Aggregation induced emission of fluorescent bioprobes and methods of using the same

Patent metadata

Field	Value
Publication number	US-9618453-B2
Application number	US-201313899070-A
Country	US
Kind code	B2
Filing date	May 21, 2013
Priority date	Oct 11, 2011
Publication date	Apr 11, 2017
Grant date	Apr 11, 2017

Abstract

Provided herein are fluorescent bioprobes comprising fluorogens that exhibit aggregation-induced emission (AIE) labeled on biomolecules. The present subject matter relates to a fluorescent bioprobe comprising one or more fluorogen labeled on chitosan. The present subject matter is also directed to methods of preparing the fluorescent bioprobes, methods of labeling and detecting DNA and/or proteins with the fluorescent bioprobe, and methods of cell imaging including live cell tracking.

First claim

The invention claimed is:

1. A method of preparing a fluorescent bioprobe comprising the following steps:
(i) synthesizing fluorogens with one or more amine- or thiol-reactive functional groups; and
(ii) labeling a biomolecule with the fluorogens via a reaction between the fluorogens and one or more amine or thiol groups of the biomolecule;

wherein the one or more amine- or thiol-reactive functional groups are selected from the group consisting of isocyanates, isothiocyanates, aldehydes, succinimidyl esters, 4-sulfotetrafluorophenyl esters, 2,4,5,6-tetrafluorophenyl esters, sulfodicholorphenol esters, carbonyl azides, sulfonyl chlorides, and haloacetamides;
wherein the fluorescent bioprobe emits fluorescence;
wherein the fluorogens exhibit aggregation-induced emission and comprise a backbone structure selected from the group consisting of:

wherein:
each R and R′ are independently selected from the group consisting of: H, C n H 2n+1 , OC n H 2n+1 , C 6 H 5 , OC 6 H 5 , and (X) n N[(CH 2 ) m CH 3 ] 2 ;
X is selected from the group consisting of (CH 2 ) n , C 6 H 5 , and O(CH 2 ) n ; and
n, m independently=an integer from 0 to 20; and

wherein the biomolecule is one or more selected from the group consisting of a nucleotide, nucleoside, oligonucleotide, and DNA, each comprising a reactive thiol or amine group, proteins, and chitosan.

2. The method of claim 1 , wherein the labeling is carried out without a fixation process.

3. The method of claim 1 , wherein the labeling step is carried out without self-quenching.

4. A method of in vitro cell imaging comprising:
(a) contacting cells with a fluorescent bioprobe; and
(b) detecting cellular fluorescence via fluorescent microscopy,

wherein the fluorescent bioprobe comprises a biomolecule covalently conjugated with one or more fluorogens;
wherein the fluorescent bioprobe emits fluorescence;
wherein the one or more fluorogens exhibit aggregation-induced emission and comprise a backbone structure selected from the group consisting of:

wherein:
each R and R′ are independently selected from the group consisting of: H, C n H 2n+1 , OC n H 2n+1 , C 6 H 5 , OC 6 H 5 , and (X) n N[(CH 2 ) m CH 3 ] 2 ;
X is selected from the group consisting of (CH 2 ) n , C 6 H 5 , and O(CH 2 ) n ; and
n, m independently=an integer from 0 to 20; and
wherein the biomolecule is selected from the group consisting of nucleosides, nucleotides, and oligonucleotides.

5. The method of claim 4 , wherein the fluorescent microscopy is used for live cell tracking.

6. The method of claim 4 , wherein the cells uptake the fluorescent bioprobe.

7. The method of claim 6 , wherein the one or more fluorogens interact with the biomolecule inside cells via electrostatic attraction, hydrogen bonding, or hydrophobic interaction.

8. A method of in vitro cellular imaging comprising:
contacting target cells with a fluorescent bioprobe comprising luminogen formed nanoparticles for cellular imaging and long term cellular tracking comprising one or more luminogens that exhibit aggregation-induced emission properties;
wherein the luminogen formed nanoparticles are fully retained inside living cells and have a fluorescence emission;
wherein the one or more luminogens are represented by the structure:

wherein
each R and R′ are independently selected from the group consisting of: H, C n H 2n+1 , OC n H 2n+1 , C 6 H 5 , OC 6 H 5 , and (X) n N[(CH 2 ) m CH 3 ] 2 ;
X is selected from the group consisting of (CH 2 ) n , C 6 H 5 , and O(CH 2 ) n ; and
n, m independently=an integer from 0 to 20;

and detecting cellular fluorescence.

9. A method of in vitro cell imaging comprising:
(a) contacting cells with a fluorescent bioprobe; and
(b) detecting cellular fluorescence via fluorescent microscopy,

wherein the fluorescent bioprobe comprises a biomolecule covalently conjugated with one or more fluorogens;
wherein the fluorescent bioprobe emits fluorescence; and
wherein the one or more fluorogens exhibit aggregation-induced emission and are represented by the structure:

Assignees

• Univ Hong Kong Science & Tech

Inventors

• Tang Benzhong
• Ng Ka Ming
• Luo Qian
• Yu Yong
• Hong Yuning
• Liu Jianzhao
• Chen Sijie
• Lam Wing Yip
• Wang Zhengke
• Qin Wei
• Kwok Tsz Kin

Classifications

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · CPC title

• G01N21/6458

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

• G01N21/6486 Primary

  Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence) · CPC title

• C07F7/0809

  Chemistry & Metallurgy · mapped topic

• 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