Real-time imaging sensor for measuring cellular thiol level

Patent metadata

Field	Value
Publication number	US-10215757-B2
Application number	US-201515527816-A
Country	US
Kind code	B2
Filing date	Nov 18, 2015
Priority date	Nov 19, 2014
Publication date	Feb 26, 2019
Grant date	Feb 26, 2019

Abstract

The present invention relates to a fluorescence sensor capable of real-time imaging for measuring a cellular thiol level. The present invention reveals that the fluorescence intensity of the fluorescent real-time SH group-tracer (FreSH-Tracer) of the present invention increases or decreases continuously, ratiometrically or reversibly depending on the thiol level in living cells, and thus can be usefully used as a biosensor which is remarkably susceptible to quantitative or qualitative real-time detection of the cellular thiol level in living cells.

First claim

The invention claimed is:

1. A method for detecting thiols in living cells, comprising a step of adding a composition comprising a compound selected from the group consisting of compounds represented by the following formulas 3 to 5, 7 and 8 or a salt thereof to the living cells:

wherein the detection of thiols is performed by obtaining the ratio of the fluorescence intensity of the living cells at 430-550 nm to the fluorescence intensity at 550-680 nm.

2. The method of claim 1 , wherein the compound shows a maximum emission wavelength at 550-680 nm in a thiol-free state, and shows a maximum emission wavelength at 430-550 nm in a thiol-bound state.

3. The method of claim 1 , wherein the fluorescence intensity of the compound at an emission wavelength changes continuously and reversibly as the level of thiols in living cells increases.

4. The method of claim 3 , wherein the fluorescence intensity at the emission wavelength changes in the range of 430 nm to 680 nm.

5. The method of claim 3 , wherein the compound shows a decrease in the fluorescence intensity at 550-680 nm and an increase in the fluorescence intensity at 430-550 nm as the level of thiols in living cells increases.

6. The method of claim 1 , wherein the ratio is a relationship between the fluorescence intensity at 430-550 nm and the fluorescence intensity at 550-680 nm.

7. The method of claim 6 , wherein the relationship is a mathematical ratio between the fluorescence intensity at 430-550 nm and the fluorescence intensity at 550-680 nm, and the mathematical ratio changes ratiometrically and reversibly depending on the amount of thiols in living cells to thereby indicate the amount of thiols in living cells in real time.

8. The method of claim 1 , wherein the detection is quantitative or qualitative detection of the thiols in the living cells.

9. The method of claim 1 , wherein the detection is real-time quantitative detection.

10. The method of claim 1 , wherein the detection of thiols in living cells indicates the oxidative stress or degree of oxidation of the cells.

11. The method of claim 1 , wherein the detection of thiols in living cells indicates the degree of aging of the cells.

12. The method of claim 1 , wherein the thiols include glutathione (GSH), homocysteine (Hcy), cysteine (Cys), or thiols in cysteine residues of proteins.

Assignees

• Cell2In Inc
• Snu R&Db Foundation
• Univ Korea Res & Bus Found

Inventors

• Kim In-Gyu
• Choi Ki Hang
• Jeong Eui Man
• Kang Heun Soo

Classifications

• G01N2800/7009

  Oxidative stress · CPC title

• G01N33/6815

  containing sulfur, e.g. cysteine, cystine, methionine, homocysteine · CPC title

• G01N33/582 Primary

  with fluorescent label · CPC title

• G01N33/581

  with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates) · CPC title

• G01N33/52

  Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper {and including single- and multilayer analytical elements (immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72)} · CPC title