Difunctional G-quadruplex allosteric biosensor for detecting beta-lactoglobulin

A biosensor, lactoglobulin technology, applied in the field of bifunctional G-quadruplex allosteric biosensor, can solve the problems of difficult real-time detection, high assembly cost, limited application, etc., to expand application breadth and improve detection The effect of sensitivity

Active Publication Date: 2022-02-22
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the β-lactoglobulin sensing applications based on nucleic acid aptamers are electrochemical biosensors, which require high assembly costs, require specific chemical group modification and the introduction of signal output components, and are mostly difficult to use for instant detection. application limited

Method used

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  • Difunctional G-quadruplex allosteric biosensor for detecting beta-lactoglobulin
  • Difunctional G-quadruplex allosteric biosensor for detecting beta-lactoglobulin
  • Difunctional G-quadruplex allosteric biosensor for detecting beta-lactoglobulin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Design of β-lactobulin fitting

[0052] 1. Exclusions of different base pairs in the center region of the beta-lactone protein fitting

[0053] At present, there is a need to introduce signal output elements based on the application of the adapter detection of β-mulldine. In order to obtain a beta-lactobulin fitting body having a target identification and signal output dual function, the original β-milk globulin is suitable The ligand is designed and the design results were verified by circular diometric spectroscopy and TMB colorimetric experiments. First, 1 pair T, A, C base is introduced in the central ring region of ORG-G4 (SEQ ID NO.1), respectively, and the sequence is named Enlg1 (SEQ ID No. 2), Enlg2 (SEQ IDN.3) ) And enlg3 (SEQ ID No.4).

[0054] The ability to form a G-tetra-chain structure in different sequences is evaluated by circular dichro spectroscopy. 20 μL 60 μmol·L -1 Different sequences and 20 μL 60 μmol·L -1 Hemin and 160 μL G-tetracene incub...

Embodiment 2

[0084] Example 2. Detection Performance Evaluation of β-Millerin Biosensor

[0085] 1. Principle of detection of beta-lactobulin biosensor

[0086] In order to construct a double function, the adaptive biosensor is used for β-lactobulin detection, by modifying the original ligand ORG-G4, the anti-parallel G-tetra-chain structure is transformed into anti-parallel / parallel mix g - Tetracemic structure, the design of the adapter ENLG2-PL3 has a double function with signal output and target recognition ( image 3 . When the beta-milkurogin is absenced in the detection system, the Enlg2-PL3 is k + Ion-induced folds into a stable mixed parallel structure, and then form a parallel G-tetracene topology in the presence of HEMIN. Only when parallel G-tetracene is assembled with chlorin chlorin, the complex has a hydrogen carboase activity, which can catalyze the TMB substrate reaction to cause blue, and after the sulfuric acid solution is terminated, the solution is yellow, basis The absor...

Embodiment 3

[0093] Example 3. Optimization of detection conditions of beta-lactobulin biosensor

[0094] Optimized the pH of the G-tetracell incubation liquid and the K + NA + The concentration was also optimized for the pH of the TMB color culture. The optimization conditions are divided into 8 groups depending on the pH and ion content of the solution into 8 groups, named M1 to M8. 10 μL 40 μmol·L -1 Enlg2-PL3 and 10 μL 60 μmol·L -1 Hemin, 10 μL 0, 10, 100, 1000 and 4000 μg · ml -1β-lactone protein and 40 μL G-tetracene incubation liquid (specific component Image 6 A) mix, 37 ° C for 20 min, 30 μL of 0.8 μmol·L -1 TMB color culture (pH 3.9 and 8.4) was added to the reaction mixture for 20 min catalytic reaction. Subsequently, 30 μL 2 mol·L -1 Hide 2 SO 4 The solution is terminated to the catalytic reaction. Finally, the absorbance was measured at 450 nm using the enzyme label. The activity change of DNA bionic enzymes before and after the addition of the experimental group and the control g...

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Abstract

The invention discloses a difunctional G-quadruplex allosteric biosensor for detecting beta-lactoglobulin. The difunctional G-quadruplex allosteric biosensor is prepared by the following steps: (1) designing a difunctional G-quadruplex aptamer of beta-lactoglobulin; (2) optimizing the reaction conditions of the beta-lactoglobulin biosensor; and (3) detecting the beta-lactoglobulin. According to the invention, the design principle is that the beta-lactoglobulin biosensor sequence is subjected to conformational change before and after a detection target is introduced, the catalase activity of G-quadruplex/hemin DNA bionic enzyme is influenced, and then beta-lactoglobulin detection is realized; and the G-quadruplex sequence responds to the target concentration, such that the solution presents the corresponding colorimetric gradient after 3, 3', 5, 5'-tetramethyl benzidine (TMB) color development is catalyzed so as to finally achieve the simple, rapid, low-cost and ultra-sensitive colorimetric detection of the beta-lactoglobulin.

Description

Technical field [0001] The present invention belongs to the field of biosensor, in particular to detect the double function G-tetrachristor converted biosensor of β-lactobulin. Background technique [0002] Food allergies have become a serious global public health problem, about 5% of adults and 8% of children allergic to food, of which about 1 / 3 is caused by boiling milk allergies, and the incidence rate has a rise. Killing is one of the eight major allergic foods identified by the United Nations Food and Agriculture Organization, FAO and the World Health Organization (WHO), and infants are the main crowd of bovy allergies. my country's latest epidemiological surveys have shown that there are about 2.69% of infants and young children with milk proteins. In food processing, bovine proteins are widely added to various foods, and there is also the possibility of milk protein pollution in food processing, storage, transportation, etc.. Thus, the risk of tap milk allergic populations...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/68G01N33/53
CPCC12N15/115G01N33/68G01N33/53C12N2310/16G01N2333/4713
Inventor 许文涛田洪涛王鑫昕陈可仁朱龙佼杜再慧田晶晶
Owner CHINA AGRI UNIV
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