in particular to a β-adrenoceptor agonist (hereinafter referred to as β-agonist) Multi-residue detection immunoelectrochemical sensor and its detection method. Background technique

[0002] β-adrenergic receptor agonists, referred to as β-agonists, are the chemical residue hazards with the highest detection rate and the most serious hazards in animal-derived foods, the most famous of which is clenbuterol (CL) , commonly known as "clenbuterol". In the 1980s, researchers from the U.S. fatty amine company accidentally discovered that "lean meat powder" can enhance the decomposition of animal fat, promote protein synthesis, greatly shorten the time to market of meat products, and significantly improve carcass lean meat rate, feed return rate and econo...

Examples

Embodiment 1

[0036] Example 1 Preparation of multi-cluster antigen (SAL-RAC-BSA conjugate)

[0037] 1) Coupling the SAL hapten on BSA by the mixed anhydride method, and then purifying:

[0038] Dissolve 0.2 mmol salbutamol sulfate in 5-10 ml absolute ethanol, add 0.2 mmol glutaric anhydride, react at room temperature for 3-5 h, and centrifuge. Dissolve the solid in the lower layer in 5-10 ml of N,N-dimethylformamide, add 0.2 mmol of tri-n-butylamine and 0.2 mmol of isobutyl chloroformate, and continue the reaction for 1-3 h. The above reaction solution was slowly added to 5-10 ml of phosphate buffer solution containing 5-10 mg / ml BSA, and reacted overnight at room temperature. The above reaction solution is dialyzed 3 to 6 times with phosphate buffer solution, and freeze-dried to obtain the freeze-dried powder of the SAL-BSA conjugate. According to the UV-Vis absorption spectra of SAL, BSA and SAL-BSA conjugates ( figure 1 ) to calculate the available coupling ratio, BSA:SAL = 1:8.5;

...

Embodiment 2

[0041] Example 2 Preparation of broad-spectrum specific antibody (anti-SAL-RAC-BSA antibody)

[0042] Healthy white rabbits weighing about 2 kg were immunized with the SAL-RAC-BSA conjugate synthesized in Example 1 as an immunogen. For the first immunization, 0.25 mg of immunogen was mixed with an equal amount of Freund's complete adjuvant, fully emulsified, and injected subcutaneously at multiple points on the back. Two weeks later, the same dose of immunogen and the same amount of Freund's incomplete adjuvant were used for emulsification and booster immunization, and booster immunization was performed every two weeks, for a total of three times. Ten days after the last immunization, blood was collected from the jugular vein of the white rabbits, placed at 4°C for 30 minutes, and then purified by ammonium sulfate multi-stage precipitation to obtain the anti-SAL-RAC-BSA polyclonal antibody.

Embodiment 3

[0043] Example 3 Electrochemical immunosensor and its preparation

[0044] A glassy carbon electrode with a diameter of 3 mm was sequentially filled with Al with a diameter of 0.3 μm and 0.05 μm 2 o 3 Grinding with polishing powder, followed by ultrasonic cleaning with absolute ethanol-distilled water and distilled water for 5 min, and then rinsed with distilled water. 4 μl of graphene / chitosan dispersion was drop-coated on the electrode, and then 2 μl of 0.2 mg / ml albuterol-ractopamine-bovine serum albumin solution was drop-coated on the surface of the electrode and dried at room temperature. Finally, the electrodes were soaked in 5% BSA solution and incubated in a 37°C oven for 30 min to block the remaining active sites.

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