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Method for determining acid phosphatase based on chitosan-platinum simulated oxidase

A technology for acid phosphatase and its determination method, which is applied in the field of determination of acid phosphatase and its inhibitors, can solve the problems of inconspicuous color development of tetramethylbenzidine hydrochloride, etc., and achieve simple and fast preparation process and simple detection steps , the effect of low detection cost

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

AI Technical Summary

Problems solved by technology

Ascorbic acid with a strong reducing form can inhibit the catalytic oxidation of oxidase to 3,3',5,5'-tetramethylbenzidine hydrochloride, so that 3,3',5,5'-tetramethylbenzidine Aniline hydrochloride color development is not obvious

Method used

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  • Method for determining acid phosphatase based on chitosan-platinum simulated oxidase
  • Method for determining acid phosphatase based on chitosan-platinum simulated oxidase
  • Method for determining acid phosphatase based on chitosan-platinum simulated oxidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Weigh 0.1 g of chitosan and dissolve it in 50 mL of acetic acid with a concentration of 1% (v / v), and stir for 15 minutes to completely dissolve the chitosan to obtain a chitosan solution with a concentration of 0.2% (m / v). Add 2 mL of 10 mmol / L chloroplatinic acid to 47 mL of 0.2% (m / v) chitosan solution, stir for 30 minutes, and then add 1 mL of freshly prepared 0.2 mol / L chitosan solution dropwise. Sodium borohydride solution (completely added within 5 minutes), placed in the dark and stirred for 90 minutes to obtain a dark brown chitosan-platinum oxidase solution. Store the product in the dark and refrigerate. All glassware used in the above process was soaked in aqua regia, washed thoroughly with double distilled water, and dried.

Embodiment 2

[0031] Add 50 μL of ascorbyl phosphate with a concentration of 4 mmol / L and 50 μL of acid phosphatase with a concentration of 0.1 U / mL into 200 μL of acetate buffer (pH 5, 50 mmol / L), mix well and place in React at 37°C for 30 minutes. Add 632.5 μL acetate buffer (pH 5, 50 mmol / L), 50 μL 3,3’,5,5’-tetramethylbenzidine hydrochloride solution with a concentration of 3 mmol / L and 17.5 μL The chitosan-platinum simulated oxidase solution prepared in Example 1 was mixed evenly and then reacted at 37°C for 5 minutes, and 200 μL of sulfuric acid solution with a concentration of 2 mol / L was added to terminate the reaction, and the color change or Measure the UV-Vis absorption spectrum of the solution. When visually observing the color change, the solution color of the control group is dark yellow, and the solution color of the experimental group becomes colorless (see figure 1 ). When measuring the ultraviolet-visible absorption spectrum, the absorption spectrum of the control group...

Embodiment 3

[0033] Add 50 μL of ascorbyl phosphate with a concentration of 4 mmol / L and 50 μL of acid phosphatase of different concentrations into 200 μL of acetate buffer (pH 5, 50 mmol / L), mix well and react at 37°C for 30 minute. Add 632.5 μL acetate buffer (pH 5, 50 mmol / L), 50 μL 3,3’,5,5’-tetramethylbenzidine hydrochloride solution with a concentration of 3 mmol / L and 17.5 μL The chitosan-platinum simulated oxidase solution prepared in Example 1 was mixed evenly and then reacted at 37°C for 5 minutes, and 200 μL of sulfuric acid solution with a concentration of 2 mol / L was added to terminate the reaction, and the absorbance value A of the solution was measured. 450 . Depend on image 3 It can be seen that with the increase of acid phosphatase concentration, the absorbance value A 450 The change value (△A 450 ) gradually increases. In the range of 0.25 ~ 2.5U / L, △A 450 It has a linear relationship with the concentration of acid phosphatase, and the lowest detection limit is 0.0...

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Abstract

The invention discloses a method for determining acid phosphatase based on chitosan-platinum simulated oxidase. The method is characterized in that acid phosphatase is utilized for hydrolyzing ascorbic acid phosphate to generate ascorbic acid so as to influence a catalytic chromogenic reaction system of chitosan-platinum simulated oxidase / 3,3',5,5'-tetramethyl benzidine hydrochloride, and ascorbic acid is directly used for determining the contents of acid phosphatase and an inhibitor thereof by combining with the change on the color of a solution and the characteristics of an ultraviolet absorption spectrum. The change value deltaA450 of absorbancy is in a linear relation with the concentration of acid phosphatase in a range of 0.25U / L-2.5U / L, and the detection limit is 0.0161U / L. The method is simple and convenient in operation and high in sensitivity and can be used as an analytical method for determining the contents of acid phosphatase and the inhibitor thereof in environment and life science systems.

Description

technical field [0001] The invention relates to a method for measuring acid phosphatase and its inhibitor in a chitosan-platinum imitation oxidase catalytic color development system, and belongs to the field of analytical chemistry and nanotechnology. Background technique [0002] Acid phosphatase is a hydrolase that catalyzes the hydrolysis of phosphate monoesters to generate inorganic phosphate under acidic conditions, and is widely distributed in some plant or mammalian tissues. In agriculture, acid phosphatase plays an important role in judging the optimum pH for crop growth. In medicine, it can be used as an important diagnostic indicator for diseases such as hepatitis, hyperparathyroidism, and red blood cell lesions. Therefore, the determination of acid phosphatase content has far-reaching significance. However, there are still many limitations in the determination of acid phosphatase at present. For example, the existing methods mainly use disodium 4-nitrophenyl pho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76G01N21/33
Inventor 陈伟邓豪华林秀玲彭花萍李柯林
Owner FUJIAN MEDICAL UNIV
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