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Probe used for detecting acetylcholin esterase and its inhibitor activity, application and preparation method

An acetylcholinesterase and inhibitor technology, which is applied in the field of probes for detecting the activity of acetylcholinesterase and its inhibitor, can solve the problems of background interference and low sensitivity, and achieves the effects of high sensitivity, fast reaction speed and great application prospect.

Inactive Publication Date: 2014-12-31
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method often has background interference during use and has low sensitivity

Method used

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  • Probe used for detecting acetylcholin esterase and its inhibitor activity, application and preparation method
  • Probe used for detecting acetylcholin esterase and its inhibitor activity, application and preparation method
  • Probe used for detecting acetylcholin esterase and its inhibitor activity, application and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of formula I probe precursor and vesicle probe

[0039] 1. Preparation of the probe precursor of formula I

[0040] The first step is to dissolve 0.375g (1mmol) of 10,12-diacetylenic acid in 20ml of dichloromethane, then add 2ml of thionyl chloride dropwise to it to obtain a reaction solution, and continue to stir the reaction under the protection of nitrogen Then, the solvent is removed by distillation under reduced pressure until the end of the reaction (usually stirring overnight) to obtain the compound of formula II as a colorless oil.

[0041] In the second step, the compound of formula II obtained in the first step is dissolved in a small amount of tetrahydrofuran to obtain a solution of compound of formula II, and then the solution of compound of formula II is added dropwise to 20 ml of tetrahydrofuran solution containing 0.18 g of triethylene glycol to obtain a reaction liquid. Under the protection of nitrogen, continue to stir the reaction solut...

Embodiment 2

[0049] Example 2 Spectral properties and color changes of the reaction of vesicle probe with myristoylcholine chloride

[0050] 1. Spectral properties of the reaction of vesicle probe with myristoylcholine chloride

[0051] Take nine 60μl 1mM vesicle probes into each quartz dish, then add 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer (10mM pH=7.4) to dilute and dilute to 3ml; then , Respectively add 0μl, 0.6μl, 1.2μl, 1.8μl, 2.4μl, 3μl, 3.6μl, 4.5μl 10mM myristoylcholine chloride (final concentration 0μM, 2μM, 4μM, 6μM, 8μM, 10μM, 12μM , 15μM), measure the fluorescence emission spectrum and ultraviolet-visible light absorption spectrum after 5 minutes of reaction. The fluorescence emission spectrum was measured at 492nm excitation, and the excitation and emission slit width was 5nm.

[0052] Fluorescence emission spectra such as figure 1 As shown, when the concentration of myristoylcholine chloride is in the range of 0-15 μM, the fluorescence intensity at about 550nm ...

Embodiment 3

[0057] Example 3 Spectral properties and color changes of the reaction product of myristoylcholine chloride and acetylcholinesterase (AChE) mixed with vesicle probe

[0058] 1. Spectral properties of the reaction product of myristoylcholine chloride and acetylcholinesterase (AChE) mixed with vesicle probe

[0059] Take seven 3.6μl 10mM myristoylcholine chloride into each quartz dish, and add 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) buffer (10mM pH=7.4) to dilute and finally dilute to 3ml ; Then, add appropriate amounts (such as 2μl) of different concentrations of acetylcholinesterase (AChE) (0U / ml, 0.025U / ml, 0.05U / ml, 0.1U / ml, 0.2U / ml, 0.3U / ml, 0.4 U / ml); After reacting for 10 minutes, add 60μl of 1mM vesicle probe to the quartz dish, and then react for 5 minutes, and finally measure the fluorescence emission spectrum and ultraviolet-visible light absorption spectrum. The fluorescence emission spectrum was measured at 492nm excitation, and the excitation and emission ...

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Abstract

The invention relates to a probe used for detecting acetylcholin esterase and its inhibitor activity, an application and a preparation method. The probe is polymerized by 10,12-diyne pentacosanoic acid and a compound in a formula I according to weight ratio of 7:3, The vesicle probe presents blue color and no fluorescence in 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid buffer, and presents red color and emit fluorescence after being reacted with Myristoylcholine chloride (the fluorescence emission wavelength is 550nm), the sensitivity is high, the operation is easy, and the fluorescence detection method of the probe has great application prospect in biological medicine field.

Description

Technical field [0001] The invention relates to a probe for detecting the activity of acetylcholinesterase and its inhibitors, application and preparation method. Background technique [0002] The development of rapid detection technology for acetylcholinesterase (AChE) activity is of great significance for promoting human health and environmental protection. [0003] The current drugs used to treat Alzheimer’s disease are mainly acetylcholinesterase inhibitors, which control the acetylcholine content in patients by inhibiting acetylcholinesterase (AChE) activity. Therefore, the development of acetylcholinesterase (AChE) activity detection agents is used to screen related drugs Has an important role. [0004] In addition, certain organophosphorus pesticides and carbamate pesticides can inhibit the activity of acetylcholinesterase (AChE). Therefore, the development of acetylcholinesterase (AChE) activity detection reagents is useful for detecting organophosphorus pesticides and carba...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07C69/606C07C67/14C08F238/00C08F2/48C09K11/06G01N21/78G01N21/64
Inventor 陈小强周国栋王芳王惠琳
Owner NANJING TECH UNIV
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