Method for detecting alkaline phosphatase activities and concentration of phosphatase inhibitors

A technology of phosphatase activity and phosphatase, which is applied in the field of analysis and testing, can solve the problems of long detection time, poor dispersion and instability of Cu-MOFs, and achieve the effect of short detection time

Active Publication Date: 2018-11-27
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method requires poor dispersion of prepared Cu-MOFs, long detection time, and the use of unstable hydrogen peroxide for oxidation of peroxidase substrates

Method used

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  • Method for detecting alkaline phosphatase activities and concentration of phosphatase inhibitors
  • Method for detecting alkaline phosphatase activities and concentration of phosphatase inhibitors
  • Method for detecting alkaline phosphatase activities and concentration of phosphatase inhibitors

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0030] This embodiment relates to a method for detecting alkaline phosphatase (ALP) activity, specifically comprising the following steps:

[0031] Add 80μL Tris-HCl (pH 7.4), 10μL 7mM PPi and 10μL different concentrations of ALP (0.5, 1, 2, 4, 6, 8, 10, 20, 40mU / mL) into a 1.5mL centrifuge tube, mix well Then place in a 37°C water bath and incubate for 1 hour. Then, add 700 μL acetate buffer solution (pH 4.0), 100 μL 2.5 mg / mL CeO 2 Nanoparticles and 100 μL 5mM TMB were incubated in a water bath at 37°C for 3 minutes and then measured by UV-Vis absorption spectrum, as shown in figure 2 shown. Using ΔA and ALP activity to carry out linear fitting, the linear equations obtained are respectively: ΔA=-0.0031+0.0293c (c is ALP activity, the unit is mU / mL)), and the linear coefficient is 0.995, such as image 3 shown. In this embodiment, the experimental conditions are optimized, including pH of acetic acid buffer solution, CeO 2 Concentrations of nanoparticles and TMB, incub...

Embodiment 2

[0033] The present embodiment relates to a kind of detection sodium vanadate (Na 3 VO 4 ) concentration method, which specifically includes the following steps: mix 70 μL Tris-HCl (pH 7.4), 10 μL 2U / mL ALP and 10 μL different concentrations of Na 3 VO 4 (0, 0.005, 0.01, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.20, 0.25mM) were sequentially added to a 1.5mL centrifuge tube, mixed evenly and incubated in a 37°C water bath for 30min; Then put 10 μL of 7mM PPi into the centrifuge tube and incubate in a 37°C water bath for 1 hour. Then, add 700 μL acetate buffer solution (pH 4.0), 100 μL 2.5 mg / mL CeO 2 Nanoparticles and 100 μL 5mM TMB were incubated in a water bath at 37°C for 3 minutes and then measured by UV-Vis absorption spectrum, as shown in Figure 4 shown. We take the suppression efficiency and Na 3 VO 4 Concentration fitting curve, such as Figure 5 shown, get its IC 50 The value is 71 μM.

[0034] Test results: The best experimental conditions are: pH of a...

Embodiment 3

[0036] This embodiment involves an anti-interference experiment, and the specific steps are:

[0037] Add 80 μL Tris-HCl (pH 7.4), 10 μL 7mM PPi and 10 μL 0.1mg / mL interfering substances (bovine serum albumin, lysozyme, pepsin, trypsin and pancreatin) into a 1.5mL centrifuge tube in turn, mix well Place in a 37°C water bath and incubate for 1 hour. Then, 700 μL of acetate buffer solution (pH 4.0), 100 μL of 2.5 mg / mL CeO 2 Nanoparticles and 100 μL of 5 mM TMB were incubated in a water bath at 37°C for 3 minutes, and then measured by UV-Vis absorption spectrum.

[0038] Add 70μL Tris-HCl (pH 7.4), 10μL 7mM PPi, 10μL 0.1mg / mL alkaline phosphatase and interfering substances (bovine serum albumin, lysozyme, pepsin, trypsin and pancreatin) to a 1.5mL centrifuge tube Mix well and incubate in a 37°C water bath for 1 hour. Then, add 700 μL acetate buffer solution (pH 4.0), 100 μL 2.5 mg / mL CeO 2 Nanoparticles and 100 μL of 5 mM TMB were incubated in a water bath at 37°C for 3 minu...

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Abstract

The invention provides a method for detecting alkaline phosphatase activities. The method comprises the following steps: A1) uniformly mixing tri(hydroxymethyl) aminomethane and pyrophosphate, and then dividing into a plurality of reaction systems, respectively adding alkaline phosphatase with different concentrations into all the reaction systems, incubating at 37 DEG C, adding an acetate buffersolution, CeO2 nanoparticles and 3,3',5,5'-tetramethyl benzidine into each reaction system in turn, incubating at 22-47 DEG C, and then respectively measuring ultraviolet-visible absorption spectrum,thus obtaining a relation equation between alkaline phosphatase activities and Delta A; A2) measuring the ultraviolet-visible absorption spectrum for the to-be-detected alkaline phosphatase accordingto the operation in the step A1, thereby acquiring Delta A, substituting the Delta A into the relation equation of alkaline phosphatase activities and Delta A acquired in the step A1, and calculatingthe activities of the to-be-detected alkaline phosphatase. The method has the beneficial effects: 1) the CeO2 nanoparticles utilized by the invention are excellently dispersed in an aqueous solution and 2) the method provided by the invention is short in detecting time.

Description

technical field [0001] The invention relates to a method for detecting the activity of alkaline phosphatase and the concentration of an alkaline phosphatase inhibitor, belonging to the technical field of analysis and testing. Background technique [0002] Tan et al. used the peroxidase activity of Cu-MOFs to realize the detection of alkaline phosphatase (AnalyticaChimicaActa, 2018, 1004, 74). Specific detection steps: First, incubate PPi and different concentrations of ALP at a constant temperature of 37°C for 40 minutes; then add Cu-MOFs, hydrogen peroxide, ABTS and HEPES buffer solution to the solution in sequence, mix well and place at a constant temperature of 37°C Incubate for 50 minutes for UV-Vis absorption spectroscopy testing. This method requires poor dispersion of the prepared Cu-MOFs, a long detection time, and the use of unstable hydrogen peroxide for the oxidation of peroxidase substrates. Contents of the invention [0003] Aiming at the defects in the prio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/33
CPCG01N21/33
Inventor 逯一中倪朋娟
Owner UNIV OF JINAN
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