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Method for measuring nano-gold mimetic peroxidase based urease and inhibitor thereof

A technology of peroxidase and determination method, which is applied in the direction of material analysis through observation of the influence on chemical indicators, preparation of test samples, analysis through chemical reaction of materials, etc., which can solve environmental pollution and economic losses and other problems, to achieve the effect of high detection sensitivity, low detection cost and simple detection steps

Inactive Publication Date: 2015-05-20
FUJIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In agriculture, when the activity of urease in the soil is too high, the urea in the fertilizer is rapidly decomposed into ammonia, which is discharged into the atmosphere, causing economic losses and environmental pollution

Method used

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  • Method for measuring nano-gold mimetic peroxidase based urease and inhibitor thereof
  • Method for measuring nano-gold mimetic peroxidase based urease and inhibitor thereof
  • Method for measuring nano-gold mimetic peroxidase based urease and inhibitor thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] 1 milliliter of 0.1 g / L chloroauric acid solution was dissolved in 100 milliliters of water, 3 milliliters of 0.1 g / L trisodium citrate solution was added rapidly after heating and boiling under reflux, the reaction solution changed from light yellow to wine red, and continued to reflux for 15 After 1 minute, the reaction solution was slowly cooled to room temperature. The obtained gold nanoparticles had a diameter of 13 nm and a concentration of 3.1 nmoL / L, and were stored at 4 °C. All glassware used in the above process was soaked in aqua regia, washed thoroughly with double distilled water, and dried.

Embodiment 2

[0036] Add 0.05 mL of 18 U / mL urease solution and 0.1 mL of 5 mmoL / L urea solution into 0.5 mL of phosphate buffer (10 mmol / L, pH=6.40), shake well and incubate at 37°C for 30 minutes. After the reaction, add 0.2 mL of 30% (m / m) hydrogen peroxide solution, 0.05 mL of 3,3',5,5'-tetramethylbenzidine hydrochloride solution with a concentration of 16 mmol / L and 0.10 mL of the nano-gold solution prepared in Example 1 was mixed evenly and then incubated at 37° C. for 10 minutes. Immediately add 0.2 mL of 20% (V / V) sulfuric acid solution to terminate the reaction, visually observe the color change or measure the absorbance value A 450 . When visually observing the color change, the color of the solution in the control group was all dark yellow, and the color of the color development system in the experimental group became light yellow (see figure 1 ). Assay A 450 , the control group A 450 Almost no change, experimental group A 450 significantly reduced (see figure 2 )

Embodiment 3

[0038] Add 0.05 mL of urease solution of different concentrations and 0.1 mL of 0.5 moL / L urea solution into 0.5 mL of phosphate buffer (10 mmol / L, pH=6.40), shake well and incubate at 37°C for 30 minutes. After the reaction, add 0.2 mL of 30% (m / m) hydrogen peroxide solution, 0.05 mL of 3,3',5,5'-tetramethylbenzidine hydrochloride solution with a concentration of 16 mmol / L and 0.10 mL of the nano-gold solution prepared in Example 1 was mixed evenly and then incubated at 37° C. for 10 minutes. Immediately add 0.2 mL of 20% (V / V) sulfuric acid solution to terminate the reaction, and measure the absorbance value A 450 . Depend on image 3 It can be seen that with the increase of urease concentration, A 450 value decreases gradually. In the range of 1.8 ~ 90 U / L, A 450 The value has a linear relationship with the concentration of urease, and the detection limit is 1.8 U / L (see Figure 4 ).

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Abstract

The invention discloses a method for measuring nano-gold mimetic peroxidase based urease and an inhibitor thereof. The method is characterized by comprising the following steps: coupling a urease catalysis urea reaction system with a nano-gold mimetic peroxidase-hydrogen peroxide-3,3',5,5'-tetramethylbenzidine dihydrochloride catalytic chromogenic reaction system, and combining with the changes of solution colors and ultraviolet absorption spectrum characteristics to directly measure the content of urease and the inhibitor thereof, wherein in a range of 1.8-90U / L, A450 and the urease concentration have a linear relationship, and the limit of detection is 1.8U / L; and by virtue of software fitting, IC50 of obtained acetohydroxamic acid of the urease inhibitor is 0.05mmoL / L. The method disclosed by the invention is simple and convenient to operate and high in sensitivity, and can be applied to the measurement of the content of urease and the inhibitor thereof in environment and life science systems as an analysis method.

Description

technical field [0001] The invention relates to a method for measuring urease and its inhibitor based on a gold nanometer simulated peroxidase catalytic color development system, and belongs to the field of analytical chemistry and nanotechnology. Background technique [0002] Urease is a nickel-containing oligomerase that can efficiently and specifically catalyze the hydrolysis of urea to generate carbon dioxide and ammonia. In medicine, bacterial urease is a pathogenic factor that cannot be ignored. It can induce many diseases, such as pyelonephritis, hepatic coma, peptic ulcer and infectious urinary tract stones. Urease inhibitors are drugs that dissolve urinary stones and stop new crystals from forming in the urine. In agriculture, when the activity of urease in the soil is too high, the urea in the fertilizer is rapidly decomposed into ammonia, which is discharged into the atmosphere, causing economic losses and environmental pollution. In order to reduce environmenta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/78G01N21/31G01N1/28
Inventor 陈伟邓豪华沈奕珉李光文刘爱林
Owner FUJIAN MEDICAL UNIV
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