Method for quantitatively analyzing fluorescent dyes labeled on extracellular vesicle by using fluorescence correlation spectroscopy, and use thereof

Patent metadata

Field	Value
Publication number	US-12584794-B2
Application number	US-202218557769-A
Country	US
Kind code	B2
Filing date	Apr 28, 2022
Priority date	Apr 29, 2021
Publication date	Mar 24, 2026
Grant date	Mar 24, 2026

Abstract

A method of quantifying fluorescent dyes labeled on extracellular vesicles, including providing an extracellular vesicle molecule labeled with a fluorescence dye molecule, and determining a brightness of each of the extracellular vesicle molecule labeled with the fluorescence dye molecule and the fluorescence dye molecule through fluorescence correlation spectroscopy (FCS) of the extracellular vesicle molecule and the fluorescence dye molecule.

First claim

The invention claimed is:

1 . A method of quantifying fluorescent dyes labeled on extracellular vesicles, the method comprising:
providing an extracellular vesicle molecule labeled with a fluorescence dye molecule; determining a brightness of each of the extracellular vesicle molecule labeled with the fluorescence dye molecule and the fluorescence dye molecule through fluorescence correlation spectroscopy (FCS) of the extracellular vesicle molecule and the fluorescence dye molecule, and determining a number of labeled fluorescence dye molecules per extracellular vesicle by determining a brightness value of the fluorescence dye molecule with respect to the brightness of the extracellular vesicle molecule labeled with a fluorescence dye, wherein the fluorescence correlation spectroscopy comprises: deriving a fluorescence auto-correlation function by irradiating a sample comprising extracellular vesicle molecules labeled with a fluorescent dye, with an excitation laser, and then measuring a generated fluorescence signal with a detector; analyzing the fluorescence auto-correlation function; and calculating the fluorescent brightness per extracellular vesicle molecule by using the fluorescent brightness of the sample and the number of extracellular vesicle molecules contained in the sample, wherein the extracellular vesicle molecule labeled with a fluorescence dye is chemically labeled with a fluorescence dye.

2 . The method of claim 1 , wherein the brightness of each of the extracellular vesicle molecule labeled with the fluorescence dye molecule and the fluorescence dye molecule is expressed in units of Hz, and is an absolute value of a specific molecule.

3 . The method of claim 1 , wherein the number of labeled fluorescence dye molecules per extracellular vesicle is an integer, and the determining comprises determining an integer rounded to a first decimal place if not an integer.

4 . The method of claim 1 , wherein the extracellular vesicle molecule is labeled with a plurality of fluorescence dye molecules having different brightness values.

5 . The method of claim 1 , further comprising deriving a correlation function by determining a brightness of molecules having different conditional variables.

6 . The method of claim 5 , wherein the different conditional variables are selected from the group consisting of factors capable of affecting a size or number of extracellular vesicle molecules, a concentration of the fluorescence dye molecule used to treat the extracellular vesicle molecule, and labeling of the fluorescence dye molecule on the extracellular vesicle molecule.

7 . The method of claim 1 , wherein the fluorescence dye molecule is selected from the group consisting of fluorescein, fluorescein chlorotriazinyl, fluorescein isothiocyanate (FITC), rhodamine green, rhodamine red, tetramethylrhodamine, Oregon green, Alexa Fluor, JOE, ROX, HEX, Texas Red, TET, TRITC, TAMRA, cyanine-based dyes, and thiadicarbocyanine dyes.

8 . The method of claim 1 , wherein the fluorescence dye molecule is at least one reactive derivative selected from the group consisting of N-hydroxysuccinimide ester (NHS ester), isothiocyanates, carboxylic acids, and sulfonyl chlorides.

9 . The method of claim 1 , wherein the extracellular vesicle molecule is selected from the group consisting of an exosome, a microvesicle, an intraluminal vesicle, a multivesicular body, a multivesicular endosome, and a vesicle isolated from an endosome or a plasma membrane.

10 . A method of quantifying extracellular vesicles, the method comprising:
inputting a brightness value of extracellular vesicle molecules labeled with a fluorescence dye in an unidentified sample into a correlation function for a brightness value of the extracellular vesicle molecule labeled with the fluorescence dye molecule at specific concentrations determined through quantifying the fluorescent dyes using the method of claim 1 , thereby quantifying the extracellular vesicle molecules in the unidentified sample.

11 . A method of evaluating a therapeutic candidate material, the method comprising:
evaluating, by using the method of claim 1 , extracellular vesicles comprising a candidate material or extracellular vesicles being the candidate material, wherein the extracellular vesicles are administered in vitro or in vivo.

12 . A method of detecting a target protein in vivo, the method comprising:
providing extracellular vesicle molecules that are isolated from a cell line expressing a target protein and a protein that binds to the target protein, and are labeled with fluorescence dyes; quantifying a number of labeled fluorescence dyes per extracellular vesicle molecule according to a concentration of the fluorescence dyes measured through fluorescence correlation spectroscopy (FCS) of the extracellular vesicle molecules labeled with fluorescence dyes; and allowing an isolated biological sample comprising the target protein to bind to the extracellular vesicle molecules in which the number of the labeled fluorescence dyes is quantified and adjusted, and then detecting fluorescence, wherein the fluorescence correlation spectroscopy comprises: deriving a fluorescence auto-correlation function by irradiating a sample comprising extracellular vesicle molecules labeled with a fluorescent dye, with an excitation laser, and then measuring a generated fluorescence signal with a detector; analyzing the fluorescence auto-correlation function; and calculating the fluorescent brightness per extracellular vesicle molecule by using the fluorescent brightness of the sample and the number of extracellular vesicle molecules contained in the sample, wherein the extracellular vesicle molecule labeled with a fluorescence dye is chemically labeled with a fluorescence dye.

Assignees

• Asan Found
• Univ Ulsan Found Ind Coop

Inventors

• Kim Sang Yeob
• Pack Chan Gi

Classifications

• G01N2021/6441

  with two or more labels · CPC title

• G01N33/68

  involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)} · CPC title

• G01N33/582

  with fluorescent label · CPC title

• G01N21/6458

  Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16) · CPC title

• G01N21/6428

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title