An immunoelectrochemical sensor for detecting seven β-adrenergic receptor agonists, its preparation method and application

A technology of receptor agonist and epinephrine, which is applied in the field of food safety detection and analytical chemistry, can solve the problems of complex operation, high cost of chromatographic detection, and prone to missed detection, and achieves huge market potential, simple and practical detection operation strong value effect

Active Publication Date: 2018-06-26
NANJING NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] Chromatography is the main method for the detection of β-agonist veterinary drug residues, but the detection cost of chromatography is high, the operation is complicated, and a large number of negative samples are repeatedly detected; while the enzyme-linked immunoassay has the advantages of strong specificity and simple sample pretreatment, but it is specific The strong characteristic also determines its fatal shortcoming: an enzyme-linked immunosorbent assay kit or test strip can only detect one kind of veterinary drug residue, but cannot detect other similar veterinary drug residues, which is the reason for missed detection ( Such as the "Shuanghui Clenbuterol" event that caused a sensation in the country in 2011); in addition, with the emergence of multiple alternatives to β-agonists, if a complete test is performed, a sample needs to use multiple test products, which greatly increases the test cost and cost. Working hours, lost the meaning of quick screening

Method used

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  • An immunoelectrochemical sensor for detecting seven β-adrenergic receptor agonists, its preparation method and application
  • An immunoelectrochemical sensor for detecting seven β-adrenergic receptor agonists, its preparation method and application
  • An immunoelectrochemical sensor for detecting seven β-adrenergic receptor agonists, its preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0026] Synthesis of Example 1β-agonist universal hapten 4-(4-(2-tert-butylamino) 1-hydroxyethyl) phenylbutyric acid (TBPBA)

[0027] (1) Benzyl tert-butylamine (NS-0)

[0028] Dissolve 4.8ml of benzaldehyde in absolute ethanol, then add 5ml of tert-butylamine, react at room temperature for 2h, then add 1.8g of sodium borohydride for 1h. After extraction with dichloromethane, it was purified by silica gel column to obtain product NS-06.2g.

[0029] (2) 4-(4-Bromoacetyl)phenylbutanoic acid (NS-1)

[0030] Dissolve 8.56g of anhydrous aluminum chloride in dichloromethane, add 1.64ml of bromoacetyl bromide to react for 0.5h, then add 2.19g of 4-phenylbutyric acid to react for 3-5h. After extraction with dichloromethane and evaporation of the solvent, 3.5 g of the product were obtained.

[0031] (3) Methyl 4-(4-bromoacetyl)phenylbutyrate (NS-2)

[0032] Dissolve 2.85g NS-1 in methanol, add concentrated sulfuric acid and heat to reflux for 3h. The methanol was distilled off and ...

Embodiment 2

[0041] Embodiment 2 uses the preparation of hapten-bovine serum albumin (TBPBA-BSA) conjugate

[0042] Dissolve 0.2 mmol TBPBA in absolute ethanol, add an equal amount of N-hydroxysuccinimide (NHS) and 1-[3-(dimethylamino)propyl]-3 ethylcarbodiimide carbonate ( EDC), react at room temperature for 3 to 5 hours. The above reaction solution was slowly added to 5-10 ml of phosphate buffer solution containing 5-10 mg / ml bovine serum albumin (BSA), and reacted overnight at room temperature. The above reaction solution is dialyzed with phosphate buffer solution for 3 to 6 times, and freeze-dried to obtain the freeze-dried powder of TBPBA-BSA conjugate.

Embodiment 3

[0043] Preparation of embodiment 3 broad-spectrum specific antibody (anti-TBPBA-BSA antibody)

[0044] Healthy white rabbits weighing about 2 kg were immunized with TBPBA-BSA conjugates as immunogens. 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. 10 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-TBPBA-BSA polyclonal antibody.

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Abstract

The invention discloses an immune-electrochemistry sensor for detecting seven beta-adrenergic receptor agonists, and a manufacture method and application of the immune-electrochemistry sensor. The immune-electrochemistry sensor comprises a substrate glass electrode, and a film layer and a detection layer which sequentially coat the surface of the substrate glass electrode, wherein the material of the detection is a beta-agonist general hapten 4-(4-(2-terttert-butylamino) 1-ethoxy) phenylbutyric acid solution. The method comprises the following steps: firstly dispensing graphene / chitosan dispersion liquid on the surface of the clean substrate glass electrode, drying to form a film formation layer, dispensing the 4-(4-(2-terttert-butylamino) 1-ethoxy) phenylbutyric acid solution on the surface of the film layer, and then drying. The sensor is based on the cross immunoreactions of a beta-agonist broad specificity antibody on various beta-agonists, and can realize the detection of seven beta-agonists including clenbuterol, salbutamol, mabuterol, brombuterol, cimbuterol, tulobuterol and terbutaline.

Description

technical field [0001] The invention belongs to the technical field of food safety detection and analytical chemistry, and specifically relates to an immunoelectrochemical sensor for detecting seven beta-adrenoceptor agonists, a preparation method and application thereof. Background technique [0002] β-adrenoceptor 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 economic benefits . Then it was rapidly promoted all over the world. In the late 1980s, it was introduced into my country and pro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/543G01N27/30G01N27/327
CPCG01N27/308G01N27/327G01N33/5438
Inventor 邵科峰黄爱兰赵波吴珺
Owner NANJING NORMAL UNIVERSITY
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