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Preparation method of enzyme immobilization carrier and application of enzyme immobilization carrier to immunoassay

A technology for immobilizing enzymes and antibodies, which can be used in the direction of immobilization on/in organic carriers, analytical materials, and material testing products, etc., which can solve the problems of decreased enzyme activity, weakened signal amplification effect of the carrier, and limited binding capacity.

Inactive Publication Date: 2014-12-10
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the process of this method is cumbersome, and the multi-step chemical modification and coupling reactions will inevitably affect the activity of the enzyme and weaken the signal amplification effect of the carrier.
[0009] The common problem of the above work is that the outer surface of the carrier can only bind a single layer of enzymes and antibodies, so the binding amount is relatively limited, and the enzyme binding process is usually accompanied by a sharp drop in enzyme activity, which further restricts the signal amplification effect of the carrier.

Method used

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  • Preparation method of enzyme immobilization carrier and application of enzyme immobilization carrier to immunoassay

Examples

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Effect test

Embodiment 1

[0048] Covalent immobilization of horseradish peroxidase in polyacrylic acid ball brushes by electrostatic adsorption followed by chemical coupling (CCEE)

[0049] First, the surface-initiated RAFT polymerization method published in "Journal of Colloid and Interface Science" 2013, Volume 398, pages 82-87 was used to prepare silica with a spherical core of 80 nanometers. The length is 145 nm and the graft density is 0.34 nm -2 polyacrylic ball brushes.

[0050] By centrifugation-resuspension, the polyacrylic acid ball brush was washed twice with 10 mM MES buffer at pH=5.0, and dispersed in the buffer by ultrasonic. At the same time, the horseradish peroxidase was dissolved in the buffer solution to prepare a protein solution, which was added to the polyacrylic acid ball brush dispersion and mixed evenly. The final concentration of the polyacrylic acid ball brush was 0.8 mg / ml, and the concentration of the horseradish peroxidase was 2 mg / ml. Incubate at room temperature for 1...

Embodiment 2

[0056] Covalent immobilization of horseradish peroxidase in poly N-(2-aminoethyl)acrylamide ball brushes by CCEE method

[0057] In this example, the polyelectrolyte ball brush used is a poly N-(2-aminoethyl) acrylamide ball brush containing amino functional groups, its ball core is 80 nanometers, and the polymer brush-like chain segment is The length in aqueous solution is 75 nm, and the graft density is 0.3 nm -2 .

[0058] By centrifugation-resuspension, the poly N-(2-aminoethyl)acrylamide ball brush was washed twice with 10 mM phosphate buffer at pH=7.4, and ultrasonically dispersed in the phosphate buffer. At the same time, the horseradish peroxidase is dissolved in the phosphate buffer solution to prepare a protein solution, which is added to the ball brush dispersion and mixed evenly. The final concentration of the poly-N-(2-aminoethyl)acrylamide ball brush was 1.2 mg / ml, and the concentration of the horseradish peroxidase was 2 mg / ml. Incubate at room temperature fo...

Embodiment 3

[0062] Covalent immobilization of anti-β-hCG antibody on the outside of polyacrylic ball brushes by NHS / EDC process

[0063] The polyacrylic acid ball brush-horseradish peroxidase complex obtained in Example 1 was dispersed in 10 mM MES buffer solution with pH=5.0 by centrifugation-resuspension at a concentration of 2 mg / ml. Dissolve NHS and EDC in MES to prepare a solution with a concentration of 50 mg / ml, add it to a centrifuge tube containing polyacrylic acid ball brush-horseradish peroxidase complex, and react at room temperature for 15-30 minutes to activate the polyacrylic acid The remaining carboxyl on the ball brush. Wash 3 times with buffer to remove excess reactants.

[0064] After discarding the supernatant, the excess anti-β-hCG antibody was dissolved in MES and added to the centrifuge tube, and reacted at room temperature for 2 hours, so that the antibody and the activated polyacrylic acid ball brush were fixed by covalent bonds. Centrifuge, discard the supernat...

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Abstract

The invention discloses a preparation method of an enzyme immobilization carrier and application of the enzyme immobilization carrier. According to the preparation method of the enzyme immobilization carrier, a polyelectrolyte ball brush is taken as a carrier, an enzyme is immobilized by using a method of carrying out electrostatic adsorption and then carrying out chemical coupling, and the prepared compound is applied to immunoassay. By virtue of the preparation method of the enzyme immobilization carrier, high binding capacity and high activity of the enzyme are achieved; the sensitivity of the enzyme-linked immunoassay can be remarkably improved when the enzyme immobilization carrier is applied to the enzyme-linked immunoassay.

Description

technical field [0001] The invention relates to the technical field of biological materials, in particular to a method for preparing an enzyme-immobilized carrier and its application in enzyme-linked immunoassay. Background technique [0002] The immobilization of enzymes is widely used in many fields such as catalysis and detection. The immobilized enzyme carrier can realize the simple separation while exerting the function of the enzyme, so as to obtain a more pure product. In order to improve the catalytic efficiency, it is generally desired that the carrier has a higher amount of enzyme binding and can better maintain the activity of the enzyme. [0003] Enzyme immobilization is usually achieved by physical adsorption or chemical reaction between the carrier surface and the enzyme. However, conventional carriers usually face the problems of low enzyme binding capacity and the large impact of the binding process on enzyme activity, which makes the catalytic efficiency of...

Claims

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Application Information

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IPC IPC(8): C12N11/08G01N33/53
CPCG01N33/531
Inventor 徐宏瞿振元古宏晨
Owner SHANGHAI JIAO TONG UNIV
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