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Alkaline phosphatase activity colorimetric detection method based on CeVO4

A phosphatase and detection method technology, applied in the field of analytical chemistry, can solve the problems of high requirements for reagent storage conditions, complex operation of detection methods, and harsh environmental requirements, and achieve the effects of mild detection process conditions, 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) The operation of the detection method is relatively complicated;
[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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  • Alkaline phosphatase activity colorimetric detection method based on CeVO4
  • Alkaline phosphatase activity colorimetric detection method based on CeVO4
  • Alkaline phosphatase activity colorimetric detection method based on CeVO4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] 1. Ammonium metavanadate (NH 4 VO 3 ) and cerium nitrate hexahydrate [Ce(NO 3 ) 3 ·6H 2 0] was dissolved in ultrapure water and stirred ultrasonically for 1 hour to form a uniform yellow solution A, in which the solid-to-liquid ratio of ammonium metavanadate, cerium nitrate hexahydrate and ultrapure water was 1:4:30.

[0049] 2. Transfer the solution A into the reaction kettle, and conduct a hydrothermal reaction at 180°C for 12 hours;

[0050] 3. Wait for the reaction product in step 2 to cool naturally, then centrifuge at 9000r / min for 5 minutes to separate the product, wash the product with ethanol, and dry it in vacuum at 35°C for 12 hours to obtain black solid nanoparticles with a particle size of about 500nm.

[0051] CaVO 4 As an application experiment of oxidase-like catalyzed oxygen oxidation TMB reaction, the following steps are included:

[0052] 1) Take 2.8mL of 0.2M acetate buffer solution (pH 4), add 0.1mL of 5mM TMB and 0.1mL of 1mg / mL CeVO in seque...

Embodiment 2

[0057] 1. Ammonium metavanadate (NH 4 VO 3 ) and cerium nitrate hexahydrate [Ce(NO 3 ) 3 ·6H 2 0] dissolved in ultrapure water, and ultrasonically stirred to form a yellow homogeneous solution A;

[0058] 2. Pour the solution A in step 1 into the reaction kettle, and heat the reaction at 120°C for 6 hours in a blast oven;

[0059] 3. Wait for the reaction product in step 2 to cool naturally, then centrifuge at 11,000r / min for 10 minutes to separate the product, wash the product with ethanol, and dry it in vacuum at 45°C for 12 hours to obtain black solid nanoparticles with a particle size of about 500nm.

[0060] CaVO 4 As an application experiment of oxidase-like catalyzed oxygen oxidation TMB reaction, the following steps are included:

[0061] 1) Take 2.8mL of acetate buffer solution or phosphate buffer solution with pH values ​​of 3, 4, 5, 6, 7, 8 and 9 respectively, and add 0.1mL of 5mM TMB and 0.1mL of 1mg / mL CeVO 4 Disperse the solution, then mix the above solut...

Embodiment 3

[0066] ALP affects TMB+CeVO 4 +(NaPO 3 ) 6 reaction system

[0067] 1) Take 2.8mL of 0.2M acetate buffer solution (pH 4), add 0.1mL of 5mM TMB and 0.1mL of 1mg / mL CeVO in sequence 4 Disperse the solution, then mix the above solution evenly, and control the time between 0.5 and 1min. The liquid is colorless or light blue. After mixing the system, react for 5min. Measure the temperature of the above mixed solution at 652nm with a UV-visible absorption spectrophotometer. absorbance at

[0068] 2) Take 0.05mL 1mM (NaPO 3 ) 6 , and then sequentially added 2.75mL 0.2M acetate buffer (pH 4), 0.1mL 1mg / mL CeVO 4 Suspension, 0.1mL 5mM TMB, mix the system and react for 5min, filter and measure the absorbance of the above mixed solution at 652nm with a UV-Vis absorption spectrophotometer;

[0069] 3) Take 0.05mL 1mM (NaPO 3 ) 6 , adding 0.15mL 400U / L alkaline phosphatase, reacted for 45min, then added 2.6mL 0.2M acetate buffer (pH 4), 0.1mL 1mg / mL CeVO 4 Suspension, 0.1mL 5mM T...

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Abstract

The invention belongs to the technical field of analytical chemistry, and relates to an alkaline phosphatase activity colorimetric detection method based on CeVO4. The alkaline phosphatase activity colorimetric detection method comprises the following steps: respectively adding alkaline phosphatase with different activities and 0.05 mL of 1 mM sodium hexametaphosphate (NaPO3) 6 into a 5 mL centrifuge tube; sequentially adding 0.1 mL of 1mg / mL CeVO4 suspension, 0.1 mL of 5mM TMB and an acetate buffer solution into the mixed solution, wherein the final total volume of the solution is 3mL; afterpassing through the membrane, recording the absorbance at the wavelength of 652 nm by using an ultraviolet-visible absorption spectrophotometer, and drawing an ALP activity-absorbance standard workingcurve; performing calibration; and measuring the absorbance of the sample to be measured, and comparing to obtain the ALP activity. The method is mild in detection process condition, low in detectioncost and simple to operate; the alkaline phosphatase activity is detected through a TMB + CeVO4 system, the detection limit is as low as 0.68 U / L, and the detectable range is as wide as 1-210 U / L; the alkaline phosphatase activity is detected by using a TMB + CeVO4 system, the response to alkaline phosphatase is sensitive, the measurement result of an actual sample is accurate and reliable, and the detection of the alkaline phosphatase activity in human serum is realized.

Description

technical field [0001] The invention belongs to the technical field of analytical chemistry, relates to the detection of alkaline phosphatase, in particular to a method based on CeVO 4 colorimetric assay for alkaline phosphatase activity. Background technique [0002] Alkaline phosphatase (ALP) is a biological enzyme widely distributed in human liver, bone, intestine, kidney and placenta. of phosphate groups. The activity range of ALP is 40~190U / 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 the upper limit, it means that active bone deposition may occur during this process. In addition, other bone diseases, such as rickets, lupus, and bone cancer, can also lea...

Claims

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Application Information

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IPC IPC(8): G01N21/78G01N21/31G01N21/33
CPCG01N21/78G01N21/31G01N21/33
Inventor 叶堃牛湘衡李欣王林杰
Owner JIANGSU UNIV
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