Detection method for activity of carboxylesterase and activity of carboxylesterase inhibitor

A carboxylesterase and detection method technology, applied in the biological field, can solve the problems of easy occurrence of false positive signals, high price, low sensitivity, etc., and achieve the effects of avoiding the generation of false positive signals, good selectivity, and high sensitivity

Active Publication Date: 2012-11-21
CHANGZHOU INST OF ENERGY STORAGE MATERIALS &DEVICES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a kind of activity of carboxylesterase and carboxylesterase in order to solve the problems of low sensitivity of existing enzyme activity detection methods, prone to false positive signals, complex methods, high price and long time consumption. Assays for the Activity of Inhibitors

Method used

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  • Detection method for activity of carboxylesterase and activity of carboxylesterase inhibitor
  • Detection method for activity of carboxylesterase and activity of carboxylesterase inhibitor
  • Detection method for activity of carboxylesterase and activity of carboxylesterase inhibitor

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Experimental program
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Effect test

preparation example Construction

[0047] Concrete preparation method is:

[0048] Step 1: Dissolve perylene anhydride in 5% KOH aqueous solution to make it dissolve, filter, adjust the pH value of the filtrate to 8-9 with dilute hydrochloric acid, add tetra-n-octyl ammonium bromide and 1,4-dibromobutane, in Reflux at 100°C for 2 hours, extract with chloroform, wash the separated organic phase with 15% NaCl aqueous solution for 3 times, distill off the solvent, and purify the crude product by chromatography to obtain compound 3,4,9,10-tetra -(4-bromobutyl-ester group)-perylene; the molar ratio of perylene anhydride, tetra-n-octyl ammonium bromide and 1,4-dibromobutane is 1:0.1:10;

[0049] Step 2: Dissolve the compound 3,4,9,10-tetrakis-(4-bromobutyl-ester)-perylene obtained in step 1 in a mixture of tetrahydrofuran and water to dissolve it, and add Trimethylamine, refluxed at 66°C for 72h, distilled off the solvent, dissolved the obtained crude product in water, extracted 3 times with chloroform, distilled th...

Embodiment 1

[0071] The detection of embodiment 1 acetylcholinesterase

[0072] 1. Sodium polyethylene sulfonate with a concentration of 0 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 5 μM, 6 μM and 7 μM was mixed with 1 μM 3, 4, 9, 10-tetra- (4-trimethylaminobutyloxy-carbonyl)-perylene mixture, measure the fluorescence spectrum under the conditions of fluorescence excitation at 442nm and detection at 460-650, such as figure 1 As shown, the fluorescence intensity of 3,4,9,10-tetrakis-(4-trimethylaminobutyloxy-carbonyl)-perylene decreased gradually with the concentration of sodium polyethylene sulfonate, and the concentration of sodium polyethylene sulfonate was 6 μM , the quenching efficiency reaches 99.9%; it is determined that when the concentration ratio of 3,4,9,10-tetrakis-(4-trimethylaminobutyloxy-carbonyl)-perylene and sodium polyethylene sulfonate is 1:6 , the quenching efficiency reaches 99.7%. Fix the ratio of the two and change their concentrations to obtain the...

Embodiment 2

[0078] The detection of embodiment 2 lipase

[0079] 1, step 1 is the same as step 1 in embodiment 1;

[0080] 2. Add 68.75μM (concentration in the hydrolysis system) thioacetylcholine and the concentrations are 0, 5mU / mL, 10mU / mL, 20mU / mL, 40mU / mL, 60mU / mL, 80mU / mL, 100mU / mL, 120mU / mL, 150mU / mL, 200mU / mL, 250mU / mL, 300mU / mL, and 400mU / mL lipase were reacted at 37°C for 1h to obtain the hydrolyzed product thiocholine;

[0081] The hydrolysis reaction system is: 400 μL system, including 27.5 μL 100 mM Tris-HAc buffer (pH 7.4), 27.5 μL 1 mM thioacetylcholine solution, 2 μL lipase solution with a concentration of 0-0.08 U / μL, and the remaining The amount is sterile water;

[0082] 3. The obtained hydrolyzate has a concentration of 50 μM (concentration in the detection system) thiocholine, a concentration of 100 μM silver nitrate solution, a concentration of 60 μM sodium polyethylene sulfonate solution and a concentration of 10 μM3,4,9,10-four -(4-trimethylaminobutyloxy-carbon...

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Abstract

The invention discloses a detection method for the activity of carboxylesterase and the activity of a carboxylesterase inhibitor, and belongs to the technical field of biology. The detection method solves the problems of lower sensitivity, frequent occurrence of false positive signals, complex method, expensive price and long consumed time in the existing enzyme activity detection method. The detection method comprises the following steps of: reacting substrate molecules and carboxylesterases with different concentrations so as to obtain a hydrolysis product; and mixing the obtained hydrolysis product, silver nitrate solution, polyanion solution and a micromolecule probe with positive charges, and carrying out fluorescence detection on the activity of the carboxylesterase. The invention also provides a detection method for the activity of the carboxylesterase inhibitor. The method has higher sensitivity, and the detection limit is 0.05mU/mL, and is lower than that of the existing certain detection method by 1 or 2 order of magnitudes; and simultaneously, experiments prove that the method has good selectivity, simpleness and convenience and low cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting the activity of carboxylesterase and the activity of carboxylesterase inhibitors. Background technique [0002] Coordination polymers generally refer to metal-organic materials with periodic network structures formed by self-assembly of metal ions and organic ligands. For example, sulfhydryl ligands-SR, amino acids, amino ligands and metal ions M (M: transition metal ions such as Cu, Ag, Au) can form coordination polymers through ordered self-assembly. The general structural formula is as follows: [0003] [0004] At present, the detection methods of enzyme activity mainly include colorimetric method, fluorescence method, electrochemical method and other methods. The traditional colorimetric method for measuring enzyme activity is convenient and can be distinguished with the naked eye, but the detection sensitivity is relatively low, and false...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 于聪廖冬丽陈健周会鹏
Owner CHANGZHOU INST OF ENERGY STORAGE MATERIALS &DEVICES
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