Fluorescence probe based on beta-glucuronidase of acridone and application of fluorescence probe

A technology of glucuronidase and aldolase, which is applied in the field of fluorescent probes of beta-glucuronidase, can solve the problems of excitation, short wavelength, background fluorescence interference, etc., achieves high sensitivity, and has a simple and easy synthesis process. line, the effect of reduced interference

Inactive Publication Date: 2018-04-17
DALIAN MEDICAL UNIVERSITY
View PDF1 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, detection based on small molecule fluorescent probes β - Glucuronidase activity is still in its infancy and is the most common assay β -The fluorescent probe for glucuronidase activity is 4-methylumbelliferone- β -Glucuronide, its excitation and occurrence wavelengths are relatively short, and it is easily interfered by background fluorescence

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Fluorescence probe based on beta-glucuronidase of acridone and application of fluorescence probe
  • Fluorescence probe based on beta-glucuronidase of acridone and application of fluorescence probe
  • Fluorescence probe based on beta-glucuronidase of acridone and application of fluorescence probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1. In vitro determination of the selectivity of different hydrolases

[0027] (1) Prepare 99 µL in vitro metabolic reaction system in advance, including pH 6.0 phosphate buffer (50 mM), different kinds of hydrolases (0.1 mg / mL), and pre-incubate at 37°C for 3 minutes with shaking;

[0028] (2) Add 1 µL of DDAO-glu at a concentration of 1 mM (final concentration 10 μM) to the reaction system to initiate the reaction;

[0029] (3) After 60 minutes, add 50 µL of glacial acetonitrile and shake vigorously to terminate the reaction;

[0030](4) After high-speed centrifugation at 4°C and 20,000×g for 20 minutes in a high-speed refrigerated centrifuge, the supernatant was collected for fluorescence detection (DDAO-glu: Ex=600 nm, Em=660 nm). The results show that only β -Glucuronidase (GLU) catalyzes the reaction, and the reaction rate is much higher than other hydrolases, indicating that β -The reaction of glucuronidase catalyzed DDAO-glu has good selectivity, and t...

Embodiment 2

[0031] Example 2. In vitro β -Inhibition experiment of glucuronidase

[0032] (1) Prepare 99 µL in vitro metabolic reaction system in advance, including pH 6.0 phosphate buffer (50 mM), β -Glucuronidase (0.1 mg / mL), and different concentrations of inhibitors (final concentrations were 50, 200, 600 μM) were pre-incubated with shaking at 37°C for 3 minutes;

[0033] (2) Add 1 µL of DDAO-glu at a concentration of 1 mM (final concentration 10 μM) to the reaction system to initiate the reaction;

[0034] (3) After 60 minutes, add 50 µL of glacial acetonitrile and shake vigorously to terminate the reaction;

[0035] (4) After high-speed centrifugation at 4°C and 20,000×g for 20 minutes in a high-speed refrigerated centrifuge, the supernatant was collected for fluorescence detection (DDAO-glu: Ex=600 nm, Em=660 nm). The results showed that only baicalin showed a dose-dependent inhibition of β - Catalyzed reaction by glucuronidase (GLU), while neither BNPP nor LAP (carboxyesterase...

Embodiment 3

[0036] Example 3 Different sources β - Activity assay of glucuronidase

[0037] (1) Prepare 99 µL in vitro metabolic reaction system in advance, including pH 6.0 phosphate buffer (50 mM), different sources β -Glucuronidase (0.1 mg / mL), pre-incubated with shaking for 3 minutes at 37°C;

[0038] (2) Add 1 µL of DDAO-glu at a concentration of 1 mM (final concentration 10 μM) to the reaction system to initiate the reaction;

[0039] (3) After 60 minutes, add 50 µL of glacial acetonitrile and shake vigorously to terminate the reaction;

[0040] (4) After high-speed centrifugation at 4°C and 20,000×g for 20 minutes in a high-speed refrigerated centrifuge, the supernatant was collected for fluorescence detection (DDAO-glu: Ex=600 nm, Em=660 nm). The results showed that different sources β -Glucuronidase, including recombinant expression single enzyme and subtype E. coli origin such as E.coli, E.coli IX and E.coli VII-A β -Glucuronidase can catalyze both HC-glu and DDAO-glu react...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention relates to a fluorescence probe based on beta-glucuronidase of acridone and application of the fluorescence probe, belonging to the technical field of biological medicines. The fluorescence probe can be used for determining the enzyme activities of beta-glucuronidase in biological systems of different sources. HC-glu is taken as a specific probe reaction substrate, beta-glucuronidasehydrolysis reaction is taken as a probe reaction by virtue of a beta-glucuronidase in-vitro reaction system, and the activity of beta-glucuronidase in each biological sample is determined by quantitatively detecting the generation amount of an aglycone metabolite in a unit time. The fluorescence probe can be applied to the qualitative and quantitative determination of the activities in beta-glucuronidase of different individual sources of human and animal tissue samples, different species of cells and cell preparation objects and various plants and microorganisms, and the evaluation of the drug treatment capacity of beta-glucuronidase and the development and screening of a beta-glucuronidase inhibitor can be realized.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, in particular to an acridone-based β - Fluorescent probes for glucuronidase and applications thereof. Background technique [0002] β -glucuronidase ( β -glucuronidase) is a glycoside hydrolase that can hydrolyze β-glucuronides into corresponding aglycones and β-glucuronic acid. Currently, it has been found in animals, plants and microorganisms β - the presence of glucuronidase. in the human body β -Glucuronidase is an endogenous enzyme, which is produced, synthesized and secreted by the body's own tissue cells, and has been isolated in various tissues and body fluids, such as macrophages, blood cells, liver, spleen, kidney, intestine , lung, muscle, bile, intestinal juice, urine and serum. In the lysosomes of the cells of internal organs, β -Glucuronidase was also abundantly expressed. As an acid hydrolase located in the lysosome of glycoprotein, it participates in the physiological...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C07H17/02C09K11/06C12Q1/34G01N21/64G01N33/573
CPCC07H17/02C09K11/06C09K2211/1029C09K2211/1088C12Q1/34G01N21/6428G01N33/573G01N2333/924
Inventor 马骁驰冯磊田象阁霍晓奎王超宁静于振龙孙成鹏
Owner DALIAN MEDICAL UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap