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Method for detecting uric acid by using mercaptopropyl agarose sphere loaded uricase and catalase

A mercaptopropyl agarose sphere, catalase technology, applied in oxidoreductase, biochemical equipment and methods, immobilized on/in organic carriers, etc., can solve the problem of low sensitivity, affecting measurement results, absorbance Small changes, etc., to achieve the effect of color concentration, elimination of product inhibition, and avoidance of external environment interference

Active Publication Date: 2020-08-25
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Chinese patent CN110108656A [A method for detecting uric acid by immobilizing uricase with mesoporous organosilicon hollow nanospheres] introduces the one-step growth-induced corrosion method mesoporous organosilicon hollow nanospheres synthesized by it, and uses the nanospheres to immobilize uricase to detect serum uric acid. The detection is a beneficial attempt and supplement to the uric acid detection system, but the change of absorbance detected by the ultraviolet spectrophotometer will be interfered by other coexisting components, thereby affecting the measurement results. In addition, the change of absorbance is small and the sensitivity is low , needs to be further improved

Method used

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  • Method for detecting uric acid by using mercaptopropyl agarose sphere loaded uricase and catalase
  • Method for detecting uric acid by using mercaptopropyl agarose sphere loaded uricase and catalase
  • Method for detecting uric acid by using mercaptopropyl agarose sphere loaded uricase and catalase

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Experimental program
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Effect test

Embodiment 1

[0061] Test strips (such as figure 1 ) preparation process:

[0062] a) First add 2ml double distilled water to 0.2ml dry mercaptopropyl agarose ball After soaking in 6B (average size 90 μm) overnight, the mercaptopropyl agarose spheres swelled with water, and their volume was about 3 times that of the dry state. The supernatant was discarded by centrifugation, and the tris(2-carboxyethyl)phosphine (TCEP) solution was reacted with the above-mentioned mercaptopropyl agarose spheres, uricase solution and catalase solution at room temperature for 1 hour to make disulfide key to turn on. The concentration of TCEP solution is 0.5mM, and solvent is 10mM Tris-HCl (pH8.0) solution, and uricase solution and catalase solution concentration are 50 μ g / ml, and solvent is 10mM PBS (pH7.4); The activity is ≥2units / mg, and the enzyme activity of catalase is ≥250units / mg.

[0063] b) The activated mercaptopropyl agarose spheres treated with TCEP were washed 5 times with 10 mM PBS (pH 7....

Embodiment 2

[0068] Test strips (such as figure 1 ) preparation process:

[0069] a) First add 2ml double distilled water to 0.2ml dry mercaptopropyl agarose ball After soaking in 6B overnight, the mercaptopropyl agarose spheres swelled with water, and their volume was about 3 times that of the dry state. The supernatant was discarded by centrifugation, and the dithiothreitol (DTT) solution was reacted with the above-mentioned mercaptopropyl agarose spheres, uricase solution and catalase solution at room temperature for 1 hour to open the disulfide bond. The concentration of DTT solution is 10mM, the solvent is 10mM Tris-HCl solution, the concentration of uricase solution and catalase solution is 30μg / ml, and the solvent is 10mM PBS (PH7.4); the enzyme activity of uricase is ≥2units / mg , the enzyme activity of catalase is ≥250units / mg.

[0070] b) The DTT-treated activated mercaptopropyl agarose spheres were washed 5 times with 10 mM PBS (pH 7.4) buffer solution, and the supernatant was...

Embodiment 3

[0075] Preparation process of enzyme-loaded mercaptopropyl agarose spheres:

[0076] a) First add 2ml double distilled water to 0.2ml dry mercaptopropyl agarose ball After soaking in 6B overnight, the mercaptopropyl agarose spheres swelled with water, and their volume was about 3 times that of the dry state. The supernatant was discarded by centrifugation, and the tris(2-carboxyethyl)phosphine (TCEP) solution was reacted with the above-mentioned mercaptopropyl agarose spheres, uricase solution and catalase solution at room temperature for 1 hour to make disulfide key to turn on. The concentration of TCEP solution is 0.5 mM, the solvent is 10 mM Tris-HCl solution, the concentration of uricase solution and catalase solution is 50 μg / ml, and the solvent is 10 mM PBS (PH7.4).

[0077] b) The activated mercaptopropyl agarose spheres treated with TCEP were washed 5 times with 10 mM PBS (pH 7.4) buffer solution, and the supernatant was removed. Then, 1.5 ml of TCEP-treated activa...

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Abstract

The invention discloses a method for detecting uric acid by using mercaptopropyl agarose sphere loaded uricase and catalase. According to the method, mercaptopropyl agarose spheres are used as carriers, uricase and catalase are loaded on the surfaces and the interiors of the mercaptopropyl agarose spheres through a chemical reaction to form a detection micro unit, then the mercaptopropyl agarose spheres loaded with the enzymes are fixed on test paper to form a uric acid detection test strip, and uric acid is detected. The method is simple in preparation, mild in reaction condition, and is capable of achieving rapid and efficient detection anytime and anywhere. In addition the method low in price, is safe to use, and has important guiding significance for clinical uric acid monitoring and disease diagnosis.

Description

technical field [0001] The invention relates to the technical field of medical in vitro diagnosis, in particular to a method for detecting uric acid by using mercaptopropyl agarose balls loaded with uricase and catalase. Background technique [0002] Uric acid is the end product of the metabolism of purine compounds, which is transported from the liver to the kidney through plasma, 70% of which is excreted through urine, and the rest is excreted through the intestines, skin and hair. The normal value of uric acid in human plasma: 150-400μM, the normal value of uric acid in urine: 1.4-4.4mM, the concentration of uric acid in plasma is greater than 420μM is called hyperuricemia, lower than 150μM is called hypouricemia disease. Hyperuricemia occurs, on the one hand, because the body produces too much uric acid, and on the other hand, because the excess uric acid cannot be excreted with urine in time. Excessive accumulation of uric acid in the plasma will lead to gout. Uric ac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/62C12Q1/30C12Q1/26C12N11/10
CPCC12Q1/62C12Q1/30C12Q1/26C12N11/10C12N9/0048C12N9/0065C12Y107/03003C12Y111/01006G01N2333/90694
Inventor 马岚薛超文
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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