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Sulfuration modified CoOx-based Alkaline phosphatase activity colorimetric detection method

A phosphatase and detection method technology, applied in the fields of analytical chemistry and simulated enzymes, can solve the problems of high requirements on reagent storage conditions, complicated operation of detection methods, harsh environmental requirements, etc., and achieve the effects of simple operation, low detection cost and sensitive response.

Active Publication Date: 2020-06-02
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] 1) These detection methods are relatively complicated to operate;
[0010] 2) Some detection methods have strict environmental requirements, relatively high cost, and high requirements for reagent storage conditions

Method used

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  • Sulfuration modified CoOx-based Alkaline phosphatase activity colorimetric detection method
  • Sulfuration modified CoOx-based Alkaline phosphatase activity colorimetric detection method
  • Sulfuration modified CoOx-based Alkaline phosphatase activity colorimetric detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] 1. Dissolve polyvinylpyrrolidone (PVP) and cobalt salt with a mass ratio of 1:2 in ethanol, stir to form solution A, and the mol ratio of cobalt salt to ethanol is 1:220;

[0055] 2. Dissolve 2-methylimidazole (2-MI) with a cobalt salt molar ratio of 1:4 in ethanol, stir to form a solution B, and the molar ratio of the cobalt salt to ethanol is 1:220;

[0056] 3. Add solution B dropwise to solution A while stirring, keep stirring for 15 minutes, and then let stand for 12 hours;

[0057] 4. Centrifuge the product at a speed of 9000r / min for 5 minutes to separate the product, wash the product with ethanol, and dry it in vacuum at 35° C. to obtain purple solid nanoparticles of rhombohedral dodecahedrons with an edge length of 200-250 nm.

[0058] 5. Pour the purple solid nanoparticles in step 4 into a porcelain boat and place it in a tube furnace, and raise the temperature at a rate of 2°C / min to 400°C under the protection of argon, and the calcination process lasts for 2h...

Embodiment 2

[0066] 1. Dissolve polyvinylpyrrolidone (PVP) and cobalt salt with a mass ratio of 1:3 in ethanol, stir to form solution A, and the mol ratio of cobalt salt to ethanol is 1:250;

[0067] 2. Dissolve 2-methylimidazole (2-MI) with a cobalt salt molar ratio of 1:4 in ethanol, and stir evenly to form solution B. The molar ratio of the cobalt salt to ethanol is 1:250;

[0068] 3. Add solution B dropwise to solution A while stirring, keep stirring for 30 minutes, and then let stand for 24 hours;

[0069] 4. Centrifuge the product at a speed of 11000r / min for 5 minutes to separate the product, wash the product with ethanol, and dry it under vacuum at 45°C to obtain purple solid nanoparticles of rhombohedral dodecahedrons with an edge length of 200-250 nm.

[0070] 5. Pour the purple solid nanoparticles in step 4 into a porcelain boat and place it in a tube furnace, and raise the temperature at a rate of 5°C / min to 500°C under the protection of argon, and the calcination process lasts...

Embodiment 3

[0078] ALP affects TMB+Sulfuration-engineered CoOx+H 2 o 2 reaction system

[0079] 1) Take 2.85mL of 0.2M acetate buffer solution (pH 4), add 0.1mL of 5mM TMB and 0.05mL of 1mg / mL Sulfuration-engineered CoOx dispersion solution to it in turn, and then mix the above solutions evenly, Control the time between 0.5 and 1 min, the liquid is colorless or light blue, react for 5 min after mixing the system, and measure the absorbance of the above mixed solution at 652 nm with an ultraviolet-visible absorption spectrophotometer;

[0080]2) Take 0.125mL 1mM HMPi, then add 2.675mL 0.2M acetate buffer (pH 4), 0.05mL 1mg / mL Sulfuration-engineered CoOx suspension, 0.1mL 5mM TMB and 0.05mL 9.8M H 2 o 2 , react for 5 minutes after mixing the system, and measure the absorbance of the above mixed solution at 652nm with a UV-Vis absorption spectrophotometer after filtration;

[0081] 3) Take 0.125mL 1mM HMPi, add 0.125mL 400U / L alkaline phosphatase, react for 45min, then add 2.6mL 0.2M ac...

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Abstract

The invention belongs to the technical field of mimic enzyme and analytical chemistry, and relates to a sulfuration modified CoOx-based alkaline phosphatase activity colorimetric detection method, which comprises respectively adding alkaline phosphatase with different activities and 1 mM HMPi to a 5 mL centrifuge tube, and reacting for 30-60 min; sequentially adding the vulcanization modified CoOxsuspension, TMB, H2O2 and an acetate buffer solution into the mixed solution, uniformly mixing the system, and reacting for 1-30 minutes; recording the absorbance at the wavelength of 652 nm by usingan ultraviolet-visible absorption spectrophotometer after passing through the film; drawing a calibrated ALP activity-absorbance standard working curve according to the absorbance measurement value of the TMBox and the corresponding ALP activity; repeating the above steps on the ALP sample to be detected, and comparing the ALP sample with the standard working curve to obtain the ALP activity. Themethod has the advantages of mild conditions in the detection process, does not need other reagents, realizes convenient and rapid detection of the ALP activity, is low in detection cost, simple in operation, has wide detectable range of 0.8-320 U / L, and realizes detection of the alkaline phosphatase activity in human serum.

Description

technical field [0001] The invention belongs to the technical field of simulated enzymes and analytical chemistry, and relates to a detection method for alkaline phosphatase, in particular to a colorimetric detection method for alkaline phosphatase activity based on sulfide-modified CoOx. Background technique [0002] Alkaline phosphatase (ALP) is a biological enzyme widely present in many human tissues and organs. It has the ability to catalyze the hydrolysis of many phosphorylated substances such as nucleic acids, proteins and small molecules. Typically, the activity of ALP in adult blood ranges from 40 to 190 U / L. A large number of studies have shown that abnormal ALP activity is closely related to a variety of diseases, including bone damage, liver dysfunction and prostate cancer. For example, ALP is an important indicator of osteoblast activity during early osteoblast differentiation, and its activity usually increases with bone activity. When its activity exceeds th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/78G01N21/31G01N21/33
CPCG01N21/78G01N21/31G01N21/33
Inventor 宋洪伟牛湘衡李欣薛强胜
Owner JIANGSU UNIV
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