Beta2 receptor biosensor as well as preparation method and application thereof

A biosensor, β2 receptor technology, applied in the field of electrochemical biosensors, to achieve high specificity and sensitivity

Inactive Publication Date: 2013-07-10
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]Existing detection technology cannot fundamentally curb the abuse of β2 agonists

Method used

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  • Beta2 receptor biosensor as well as preparation method and application thereof
  • Beta2 receptor biosensor as well as preparation method and application thereof
  • Beta2 receptor biosensor as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Purification and preparation of solubilized β2 receptor

[0033] For the cloning and expression of the human β2 receptor gene in Pichia pastoris, and the preparation of cell membranes of recombinant engineered bacteria, see "Research on the establishment of a new detection system for β2-agonists" (Dai Xiaofeng, master's thesis of Shanghai University, 2004 ), and "An electrochemical receptor biosensor and its application" (Chen Yuguang et al., Invention Patent Application No. 200910196550.4, 2009).

[0034] Preparation of solubilized β2 receptor protein: Solubilize the cell membrane of the above-mentioned recombinant engineering bacteria with 1% DDM (N-dodecyl-β-D-maltoside)-PBS buffer solution, 0??C 45min, 18000g After centrifugation for 20 min, the supernatant was obtained as homogeneous solubilized β2 receptor protein.

[0035] Partial purification of solubilized β2 receptor protein: Add the above solubilized β2 receptor solution to Ni 2+ -Adsorption o...

Embodiment 2

[0036] Example 2: Preparation of β2 receptor working electrode

[0037]Pretreatment of the gold electrode: the gold electrode is firstly ground on 5000-mesh metallographic sandpaper, and then polished with a suspension of alumina powder with a particle size ranging from 1.0 μm, 0.3 μm, and 0.05 μm to make the surface of the gold electrode smooth mirror surface, take it out and ultrasonicate it in absolute ethanol and ultrapure water for 5 minutes, then put the gold electrode into 0.5 M H 2 SO 4 Cyclic voltammetry scan (0 - 1.5 V voltage range) was carried out in , the scan rate was set at 100 mV / s, and the stability was reached in about 20 cycles. The electrodes were then blown dry in nitrogen to obtain bare gold electrodes with clean surfaces.

[0038] Preparation of β2 receptor working electrode:

[0039] a. Immerse the above bare gold electrode in 1 mM mercaptopropionic acid (MPA) solution overnight;

[0040] b. Immerse the above (a) modified gold electrode in a solutio...

Embodiment 3

[0044] Example 3: Electrochemical β2 receptor biosensor applied to the detection of β2 agonist (clenbuterol)

[0045] Operating procedure: In the system of the three-electrode electrochemical sensor

[0046] a. Immerse the β2 receptor working electrode obtained above into the test sample solution or negative control solution containing 100 μL β2 agonist, wash at 0°C for 30 min, and wash with ultrapure water;

[0047] b. Immerse the β2 receptor working electrode after the above a. reaction into 5.0 mL detection buffer solution (20??C), the detection buffer solution is: 0.5 M K3Fe(CN)6 / K4Fe(CN)6 phosphate buffer (50 mM, pH 7.4).

[0048] c. Electrochemical scanning by cyclic voltammetry; parameter settings: scanning range -0.1~0.55 V, scanning rate 100 mV / s.

[0049] Beta2 agonist (clenbuterol) detection characteristics:

[0050] Six serial concentrations (0, 1, 10, 100, 1000, 10000 pM) of clenbuterol (CLE) were prepared separately.

[0051] Three kinds of working electrode...

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Abstract

The invention relates to a beta2 acceptor biosensor, as well as a preparation method and an application thereof. The beta2 receptor biosensor is a three-electrode system sensor, wherein a counter electrode is a platinum electrode, a reference electrode is a saturated calomel electrode, and a work electrode is a gold electrode. The beta2 acceptor biosensor is characterized in that a recombinant beta2 epinephrine purinergic receptor is modified on the gold electrode, and the recombinant beta2 epinephrine purinergic receptor is a base sequence as described in SEQ ID NO: 1. After the beta2 acceptor biosensor is used, the test result that the electrochemical biosensor has false negative can be reduced, the test sensitivity can be further improved, the urgent need to the test of a beta2 agonist field in the field can be preferably met, and a novel technical platform is provided for the food safety test; and therefore, the beta2 acceptor biosensor can be used for the field test for the import and export inspection and quarantine, the food hygiene supervision, a livestock rearing farm and the like, thereby being wide in market application prospect, and relatively good in economic benefit and social benefit.

Description

technical field [0001] The invention relates to an electrochemical biosensor, a preparation method and its application, in particular to a β2 receptor biosensor, a preparation method and its application. Background of the invention [0002] β2 adrenergic agonist (referred to as β2 agonist or β2 ligand, commonly known as clenbuterol) is a class of synthetic hormone drugs, with dozens of family members. Their common mechanism of action is: β2 agonists first bind to β2 adrenergic receptors on the cell membrane (abbreviated as β2 receptors), exert normal physiological effects through the G protein-coupled cAMP signal transduction pathway, and at the same time enhance muscle, The physiological effect of reducing fat has been abused as a growth promoter to increase the lean meat rate of livestock, and the drug residues in meat products seriously endanger people's health. [0003] Existing detection technologies cannot fundamentally curb the abuse of β2 agonists. [0004] Bioele...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327G01N27/48C07K14/72C07K1/22
Inventor 苏洁沈敏戴小峰郭敏顾鸣陈宇光
Owner SHANGHAI UNIV
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