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Method for determining nano-material inhibitory enzyme activity

A technology of nanomaterials and determination methods, applied in the field of activity determination, can solve problems affecting the accuracy of determination, limit nanomaterials, and difficulty in judging titration end points, etc., and achieve the effects of wide applicability, improved accuracy and reproducibility

Inactive Publication Date: 2008-12-24
OCEAN UNIV OF CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the special adsorption effect of nanomaterials, the substances in the reaction system will be adsorbed to varying degrees, which will affect the accuracy of the measurement; it should also be emphasized that when using the existing method, due to the instability of the nanomaterial suspension, Some assay methods that measure the activity of the enzyme by measuring the change of the enzymatic reaction rate by a spectrophotometer cannot be used to measure the inhibitory rate of the nanomaterial to the enzyme activity; in addition, because the nanomaterial suspension is not a clear and transparent solution, some It is difficult to determine the end point of titration by titration method to determine the activity of enzyme, and it cannot be used to determine the inhibition rate of nanomaterials on enzyme activity.
Therefore, in order to determine the inhibitory effect of nanomaterials on enzyme activity, that is, to measure the inhibition rate of nanomaterials on enzyme activity, it is often impossible to analyze according to the existing general assay methods, which greatly limits the activity of nanomaterials on enzymes. Determination of the inhibition rate

Method used

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  • Method for determining nano-material inhibitory enzyme activity
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  • Method for determining nano-material inhibitory enzyme activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Take acetylcholinesterase as an example

[0016] Existing methods:

[0017] Prepare the acetylcholinesterase solution of 5U / ml and the nanomaterial suspension of 250mg / L, 25mg / L, 12.5mg / L with the phosphate buffer solution of pH8.0, select following eight kinds of nanomaterials for use, comprise multi-walled carbon nanotube ( L-MWNTs-1020), single-walled carbon nanotubes (L-SWNTs), Cu, Al, carbon-coated copper (Cu-C), TiO 2 , SiO 2 and Al 2 o 3 , add acetylcholinesterase solution to each kind of nanomaterial suspension, mix evenly, make nanomaterial and acetylcholinesterase react after 15min, then add the substrate of acetylcholinesterase, measure the activity of enzyme, and then obtain nanomaterial to acetylcholinesterase Enzyme inhibition rate.

[0018] The inventive method:

[0019] Prepare the acetylcholinesterase solution of 5U / ml and the nanomaterial suspension of 250mg / L, 25mg / L, 12.5mg / L with the phosphate buffer solution of pH8.0, select following eight k...

Embodiment 2

[0024] Take nitrate reductase as an example

[0025] Existing methods:

[0026] Prepare 5U / ml of nitrate reductase solution and 50mg / L, 25mg / L, 12.5mg / L nanomaterial suspensions with phosphate buffer solution of pH 7.5, select the following eight nanomaterials for use, including multi-walled carbon nanotubes, Single-walled carbon nanotubes, Cu, Al, carbon-coated copper (Cu-C), TiO 2 , SiO 2 and Al 2 o 3 , add nitrate reductase solution to each nanomaterial suspension, mix well, let the nanomaterial and nitrate reductase react for 30 minutes, then add the substrate of nitrate reductase, measure the activity of the enzyme by spectrophotometry, and then get the nanometer Inhibition rate of materials to nitrate reductase.

[0027] The inventive method:

[0028]Prepare the nitrate reductase solution of 5U / ml and the nanomaterial suspension of 50mg / L, 25mg / L, 12.5mg / L with the phosphate buffer solution of pH7.5, select the following eight kinds of nanomaterials for use, includ...

Embodiment 3

[0032] Take peroxidase as an example

[0033] Existing methods:

[0034] Prepare the peroxidase solution of 5U / ml and the nanomaterial suspension of 100mg / L, 50mg / L, 10mg / L with the phosphate buffer solution of pH7.0, select following eight kinds of nanomaterials for use, comprise multi-walled carbon nanotube, Single-walled carbon nanotubes, Cu, Al, carbon-coated copper (Cu-C), TiO 2 , SiO 2 and Al 2 o 3 , add peroxidase solution to each nanomaterial suspension, mix evenly, make the nanomaterial and peroxidase react for 30min, then add the substrate of peroxidase, measure the activity of the enzyme by spectrophotometry The rate of change of time is used to obtain the inhibition rate of nanomaterials to peroxidase.

[0035] The inventive method:

[0036] Prepare the peroxidase solution of 5U / ml and the nanomaterial suspension of 100mg / L, 50mg / L, 10mg / L with the phosphate buffer solution of pH7.0, select following eight kinds of nanomaterials for use, comprise multi-walled...

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Abstract

The invention relates to a measuring method for the activity of nanometer material inhibitive enzyme. The measuring method is characterized in that an enzyme solution and a nanometer material suspension are mixed and centrifuged; subsequently, the activity of enzyme in the supernate is measured so as to obtain the adsorption rate E of the nanometer material on the enzyme; subsequently, the buffer solution of the enzyme is added into the precipitate, sufficiently desorbed and centrifuged; the desorption rate D of the nanometer material on the enzyme is calculated; finally, the inhibition rate IE of the nanometer material on the activity of the enzyme is calculated according to a formula: IE is equal to E minus D. The measuring method of the invention overcomes the effects of the nanometer material on the subsequently added matters and the adsorption effect of the reaction outcome; by measuring the activity of the enzyme adsorbed by the nanometer material, the precision and repeatability for measuring the activity of the nanometer material inhibitive enzyme are greatly improved; the method of the invention is suitable for all artificial nanometer materials which can not be dissolved in water; wherein, the enzymes are a plurality of enzymes with the activity measured by a spectrophotometry or a titrimetry; therefore, the method of the invention has wide applicability and is expected to be a standard measuring method for measuring the activity of the nanometer material inhibitive enzyme.

Description

technical field [0001] The invention belongs to the field of environmental toxicology measurement of nanometer materials, and in particular relates to a method for measuring the activity of nanomaterials inhibiting enzymes. Background technique [0002] Micron technology is a symbol of science and technology in the 20th century, while nanotechnology is a symbol of science and technology in the 21st century. Due to the unique physical and chemical properties of nanomaterials, such as small size effect, large specific surface area, extremely high reactivity, quantum effect, etc., resulting in abnormal adsorption capacity, chemical reaction ability, dispersion and aggregation ability, etc., making nanoscience and life Science and information science have become the three pillars of science in today's world. In recent years, with the rapid development of nanotechnology, countless artificial nanomaterials (materials with at least one dimension less than 100nm) have been created,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/25G01N33/48C12Q1/00
Inventor 高冬梅王震宇赵建李锋民
Owner OCEAN UNIV OF CHINA
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