Eureka AIR delivers breakthrough ideas for toughest innovation challenges, trusted by R&D personnel around the world.

Method for testing hydrogen peroxide in cell based on horseradish peroxidase-attapulgite nanometer composite material

A technology of horseradish peroxidase and nanocomposite materials, which is applied in the fields of biosensing and cell electrochemistry, can solve the problems of being easily decomposed by microorganisms, the loss of biological activity of proteins, and the loss of original conformation, etc., to achieve good electrochemistry Chemical properties, low detection limit, fast response effect

Inactive Publication Date: 2011-08-10
NANJING NORMAL UNIVERSITY
View PDF4 Cites 12 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These immobilized carriers all have some shortcomings. For example, although carbon materials have good electrical conductivity and strong adsorption capacity, whether they will have toxic effects on biomacromolecules and affect the bioactivity of biomacromolecules is still unclear. It is not very clear; while metal oxides have the advantages of good stability, high mechanical strength, and low cost, but biomacromolecules have lost their original conformation on their surfaces, resulting in denaturation, resulting in the loss of proteins (enzymes) immobilized on their surfaces. Biological activity; the biggest feature of natural polymer carriers is non-toxicity, but they have disadvantages such as low strength, poor electrical conductivity, easy to be decomposed by microorganisms under anaerobic conditions, and low service life; synthetic organic polymer materials are due to their The chemical and physical properties have great variability. In theory, it can act as a carrier for any enzyme, and it has higher strength than natural polymer compounds, but it has the disadvantage of poor electrical conductivity, which is the biggest disadvantage of electrochemical research. one of the factors

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for testing hydrogen peroxide in cell based on horseradish peroxidase-attapulgite nanometer composite material
  • Method for testing hydrogen peroxide in cell based on horseradish peroxidase-attapulgite nanometer composite material
  • Method for testing hydrogen peroxide in cell based on horseradish peroxidase-attapulgite nanometer composite material

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Grind 10 g of attapulgite in a grinder for 2 hours at a speed of 800 rpm; weigh 1 g of attapulgite ground, put it into 100 mL of ultrapure water for ultrasonic dispersion for 2 hours, and centrifuge it in a high-speed centrifuge ( 5000 rpm), washed the settled attapulgite with deionized water; repeated ultrasonic dispersion and centrifugation three times, and dried the washed attapulgite at 80 °C for 12 h to obtain purified attapulgite.

[0031] Add 0.5 mg of purified attapulgite into 1 mL of 0.1 mol / LPBS (pH 7.4) solution, and disperse ultrasonically for 0.5–2 hours to obtain a 0.5 mg / mL attapulgite dispersion; mix the attapulgite suspension with an equal volume Mix HRP in PBS (1 mg / mL, pH 7.4) and stir continuously at 4°C for 0.5 h. The mixture was centrifuged at 18,000 rpm for 30 min, and washed thoroughly with secondary water three times to remove the HRP molecules that were not firmly adsorbed, and the material was dried in a vacuum desiccator to obtain the HRP-Att...

Embodiment 2

[0034] The attapulgite purification method is the same as in Example 1. Add 2 mg of purified attapulgite into 1 mL of 0.1 mol / LPBS (pH 7.4) solution, and disperse ultrasonically for 0.5–2 hours to obtain a 2 mg / mL attapulgite dispersion; mix the attapulgite suspension with an equal volume of HRP PBS solution (5 mg / mL, pH 7.4) was mixed and stirred continuously at 4°C for 1 hour. The mixture was centrifuged at 10,000 rpm for 20 min, and washed thoroughly with secondary water three times to remove the HRP molecules that were not firmly adsorbed, and the material was dried in a vacuum desiccator to obtain the HRP-Attapulgite complex.

[0035] Glassy carbon electrodes (GC, 3 mm in diameter) were coated with No. 6 sandpaper, 0.3 μm and 0.05 μm Al 2 o 3 Polish to a mirror surface, then ultrasonically clean in ultrapure water for 30 seconds, and dry for use. Weigh 1 mg of HRP–Attapulgite complex and disperse it in 1 mL of 0.1 mol / L PBS (pH 7.4) solution, use a microsampler to tak...

Embodiment 3

[0037] The attapulgite purification method is the same as in Example 1. Add 5 mg of purified attapulgite into 1 mL of 0.1 mol / L PBS (pH 7.4) solution, and ultrasonically disperse for 0.5–2 hours to obtain a 5 mg / mL attapulgite dispersion; mix the attapulgite suspension with an equal volume of A solution of HRP in PBS (10 mg / mL, pH 7.4) was mixed and stirred continuously for 1 hour at 4 °C. The mixture was centrifuged at 5000 rpm for 15 min, and washed thoroughly with secondary water three times to remove the HRP molecules that were not firmly adsorbed, and the material was dried in a vacuum desiccator to obtain the HRP-Attapulgite complex.

[0038] Glassy carbon electrodes (GC, 3 mm in diameter) were coated with No. 6 sandpaper, 0.3 μm and 0.05 μm Al 2 o 3 Polish to a mirror surface, then ultrasonically clean in ultrapure water for 30 seconds, and dry for use. Weigh 1 mg of HRP–Attapulgite complex and disperse it in 1 mL of 0.1 mol / L PBS (pH 7.4) solution, use a microsampl...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention relates to a method for testing hydrogen peroxide in a cell based on a horseradish peroxidase-attapulgite nanometer composite material; the method comprises the following steps: horseradish peroxidase is adsorbed on the surface of purified attapulgite so as to prepare the horseradish peroxidase-attapulgite nanometer composite material; a dispensing method is used for applying the composite material to the surface of a glassy carbon electrode so as to construct a horseradish peroxidase-attapulgite nanometer composite material biological sensor; the sensor is used as a working electrode; and a chronoamperometry is used for detecting H2O2 in the cell. The biological sensor adopted by the method for testing the hydrogen peroxide in the cell has good electrical catalytic activity on hydrogen peroxide, can be applied to the detection of H2O2 in the cell and has the advantages of fast response, wide linear range, low detection limit, good repeatability and the like; and a new electrochemistry method based on enzyme sensing for detecting H2O2 in the cell is established.

Description

technical field [0001] The invention relates to an electrochemical detection method of intracellular hydrogen peroxide based on enzyme sensing, in particular to a detection method of intracellular hydrogen peroxide based on horseradish peroxidase-attapulgite nanocomposite materials, belonging to Biosensing and cell electrochemistry technology field. Background technique [0002] During the metabolic process, cells continuously produce various reactive oxygen species (reactive oxygen species, ROS), such as superoxide anion free radicals (O 2 -· ), hydroxyl radicals (·OH), lipid radicals (ROO·), hydrogen peroxide (H 2 o 2 )wait. Hydrogen peroxide is one of the most stable, and its concentration and O 2 -· , OH and other ROS molecules are closely related. The latest research has found that hydrogen peroxide plays an important regulatory role in the cellular metabolism of living systems. Hydrogen peroxide can be used as a signal molecule or second messenger to participate...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): G01N27/26G01N27/327B82Y40/00
Inventor 吴萍蔡称心张卉屠蕴秋
Owner NANJING NORMAL UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com