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Method for determining urease based on nano copper oxide

A technology of nano-copper oxide and urease, which is applied in the field of analytical chemistry and nanometers to achieve the effects of less sample demand, good reproducibility and low cost

Inactive Publication Date: 2017-05-10
FUJIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, few studies have been performed under alkaline conditions

Method used

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  • Method for determining urease based on nano copper oxide
  • Method for determining urease based on nano copper oxide
  • Method for determining urease based on nano copper oxide

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0045] The specific preparation steps of nano-copper oxide are as follows: (1) Take 150 ml of 0.02 mol / L copper acetate solution and 0.5 ml of glacial acetic acid into a three-necked flask equipped with a condenser, stir and heat to boiling; (2) Quickly add 0.04 g / ml sodium hydroxide solution 10 ml, continue to stir for 5 minutes after the addition, to obtain a black copper oxide precipitate; (3) centrifuge the black copper oxide precipitate obtained from the reaction, wash with absolute ethanol three times, and dry under reduced pressure, that is A nano-copper oxide powder with a diameter of 6 nm was obtained.

example 2

[0047] 50 µL concentration of 120 mmol / L hydrogen peroxide solution, 50 µL concentration of 80 mmol / L p-hydroxyphenylpropionic acid and 200 µL concentration of 2 mg / L nano-copper oxide solution prepared in Example 1 were added to 700 µL In a phosphate buffer solution (pH 10.25) with a concentration of 100mmol / L, mix and shake well, place in a warm bath at 65°C, and measure the fluorescence spectrum after 15 minutes. Such as figure 1 As shown, the excitation wavelength of the fluorescent product is 320 nm, and the emission wavelength is 409 nm.

example 3

[0049] Add 50 µL concentration of 120 mmol / L hydrogen peroxide solution, 50 µL concentration of 80 mmol / L p-hydroxyphenylpropionic acid and 200 µL concentration of 2 mg / L nano-copper oxide prepared in Example 1 to a concentration of 700 µL 100mmol / L and different pH phosphate buffer solutions (pH 9-11), mix and shake well and place in a warm bath at 65°C, measure the fluorescence intensity at 409 nm after 15 minutes (excitation wavelength is 320 nm). Such as figure 2 As shown, the fluorescence intensity reaches the maximum value at pH 10.1~10.3.

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Abstract

The invention discloses a method for determining urease based on nano copper oxide. The method comprises the following steps: simulating enzymatic activity of alkaline peroxide based on the nano copper oxide, selectively hydrolyzing urea to generate ammonia and carbon dioxide in the existence of the urease, inhibiting the process in which the nano cooper oxide catalyzes the oxidation of hydrogen peroxide for p-hydroxyphenylpropionic acid by virtue of the ammonia, so that the generation of dipolymer fluorescent products is reduced, and a fluorescent analysis method adopting the nano copper oxide as a catalyst to determine the urease is provided. Urea, urease and a phosphate buffer are mixed to perform urease enzymatic reaction in a warm bath, then the p-hydroxyphenylpropionic acid, the hydrogen peroxide, the nano copper oxide and the phosphate buffer are added to continuously perform fluorescent reaction in the warm bath, a maximum excitation wavelength and a maximum emission wavelength of a fluorescent reaction product are respectively 320 nm and 409 nm. A linear range for determining the urease is 0.003 to 0.04 U / mL, and a detection limit is 2.6 U / L. The method can be used for determining the urease in soil.

Description

technical field [0001] The invention relates to urease catalyzing the hydrolysis of urea to produce ammonia, inhibiting the activity of nano-copper oxide simulating alkaline peroxidase in catalyzing the oxidation of p-hydroxyphenylpropionic acid by hydrogen peroxide under alkaline conditions to form fluorescent products, and establishing a fluorescence analysis method for measuring urease. It belongs to the field of analytical chemistry and nanotechnology. Background technique [0002] Peroxidase mainly exists in the peroxisomes of cells, which can catalyze the oxidation of phenols and amines by hydrogen peroxide, and then remove the toxic effects of hydrogen peroxide, phenols and amines. Natural peroxidase has less content, is expensive, and is easily disturbed by the external environment to lose its catalytic activity. Nano-artificial enzymes have been widely used in biological analysis due to their stable structure, economy, easy large-scale preparation, and adjustable ca...

Claims

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 陈伟郑晓晴邓豪华彭花萍庄琼琼
Owner FUJIAN MEDICAL UNIV
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