Ethanol oxidizing enzyme film using polycarbonate film as substrate and method for making same

A technology of polycarbonate membrane and alcohol oxidase, applied in biochemical equipment and methods, fixed on/in organic carrier, microbial measurement/inspection, etc., can solve complex enzyme membrane and increase mass transfer resistance , unfavorable reactant diffusion and other issues, to achieve the effect of high sensitivity and short response time

Inactive Publication Date: 2009-03-11
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the pretreatment of polycarbonate membrane by plasma polymerization technology makes the preparation of enzyme membrane too complicated. In addition, the use of polycarbonate membrane with smaller pore size increases the mass transfer resistance, which is not conducive to the diffusion of reactants.

Method used

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  • Ethanol oxidizing enzyme film using polycarbonate film as substrate and method for making same
  • Ethanol oxidizing enzyme film using polycarbonate film as substrate and method for making same
  • Ethanol oxidizing enzyme film using polycarbonate film as substrate and method for making same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] (1) Cut the polycarbonate film with an average pore diameter of 0.45μm and a thickness of 10μm into a disc with a diameter of 10mm, and then stick the cut polycarbonate film on the O-shaped rubber ring and dry it for use;

[0023] (2) Immerse the polycarbonate film with the O-shaped rubber ring in a phosphate buffer solution with a pH of 7.5 for 6 hours, then take it out and blow dry with high-purity nitrogen;

[0024] (3) Spread 5μL of ethanol oxidase solution with a concentration of 1.02U / μL on the treated polycarbonate membrane to form a round spot of the enzyme layer with a diameter of 5mm. Dry the enzyme membrane in a refrigerator at 4°C. hour;

[0025] (4) Hang the enzyme membrane in a closed container containing a 50% glutaraldehyde aqueous solution, and cross-link it in a refrigerator at 4°C for 12 hours;

[0026] (5) Immerse the enzyme membrane in a phosphate buffer with a pH of 7.5 to wash away the weakly bound alcohol oxidase, that is, form an alcohol oxidase mem...

Embodiment 2

[0029](1) Cut the polycarbonate film with an average pore diameter of 0.10μm and a thickness of 30μm into a disc with a diameter of 10mm, and then stick the cut polycarbonate film on the O-shaped rubber ring and dry it for use;

[0030] (2) Immerse the polycarbonate membrane with the O-shaped rubber ring in a phosphate buffer solution with a pH of 6.0 for 12 hours, then take it out and blow dry with high-purity nitrogen;

[0031] (3) Drip 8μL of ethanol oxidase solution with a concentration of 1.1U / μL on the treated polycarbonate membrane to form an enzyme layer round spot with a diameter of 6mm, and dry the enzyme membrane in a refrigerator at 0°C 2 hour;

[0032] (4) Hang the enzyme membrane in a closed container containing a 60% mass fraction of glutaraldehyde aqueous solution, and cross-link it in a refrigerator at 0°C for 18 hours;

[0033] (5) Immerse the enzyme membrane in a phosphate buffer with a pH of 6.0 to wash away the weakly bound alcohol oxidase, that is, form an al...

Embodiment 3

[0036] (1) Cut the polycarbonate film with an average pore diameter of 0.80μm and a thickness of 20μm into a 10mm diameter disc, and then glue the cut polycarbonate film on the O-shaped rubber ring and dry it for use;

[0037] (2) Immerse the polycarbonate membrane with the O-shaped rubber ring in a phosphate buffer solution with a pH of 9.0 for 10 hours, then take it out and blow dry with high-purity nitrogen;

[0038] (3) Spread 2 μL of ethanol oxidase solution with a concentration of 0.8 U / μL on the treated polycarbonate membrane to form a round spot of the enzyme layer with a diameter of 4 mm, and dry the enzyme membrane in a refrigerator at 0°C for 0.5 hour;

[0039] (4) Hang the enzyme membrane in a closed container containing 40% glutaraldehyde aqueous solution, and cross-link it in a refrigerator at 2°C for 6 hours;

[0040] (5) Immerse the enzyme membrane in a phosphate buffer with a pH of 9.0 to wash away the weakly bound alcohol oxidase, that is, form an alcohol oxidase...

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Abstract

The invention provides an ethanol oxidase membrane which takes a polycarbonate membrane as a matrix and a preparation method thereof, pertaining to the technical field of electrochemical biosensors and the preparation thereof. The ethanol oxidase membrane consists of a polycarbonate matrix membrane, the glutaraldehyde of ethanol oxidase and immobilized enzyme. The preparation method of the ethanol oxidase membrane comprises the following steps: the polycarbonate membrane is pasted on an O-shaped rubber ring and soaked into phosphate buffer solution after drying; the polycarbonate membrane is blow-dried by nitrogen and then the ethanol oxidase is dropped on the membrane; and glutaraldehyde steam is utilized to fix the ethanol oxidase by crosslinking. The ethanol oxidase has the advantages of shorter response time, higher sensitivity, wider linear response range, good operation stability and storage stability; in addition, the preparation method of the ethanol oxidase is simple and has low cost.

Description

Technical field [0001] The invention belongs to the technical field of electrochemical biosensors and their preparation, and particularly relates to an alcohol oxidase membrane with a polycarbonate membrane as a matrix and a preparation method thereof. Background technique [0002] Accurate and rapid detection of ethanol content has important application value in food detection, fermentation control and clinical diagnosis. There are many methods for analysis and detection of ethanol, among which gas chromatography, high performance liquid chromatography and spectrophotometry are the most commonly used, but these methods require more expensive instruments and equipment, and the detection steps are more complicated. In recent years, people have begun to use electrochemical biosensors to detect ethanol. This method is simple, fast and highly selective. [0003] The electrochemical biosensor prepared with ethanol oxidase as a biologically active substance can be used for the detectio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/327C12N11/08C12Q1/26
Inventor 杨文胜杨利刘长霞
Owner BEIJING UNIV OF CHEM TECH
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