Preparation method and application of double-rate-type multifunctional high-sensitivity florescent probe for carboxylesterase detection

A functional carboxylate, fluorescent probe technology, applied in the field of analytical chemistry, can solve the problems of no double ratio, single detection method, unfavorable visual monitoring, etc., and achieve the effect of sensitive detection

Inactive Publication Date: 2017-04-26
QUFU NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although these methods have their own advantages, in general, their detection limit (0.5-1 μg / ml) needs to be improved, and the quantification methods are all fluorescence single quantification, and there is no function of double ratio correction
This will lead to problems such as low detection limit of existing detection methods, inaccurate quantitative results, single detection method, and unfavorable visual monitoring.
These issues are very limiting for the detection of trace carboxylesterases in cells

Method used

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  • Preparation method and application of double-rate-type multifunctional high-sensitivity florescent probe for carboxylesterase detection
  • Preparation method and application of double-rate-type multifunctional high-sensitivity florescent probe for carboxylesterase detection
  • Preparation method and application of double-rate-type multifunctional high-sensitivity florescent probe for carboxylesterase detection

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Preparation of small molecule carboxylesterase ratiometric probes

[0063] (1) Dissolve 2,4-Dihydroxybenzaldehyde (2,4-Dihydroxybenzaldehyde) in ethanol at a concentration of 0.048g / mL, then add Diethyl glutaconate at a concentration of 0.067g / mL mL, after mixing evenly, add 4 mL of anhydrous piperidine dropwise, reflux for 24 hours, cool, and a yellow solid precipitates, which is recrystallized with absolute ethanol to obtain product 1;

[0064] (2) Dissolve the product 1 in anhydrous pyridine at a concentration of 0.025g / mL, then add acetic anhydride at a concentration of 1mol / L, stir for 0.5h, add 100g of crushed ice, and stir for 10min to precipitate an off-white solid. Elute with acetonitrile-dichloromethane eluent with a volume ratio of 1:5, and spin evaporate to obtain product 2;

[0065] (3) Dissolve the product 2 in tetrahydrofuran at a concentration of 0.011 g / mL, then add 4% osmium tetroxide aqueous solution, stir for 0.5 h, then add sodium period...

Embodiment 2

[0076] Example 2: The dual-ratio multifunctional carboxylesterase detection fluorescent probe prepared in Example 1 quantitatively analyzes carboxylesterase in biological samples: detection of carboxylesterase content in serum

[0077] 1) Prepare the solution

[0078] Probe stock solution: Accurately weigh the dual-ratio multifunctional carboxylesterase detection fluorescent probe and dissolve it in anhydrous acetonitrile to prepare a probe stock solution with a concentration of 60 µM;

[0079] Carboxylesterase stock solution: Accurately weigh 0.0050g carboxylesterase of the target object to be tested and dissolve it in 1000ml distilled water to prepare a concentration of 5×10 -5 g / mL carboxylesterase stock solution;

[0080] 2) Establish a linear equation for the serum-carboxyesterase standard

[0081]Dilute the carboxylesterase stock solution prepared in step 1) with distilled water to obtain a carboxylesterase standard solution with a gradient concentration of 0-15g / mL, t...

Embodiment 3

[0087] Example 3: Qualitative detection of small molecule carboxylesterases in biological samples

[0088] The detection method of carboxylesterase in serum samples is as follows: after the serum sample to be tested is mixed with anhydrous acetonitrile at a volume ratio of 5:1, centrifuged at 5000 rpm for 20 minutes, the supernatant is taken out and processed through a dialysis membrane, and then 200 μL of dialysis is taken Add 100 μL of probe stock solution and 200 µL of carboxylesterase stock solution to the supernatant in turn, make the volume to 1000 μL with Tris-hydrochloric acid buffer solution with pH 7.46, store at 25°C for 50 min, the color is yellow-green under ultraviolet light, It can be judged that the serum sample contains carboxylesterase.

[0089] The probe stock solution and carboxylesterase stock solution in this example are the same as step 1) in Example 2. The color change of the probe with the increase of the concentration of the analyte under fluorescent...

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Abstract

The invention relates to a preparation method and an application of a double-rate-type (ultraviolet/fluorescence) multifunctional (calorimetric/fluorescence/ultraviolet) high-sensitivity florescent probe for carboxylesterase detection. Firstly, 2,4-dihydroxybenzaldehyde, diethyl glutaconate and anhydrous piperidine react in ethyl alcohol to produce a product 1, the product 1 and acetic anhydride react in anhydrous pyridine to produce a product 2, the product 2 reacts with osmium tetroxide and sodium periodate in tetrahydrofuran to produce a product 3, and the product 3 reacts with anhydrous potassium carbonate in methyl alcohol to produce a product 4; then methylpyridine and methyl iodide react in absolute ether to produce a product 5; next, the product 4 and the product 5 are dissolved in absolute ethanol, piperidine is added for a reaction, and a product 6 is obtained; finally, the product 6 is dissolved in acetic anhydride, anhydrous sodium acetate is added, and the double-rate-type multifunctional florescent probe for carboxylesterase detection is obtained. The probe is applicable to qualitative and quantitative analysis of carboxylesterase in biological samples, is sensitive, accurate and quick in detection, and can be applied to related fields of analytical chemistry, life organic analytical chemistry, disease pre-diagnosis, clinical medical examination and the like.

Description

technical field [0001] The invention belongs to the field of analytical chemistry, and relates to a preparation method and application of a double-ratio type multifunctional carboxylesterase detection fluorescent probe. Background technique [0002] Carboxylesterase plays an important role in physiological regulation in organisms, especially in cells, and its concentration can reflect the disease state of the body or some cells, and is also closely related to some diseases or metabolic disorders. At present, there are few studies on carboxylesterase detection methods, and most of the reported detection methods are fluorescent molecular probe detection methods. Although these methods have their own advantages, in general, their detection limit (0.5-1 μg / ml) needs to be improved, and the quantification methods are all fluorescence single quantification, and there is no double ratio correction function. This will lead to problems such as low detection limit of existing detecti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D311/16C09K11/06G01N21/64
CPCC07D311/16C09K11/06C09K2211/1088G01N21/6486
Inventor 陈光姜翱胡金莲付强刘玉霞王桦尤进茂
Owner QUFU NORMAL UNIV
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