A Simple Detection Method of Alkaline Phosphatase Activity

Abstract

The invention discloses a simple and low cost method for detecting the activity of alkaline phosphatase. According to the method, after the alkaline phosphatase analyte in a sample solution catalyzes the pyrophosphoric acid hydrolysis, Cu2+ is not complexed with pyrophosphoric acid and thus can high efficiently inhibit the activity of glucoamylase, and starch with low water solubility cannot be hydrolyzed by f glucoamylase. Starch in the reaction solution is dropwise added on a paper micro-fluidic analysis device, the corresponding paper carries out wettability alteration from hydrophilic property to hydrophobic property, the volume of a detection reagent solution that can flow through the paper is reduced, and finally the flow length of the detection reagent solution in the paper device is shortened. The flow length of the detection reagent solution in the paper device and the activity of the alkaline phosphatase in the sample are in a inversely proportional relationship, and thus the quantitative detection of alkaline phosphatase activity can be realized. The method is simple and easy to perform, even an untrained operator can carry out experiments, the quantitative signals can be read without using any instrument, the analysis cost is largely reduced, and the method has a wide application prospect.

Description

technical field

[0001] The invention belongs to the technical field of biochemical analysis and biosensing, and in particular relates to a simple and low-cost alkaline phosphatase activity detection method. Background technique

[0002] Alkaline phosphatase, also known as alkaline phosphatase, can catalyze the hydrolysis of phosphate bonds under alkaline conditions. In mammals, alkaline phosphatase activity is relatively high in liver, bile duct, kidney, bone and placenta. Common alkaline phosphatases include intestinal alkaline phosphatase, non-tissue-specific alkaline phosphatase, and placental alkaline phosphatase. High or low alkaline phosphatase activity is associated with many diseases. For example, the activity of alkaline phosphatase in differentiated colon cancer cells will be significantly increased, which is used as a qualitative and quantitative indicator of the degree of differentiation of colon cancer cells. Elevated alkaline phosphatase in serum, known as h...

Examples

Embodiment 1

[0019] Use a simple and low-cost alkaline phosphatase activity detection method to detect 5U / mL alkaline phosphatase sample solution and blank sample (buffer solution without analyte).

[0020] Such as figure 1 As shown, the specific implementation process is as follows: step 1, add 15 μ L 5U / mL alkaline phosphatase sample solution (prepared by 10 mM tris-HCl buffer solution containing 100 mM NaCl, pH 7.4) in a 1.5 mL plastic detection test tube, and then add 15 μL of 200 μM pyrophosphate solution (prepared from 10 mM tris-HCl buffer solution containing 100 mM NaCl, pH 7.4) was mixed with it, and placed at 37 ° C for 60 min, and continued to add 15 μ L of 50 μM copper nitrate aqueous solution, reacted at room temperature for 15 min, and then continued Add 15 μL of 1 mg / mL glucoamylase solution (prepared from 20 mM acetic acid-sodium acetate buffer solution, pH 4.5), react at room temperature for 70 min, then add 15 μL of 2% soluble starch solution (heated by ultrapure water wi...

Embodiment 2

[0023] A simple and low-cost alkaline phosphatase activity detection method is used to detect the alkaline phosphatase sample solution with an activity concentration ranging from 0.0375 to 5 U / mL.

[0024] Such as figure 1 As shown, the concrete steps of detecting each alkaline phosphatase sample solution in the present embodiment are: Step 1, add 15 μ L of alkaline phosphatase sample solution (containing 100mMNaCl 10mM tris-HCl buffer solution, pH 7.4), then add 15μL 200μM pyrophosphate solution (prepared from 10mM tris-HCl buffer solution containing 100mM NaCl, pH 7.4) to mix with it, and place it at 37℃ for 60min, Continue to add 15 μL of 50 μM copper nitrate aqueous solution, react at room temperature for 15 minutes, then continue to add 15 μL of 1 mg / mL glucoamylase solution (prepared from 20 mM acetic acid-sodium acetate buffer solution, pH 4.5), react at room temperature for 70 minutes, and then add 15 μL of 2% soluble starch Solution (prepared by heating ultrapure wat...

technical field

[0001] The invention belongs to the technical field of biochemical analysis and biosensing, and in particular relates to a simple and low-cost alkaline phosphatase activity detection method. Background technique

[0002] Alkaline phosphatase, also known as alkaline phosphatase, can catalyze the hydrolysis of phosphate bonds under alkaline conditions. In mammals, alkaline phosphatase activity is relatively high in liver, bile duct, kidney, bone and placenta. Common alkaline phosphatases include intestinal alkaline phosphatase, non-tissue-specific alkaline phosphatase, and placental alkaline phosphatase. High or low alkaline phosphatase activity is associated with many diseases. For example, the activity of alkaline phosphatase in differentiated colon cancer cells will be significantly increased, which is used as a qualitative and quantitative indicator of the degree of differentiation of colon cancer cells. Elevated alkaline phosphatase in serum, known as h...

Examples

Embodiment 1

[0019] Use a simple and low-cost alkaline phosphatase activity detection method to detect 5U / mL alkaline phosphatase sample solution and blank sample (buffer solution without analyte).

[0020] Such as figure 1 As shown, the specific implementation process is as follows: step 1, add 15 μ L 5U / mL alkaline phosphatase sample solution (prepared by 10 mM tris-HCl buffer solution containing 100 mM NaCl, pH 7.4) in a 1.5 mL plastic detection test tube, and then add 15 μL of 200 μM pyrophosphate solution (prepared from 10 mM tris-HCl buffer solution containing 100 mM NaCl, pH 7.4) was mixed with it, and placed at 37 ° C for 60 min, and continued to add 15 μ L of 50 μM copper nitrate aqueous solution, reacted at room temperature for 15 min, and then continued Add 15 μL of 1 mg / mL glucoamylase solution (prepared from 20 mM acetic acid-sodium acetate buffer solution, pH 4.5), react at room temperature for 70 min, then add 15 μL of 2% soluble starch solution (heated by ultrapure water wi...

Embodiment 2

[0023] A simple and low-cost alkaline phosphatase activity detection method is used to detect the alkaline phosphatase sample solution with an activity concentration ranging from 0.0375 to 5 U / mL.

[0024] Such as figure 1 As shown, the concrete steps of detecting each alkaline phosphatase sample solution in the present embodiment are: Step 1, add 15 μ L of alkaline phosphatase sample solution (containing 100mMNaCl 10mM tris-HCl buffer solution, pH 7.4), then add 15μL 200μM pyrophosphate solution (prepared from 10mM tris-HCl buffer solution containing 100mM NaCl, pH 7.4) to mix with it, and place it at 37℃ for 60min, Continue to add 15 μL of 50 μM copper nitrate aqueous solution, react at room temperature for 15 minutes, then continue to add 15 μL of 1 mg / mL glucoamylase solution (prepared from 20 mM acetic acid-sodium acetate buffer solution, pH 4.5), react at room temperature for 70 minutes, and then add 15 μL of 2% soluble starch Solution (prepared by heating ultrapure wat...

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