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A kind of fluorescent detection method of alkaline phosphatase activity

A technology for phosphatase activity and fluorescence detection, which is applied in the field of analysis and detection, can solve the problems of large isotope labels, time-consuming, inefficient, and complicated operations, and achieve the effects of high selectivity, simple drugs, and high sensitivity

Active Publication Date: 2020-11-17
CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional methods for detecting alkaline phosphatase activity include isotope labeling, which is complex, time-consuming and inefficient, and requires hazardous radioactive isotope labels

Method used

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  • A kind of fluorescent detection method of alkaline phosphatase activity
  • A kind of fluorescent detection method of alkaline phosphatase activity
  • A kind of fluorescent detection method of alkaline phosphatase activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Principle and feasibility verification of the inventive method

[0034] attached figure 1 It is a schematic diagram of the principle of the fluorescence detection method for alkaline phosphatase activity of the present invention. First verify the feasibility of the present invention, as figure 2 As shown, the mixed solution of 1,4-benzenediol HQ and DAMO was incubated at 37°C for 60 minutes, and the fluorescence of the system was measured with a fluorescence spectrophotometer, excited at 370nm, and there was obvious fluorescence emission at 520nm (f); while 4 -HPP aqueous solution (a) has no fluorescence emission near 520nm; DAMO (b) or alkaline phosphatase (d) is added alone to 4-HPP solution, and incubated at 37°C for 60 minutes, there is no obvious fluorescence; DAMO and alkaline phosphoric acid The enzyme mixed solution (c) also has no fluorescence after incubation; only when 4-HPP and alkaline phosphatase are incubated together, then adding DAMO for incubation c...

Embodiment 2

[0036] Synthesis and Characterization of Fluorescent Silicon Nanoparticles

[0037] Dissolve 100mg of HQ into 95mL of ultrapure water, add 5mL of DAMO, mix well and incubate at room temperature (~25°C) for 80min. The supernatant was lyophilized and dissolved in ultrapure water and placed in a refrigerator at 4°C for use. Such as image 3 As shown, the synthesized silicon nanoparticles have obvious absorption spectra different from those of the two initial reactants HQ and DAMO, and have obvious fluorescence. The maximum fluorescence excitation peak and emission peak are located near 370nm and 520nm, respectively. Transmission electron microscope showed that the morphology of silicon nanoparticles was spherical with an average particle diameter of about 70 nanometers. In addition, the test results of infrared spectroscopy and X-ray photoelectron spectroscopy also confirmed the existence of fluorescent silicon nanoparticles.

Embodiment 3

[0039] A kind of fluorescent detection method of alkaline phosphatase activity

[0040] First, 225 μL of 2.0 mM 4-HPP and 405 μL of ultrapure water were added to 180 μL of Tris-HCl (50 mM, pH 8.5, containing 50 μM MgCl 2) buffer solution, and then mixed the prepared 4-HPP mixed solution with 90 μL of different active alkaline phosphatase standard solutions (0, 0.1mU / mL, 0.2mU / mL, 0.3mU / mL, 0.5mU / mL, 0.7mU / mL, 0.9mU / mL, 1mU / mL, 2mU / mL, 3mU / mL, 5mU / mL, 10mU / mL, 15mU / mL, 20mU / mL, 30mU / mL, 40mU / mL, 50mU / mL) , and the alkaline phosphatase solution to be tested were incubated at 37°C for 60 minutes, and then 100 μL of DAMO (50% aqueous solution) was added, and the resulting mixed solution was incubated for 80 minutes at room temperature (~25°C), and then analyzed by fluorescence spectrometry. A luminometer measures the fluorescence of the solution. Such as Figure 4 As shown, the fluorescence emission spectrum intensity of the obtained solution increases with the enhancement of a...

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Abstract

The invention relates to a fluorescence detection method for the activity of alkaline phosphatase, which is characterized in that sodium 4-hydroxyphenyl phosphate (4-HPP) is used as a substrate, molecules of the substrate are hydrolyzed into 1,4-benzenediol (HQ) under the catalysis of alkaline phosphatase in an aqueous solution, 1,4-benzenediol can react with subsequently added 3-(2-aminoethylamino)propyltrimethoxysilane (DAMO) to generate silicon nanoparticles (SiNPs) capable of emitting green fluorescence. The higher the activity of alkaline phosphatase in the system is, the more the generated fluorescent silicon nanoparticles are, and the higher the fluorescence intensity is. Quantitative analysis and detection of the activity of alkaline phosphatase are carried out according to the fluorescence intensity change of the detection solution. Compared with the prior art, the method has the advantages that required reagents and medicines are simple and easy to obtain, the operation is convenient, quick and accurate, and a sensitive and reliable method is provided for quantitative detection of the activity of alkaline phosphatase in a clinical sample. According to the fluorescence detection method, the detection limit is 0.1mU / mL, and the fluorescence detection method has the advantages of high sensitivity, high selectivity, wide linear range and the like.

Description

technical field [0001] The invention belongs to the technical field of analysis and detection, and in particular relates to a fluorescence detection method for alkaline phosphatase activity. Background technique [0002] Alkaline phosphatase (alkaline phosphatase, ALP, EC 3.1.3.1) is an enzyme that is widely distributed in human liver, bone, intestine, kidney, placenta and other tissues and excreted from the liver to the gallbladder. It plays an extremely important role in cell growth, signal transduction and intracellular regulation in the process of apoptosis. Alkaline phosphatase is an important indicator to reflect systemic diseases such as liver, gallbladder and bones. When the human body suffers from jaundice, liver cancer, cholestatic hepatitis and other diseases, the content of alkaline phosphatase in serum will increase significantly; Or when suffering from osteomalacia, rickets, bone cell cancer, osteoporosis and other bone diseases, the alkaline phosphatase conte...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64G01N1/38
CPCG01N1/38G01N21/6428
Inventor 孙健杨秀荣刘国永赵佳会卢沙沙
Owner CHANGCHUN INST OF APPLIED CHEMISTRY - CHINESE ACAD OF SCI
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