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Application of fluorescent probe for detecting enzyme activity and screening passivating agent

A fluorescent probe and enzyme detection technology, applied in the field of enzyme analysis, can solve the problems of difficult double-label modification, impact on enzyme activity, and high cost of double-labeling

Inactive Publication Date: 2009-03-25
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • 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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  • Application of fluorescent probe for detecting enzyme activity and screening passivating agent
  • Application of fluorescent probe for detecting enzyme activity and screening passivating agent
  • Application of fluorescent probe for detecting enzyme activity and screening passivating agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Embodiment 1, fluorescent probe PFP-SO 3 - Application in Enzyme Activity Analysis and Inhibitor Screening

[0077] Preparation of chemically modified enzyme substrate: under nitrogen protection, add 6-(p-methyl red) aminocaproic acid (38mg, 0.1mmol) and Carbonyldiimidazole (24 mg, 0.15 mmol), magnetically stirred at room temperature for 30 minutes. The reaction solution was transferred by syringe to a 5 mL single-necked bottle containing acetylcholine bromide (10 mg, 0.08 mmol), diazabicycloundec-7-ene (21 μL, 0.14 mmol) and dimethyl sulfoxide (0.25 mL), 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 fluorescent probe PFP-SO is attached to the enzyme substrate 3 - Matched quencher group.

[0078] Formula V

[0079] Drawi...

Embodiment 2

[0082] Embodiment 2, the application of fluorescent probe PFP-COOH in enzyme activity analysis

[0083] First prepare the same chemically modified enzyme substrate according to the method in Example 1.

[0084] Draw a standard detection curve: add 1.0mL phosphate buffer solution (25mM, pH8) to a 3mL colorimetric cell, add polymer PFP-COOH (formula VI) to 2.0μM, and measure its fluorescence emission spectrum at an excitation wavelength of 376nm. Then the above-mentioned modified enzyme substrate is continuously added dropwise, and the fluorescence emission spectrum is measured after each drop of the above-mentioned modified enzyme substrate. Record the fluorescence intensity value at 424nm of the fluorescence emission spectrum, expressed as (F 0 / F-1) is the ordinate, [Q] is the abscissa, and draws the Stern-Volmer curve as a standard detection curve, such as Figure 7 shown.

[0085] Formula VI

[0086] AChE activity analysis: add 1.0mL phosphate buffer solution (25mM, ...

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Abstract

The invention relates to application of fluorescent probes to the detecting of the enzymatic activity and sieving of an inhibitor. The invention applies a series of water-soluble conjugated polymers, conjugated oligomers and non-conjugated polymers or oligomers provided with chromophores as the fluorescent probes. By utilization of the polymers or the oligomers with high fluorescent quantum yield as the fluorescent probes which then perform single labeling on substrates of enzymes, the invention has the advantages of high efficiency, sensitivity, quantification, directness, simplicity, convenience, quickness, universality and so on when applied to the detecting of the enzymatic activity and sieving of the inhibitor.

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 for detecting enzyme activity and screening inhibitors. Background technique [0002] The traditional method for quantitative detection of enzyme activity is spectrophotometry. This method uses the change of the substrate absorbance before and after the enzymatic reaction to obtain the change value of the substrate concentration, so as to obtain the enzyme activity. Because this method needs to rely on ultraviolet detection, its sensitivity is low, and the consumption of enzyme and substrate is large, so it cannot detect enzyme activity at low concentration. [0003] Other methods for quantitative detection of enzyme activity include HPLC analysis, electrochemical analysis, chemiluminescence analysis, and the like. Although HPLC analysis has the advantages of quantitative analysis, it requires a large...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76C09K11/06G06F17/11C12Q1/00
CPCC08G2261/19C08G2261/3142C08G2261/5222
Inventor 王树冯福德
Owner INST OF CHEM CHINESE ACAD OF SCI
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