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A method for detecting enzyme activity with a fluorescent probe

A fluorescent probe and enzyme detection technology, applied in the field of enzyme analysis, can solve the problems of inability to be widely used, high cost of double-labeling, and difficulty in double-labeling modification, etc., and achieve the effects of improving the recovery times of fluorescent signals, simple operation, and improved sensitivity.

Inactive Publication Date: 2017-05-10
BEIJING FLUORESCENCE BIOTECHNOLOGY CO LTD
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  • Claims
  • Application Information

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Problems solved by technology

However, the cost of double labeling is high, and most substrate molecules are difficult to double label modification, and the activity of the enzyme may also be greatly affected, so this method cannot be widely used.

Method used

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  • A method for detecting enzyme activity with a fluorescent probe
  • A method for detecting enzyme activity with a fluorescent probe
  • A method for detecting enzyme activity with a fluorescent probe

Examples

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Effect test

Embodiment 1）

[0052] Application of fluorescent probe PCP-SO3- in enzyme activity analysis and inhibitor screening:

[0053] (1) Preparation of chemically modified enzyme substrate: under nitrogen protection, add 6-(p-methyl red)aminocaproic acid (38 mg , 0.1 mmol) and carbonyldiimidazole (24 mg, 0.15 mmol), and magnetically stirred at room temperature for 30 minutes. Transfer the reaction by syringe to a 5 mL single port containing acetylcholine bromide (10 mg, 0.08 mmol), diazabicycloundec-7-ene (21 μL, 0.14 mmol) and dimethyl sulfoxide (0.25 mL) In a bottle, the reaction was stirred at 40°C for 24 hours. After concentration under reduced pressure, it was separated by silica gel column (eluent: dichloromethane / methanol / water=65 / 25 / 4, v / v / v) to obtain the target product 6-(p-methyl red)aminocaproyl chol Alkali bromide (15 mg, 34%), that is, the quencher group matching the fluorescent probe PCP-SO3 was attached to the enzyme substrate.

[0054] (2) The structure of PCP-SO3 is shown in fo...

Embodiment 2）

[0059] Application of Fluorescent Probe PCY-COOH in Enzyme Activity Analysis

[0060] (1) First prepare the same chemically modified enzyme substrate according to the method in Example 1.

[0061] (2) The structure of PCY-COOH is shown in formula (IV), and the synthesis method is as follows: add 2,7-dibromo substituted by 3,6,9,12-tetraoxapentadecan-15-oic acid to a 100 ml single-necked bottle Carbazole (0.8g), 1,4-p-phenylene (0.2g), 10 milliliters of water and 10 milliliters of DMF, after mixing evenly, pass nitrogen into the solvent to remove the oxygen in the solvent, add 52.0 milligrams of Pd (PPh3 )4 and 10.0 mg of CuI in 10 ml of diisopropylamine and DMF (5 ml each), heated to 55° C. for 14 hours. After the reaction solution was cooled to room temperature, 20 ml of water was added and stirred. The solution was dialyzed in water with a dialysis bag (molecular weight greater than 3500). After removing water, 525 mg of polymer PCY-COOH was obtained, Mn = 1.5×104. 1H NMR (...

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Abstract

The invention relates to a method for detecting the activity of an enzyme by a fluorescence probe. A series of water-soluble conjugated polymers and conjugated oligomers are used as the fluorescence probe. The polymers or oligomers having a high fluorescence quantum yield are used as the fluorescence probe, and the substrate of the enzyme is singly labeled, so the method has the advantages of high efficiency, sensitiveness, quantity, directness, simplicity, rapidness, universality and the like when the method is used to detect the activity of the enzyme.

Description

technical field [0001] The invention belongs to the technical field of enzyme analysis, and in particular relates to the application of a fluorescent probe, which can be used to detect enzyme activity. Background technique [0002] Enzymes are biocatalysts produced in living cells in organisms, which can efficiently catalyze various biochemical reactions in the body and promote the metabolism of organisms. Enzymes are the basis for the survival of cells, and almost all chemical reactions involved in cell metabolism are catalyzed by enzymes. Therefore, the detection of enzyme activity is of great significance in the fields of biotechnology, biochemistry and clinical diagnosis. [0003] Currently common methods for detecting enzyme activity include spectrophotometry, HPLC analysis, electrochemical analysis, chemiluminescence analysis, radiation method, and the like. Spectrophotometry is to use the change of substrate absorbance before and after enzymatic reaction to obtain t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64C09K11/06
Inventor 王树王勇
Owner BEIJING FLUORESCENCE BIOTECHNOLOGY CO LTD
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