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Modified hydrocyanine dyes for the detection of reactive oxygen species

A dye compound, alkyl technology, applied in the direction of anthracene dyes, organic dyes, azo dyes, etc.

Active Publication Date: 2013-07-17
LIFE TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, DHE has limited applications due to its spontaneous auto-oxidation, rapid photobleaching, high toxicity and multiple reaction products with ROS (7-8)
What's more, the lower emission wavelength of DHE makes its application in vivo problematic
Dihydrorhodamine (DHR), another vat dye that has been investigated for ROS detection (9), suffers from a high oxidation rate, thereby limiting its application
These probes, typically requiring time-consuming and expensive multi-step synthetic procedures, undergo rapid hydrolysis, thereby limiting their applications

Method used

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  • Modified hydrocyanine dyes for the detection of reactive oxygen species
  • Modified hydrocyanine dyes for the detection of reactive oxygen species
  • Modified hydrocyanine dyes for the detection of reactive oxygen species

Examples

Experimental program
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Effect test

Embodiment 1

[0284] Bovine pulmonary artery endothelial (BPAE) cells were grown overnight at a density of 7500 cells / well, treated with or without 100 μM menadione for 1 h, and then stained with 5 μM Red ROS probe for 30 min. Cells were then washed 3 times with 1X PBS and imaged using a Thermo Scientific Cellomics ArrayScan™ VTI HCS Reader (Thermo Fisher Scientific, Pittsburgh, PA) after staining with Hoechst nuclear stain. Menadione-treated cells showed a positive reaction due to the reactivity of the red ROS (Red ROS) probe to reactive oxygen species generated in the cells (see Figure 1).

Embodiment 2

[0286] RAW macrophages were plated in a 96-well plate at a density of 10,000 cells / well, treated with 500 ng / ml lipopolysaccharide (LPS) for 18 h, and then stained with 5 μM red ROS (Red ROS) probe for 30 minutes . Cells were washed 3 times with 1X PBS, stained with Hoechst nuclear stain, and then imaged using Thermo Scientific Cellomics ArrayScan™ VTI HCS Reader. LPS-treated cells showed enhanced fluorescence as a result of the reactivity of the red ROS (Red ROS) probe to reactive oxygen species produced in LPS-treated cells; no signal was seen in the control cell population ( See Figure 2).

Embodiment 3

[0288] Human hepatocellular carcinoma (HepG2) cells were plated at a density of 7500 cells / well in collagen I-coated 96-well plates. Cells were then treated with 50 μM nefazodone for 24 h and stained with 5 μM red ROS (Red ROS) probe for 30 min. Cells were washed 3 times with 1X PBS, stained with Hoechst nuclear stain, and then imaged using Thermo Scientific Cellomics ArrayScan™ VTI HCS Reader. There was increased fluorescence intensity after nefazodone treatment, indicating that the red ROS (Red ROS) probe was reacting with nefazodone-induced reactive oxygen species in the cells (see Figure 3).

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Abstract

ABSTRACT Described herein are compounds, compositions, methods, and kits for detecting reactive oxygen species (ROS) by conventional fluorescence microscopy, fluorescence spectroscopy, flow cytometry, and / or high content imaging. The compounds disclosed herein are novel reduced dyes, including Cy-based hydrocyanine dyes and Cy-based deuterocyanine dyes, which dyes are probes for detecting ROS and measuring oxidative stress in cells either in vitro and / or in vivo. Also described herein are processes for preparing novel reduced dyes, i.e., ROS probes, for use in the disclosed compositions, methods and kits.

Description

field of invention [0001] The present invention relates to probes for detecting reactive oxygen species (reactive oxygen species, ROS), especially related to reduced cyanine dye probes (cyanine dye probes), and the application of such probes in vitro or in vivo. Background of the invention [0002] Oxidative stress arises from an imbalance between the production of reactive oxygen species (ROS) and the ability of cells to scavenge such free radicals. Oxidative stress can be caused by many different pathways, intrinsic or extrinsic, either mediated by mitochondrial respiration or by membrane-bound NADPH oxidase. ROS play an important role in the progression of a variety of diseases including, but not limited to, inflammation, atherosclerosis, aging, and age-related degenerative disorders. Probes that can detect ROS in serum samples, biopsy explants, cell cultures, and in vivo have potential use as research tools for medical diagnosis and diagnosis of diseases characterized ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61Q5/10A61K8/41
CPCC09B23/08C09B23/06C09B57/00G01N21/6486C09B1/00C09B23/083
Inventor 姜熙喆K.吉B.曼达维利L-Q.赢B.布兰肖
Owner LIFE TECH CORP
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