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A preparation method of double-enzyme co-immobilized copper nanoflower material and its application in glucose detection

A copper nanometer and nanoflower technology, applied in the field of glucose detection, can solve the problems of affecting sensitivity, time-consuming and cumbersome operations, restricting the development of glucose detection, etc., and achieve the effects of improving efficiency, sensitivity and high peroxidase activity.

Active Publication Date: 2021-11-09
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] However, because the enzymes that catalyze the two reaction steps require different optimum temperatures and pHs, the current glucose concentration detection requires multi-step operations, which are time-consuming and cumbersome, and the hydrogen peroxide produced by glucose oxidation may be catalyzed in two steps. Decomposition occurs during the reaction, which affects the sensitivity of detection, which restricts the further development of glucose detection

Method used

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  • A preparation method of double-enzyme co-immobilized copper nanoflower material and its application in glucose detection
  • A preparation method of double-enzyme co-immobilized copper nanoflower material and its application in glucose detection
  • A preparation method of double-enzyme co-immobilized copper nanoflower material and its application in glucose detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Solid phase synthesis of Dh-β-Ala-His-Glu

[0041] (1) Resin swelling: weigh 0.1mmol Rink-NH 2 Resin, add dichloromethane reagent to fully infiltrate the resin, swell for 1 hour, remove the dichloromethane by suction filtration, and wash the resin 6 times with dimethylformamide (DMF).

[0042] (2) Deprotection: to step (1) processed Rink-NH 2 Add DEP (DMF containing 20% ​​piperidine) solution to the resin, shake at room temperature for 15-30min, filter with suction, wash the resin 6 times with DMF, and use the color developer (A: 5% ninhydrin ethanol solution B: 80% Phenol ethanol solution) to detect the deprotection of the amino group, when the resin particles all turn blue-purple, the deprotection is complete.

[0043] (3) Linking amino acids: Fmoc-Glu(otBu)-OH, PyBOP (benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate), HOBT (1-Hydroxybenzotriazole), NMM (N- methylmorpholine) and Rink-NH 2 The resin is added to the reactor sequentially a...

Embodiment 2

[0050] Embodiment 2: The solid-phase synthesis of Dh-β-Ala-His-Lys

[0051] (1) Repeat step (1) of Example 1.

[0052] (2) Repeat step (2) of Example 1.

[0053] (3) Connecting amino acids: Fmoc-Lys(Boc)-OH, PyBOP (benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate), HOBT (1-Hydroxybenzotriazole), NMM (N- methylmorpholine) and Rink-NH 2 The resin is added to the reactor sequentially at a molar ratio of 3:3:3:6:1, and about 2 / 3 of the container volume of dimethylformamide is added to shake at room temperature for 1.5-2 hours, filtered with suction, and the resin is washed with dimethylformamide 6 times, use a chromogenic reagent to detect the amino acid connection, and the resin particles all become colorless, indicating that the reaction is complete.

[0054] (4) Repeat step (4) of Example 1.

[0055] (5) Repeat step (5) of Example 1.

[0056] (6) Repeat step (6) of Example 1.

[0057] (7) Repeat step (7) of Example 1.

[0058] (8) Repeat step (8) of Ex...

Embodiment 3

[0060] Example 3: Solid phase synthesis of Dh-β-Ala-His-Asp.

[0061] (1) Repeat step (1) of Example 1.

[0062] (2) Repeat step (2) of Example 1.

[0063] (3) Connecting amino acids: Fmoc-Asp(OtBu)-OH, PyBOP (benzotriazol-1-yl-oxytripyrrolidinylphosphonium hexafluorophosphate), HOBT(1-Hydroxybenzotriazole), NMM(N- methylmorpholine) and Rink-NH 2 The resin is added to the reactor sequentially at a molar ratio of 3:3:3:6:1, and about 2 / 3 of the container volume of dimethylformamide is added to shake at room temperature for 1.5-2 hours, filtered with suction, and the resin is washed with dimethylformamide 6 times, use a chromogenic reagent to detect the amino acid connection, and the resin particles all become colorless, indicating that the reaction is complete.

[0064] (4) Repeat step (4) of Example 1.

[0065] (5) Repeat step (5) of Example 1.

[0066] (6) Repeat step (6) of Example 1.

[0067] (7) Repeat step (7) of Example 1.

[0068] (8) Repeat step (8) of Example 1...

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Abstract

The invention provides a preparation method of double-enzyme co-immobilized copper nanoflower material and its application in glucose detection. The preparation method of copper nanoflower material comprises the following steps: (1) adding heme short-term dissolved in the reaction vessel Phosphate buffer solution of peptide compound and glucose oxidase, placed at room temperature; (2) Slowly add CuSO 4 aqueous solution, so that the CuSO in the solution 4 (3) leave it alone; (4) centrifuge, discard the supernatant, and leave the precipitate; (5) add ultrapure water and absolute ethanol in turn to wash the precipitate; (6) precipitate Put it in a reaction vessel, heat it in a water bath until the absolute ethanol is completely evaporated, and obtain it; the novel hybrid nanometer prepared by the present invention immobilizes glucose oxidase and hypoheme short peptide compound at the same time, and realizes the construction of a double-enzyme system for glucose detection. It has not only the activity of glucose oxidase, but also the activity of peroxidase, so as to realize the cascade detection of glucose.

Description

technical field [0001] The invention relates to the technical field of glucose detection, in particular to a preparation method of a dual-enzyme co-immobilized copper nanoflower material and its application in glucose detection. Background technique [0002] As an important research field in bioanalysis, glucose detection has been widely concerned because of its important application value in biology, clinical medicine, and food production. [0003] At present, the commonly used glucose detection method based on glucose oxidase and peroxidase is divided into two steps: first, glucose is oxidized by glucose oxidase, and glucose oxidase can catalyze the oxidation of glucose to produce gluconic acid and H in the presence of oxygen. 2 o 2 . Then, the H obtained from the previous step reaction 2 o 2 Oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) under the catalysis of peroxidase produces blue oxidized TMB polymer (oxTMB). oxTMB has a maximum light absorption value at 652nm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/33G01N30/02
CPCG01N21/33G01N30/02
Inventor 王丽萍袁野付振东赵祯毓
Owner JILIN UNIV
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