Protein fluorescent nanoparticles and methods of synthesis thereof

Patent metadata

Field	Value
Publication number	US-10254287-B2
Application number	US-201615216420-A
Country	US
Kind code	B2
Filing date	Jul 21, 2016
Priority date	Jul 21, 2015
Publication date	Apr 9, 2019
Grant date	Apr 9, 2019

Abstract

Disclosed herein are stable and versatile protein nanoparticles having a range of tunable fluorescent properties. Such nanoparticles may find utility in biological imaging. Methods of synthesis of such nanoparticles are also disclosed.

First claim

The invention claimed is:

1. A method of synthesizing a white-emitting protein fluorescent nanoparticle, the method comprising:
labeling a protein by covalent linkage with at least three different fluorescent dye reagents selected from the group consisting of fluoresceins, rhodamines, coumarins, pyrenes, cyanines, squaraines, and boron-dipyrromethenes; and
crosslinking the protein with a crosslinking agent,
to thereby form the white-emitting protein fluorescent nanoparticle, wherein the secondary structure of the protein is retained,
wherein the protein is labeled with the fluorescent dyes either before or after the crosslinking step.

2. The method of claim 1 , wherein the protein is labeled with the fluorescent dye reagent before the crosslinking step.

3. The method of claim 1 , wherein the protein is labeled with the fluorescent dye reagent after the crosslinking step.

4. The method of claim 1 , wherein the protein is selected from the group consisting of bovine serum albumin, glucose oxidase, horseradish peroxidase, catalase, lipase, hemoglobin, and lysozyme, and any combination thereof.

5. The method of claim 1 , wherein the crosslinking agent is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.

6. The method of claim 1 , wherein the three fluorescent dye reagents are selected from the group consisting of: 1-pyrenebutanoic acid N-succinimidyl ester; 7-methoxycoumarin-3-carboxylic acid N-succinimidyl ester; 7-diethylaminocoumarin-3-carboxylic acid N-succinimidyl ester; fluorescein isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; and 5(6)-carboxy-X-rhodamine N-succinimidyl ester.

7. The method of claim 1 , wherein the protein is bovine serum albumin, and the protein is labeled with three or four different fluorescent dye reagents selected from 7-methoxycoumarin-3-carboxylic acid N-succinimidyl ester, 7-diethylaminocoumarin-3-carboxylic acid N-succinimidyl ester, fluorescein isothiocyanate, and 5(6)-carboxy-X-rhodamine N-succinimidyl ester.

8. The method of claim 1 , wherein the nanoparticle has a diameter in a range of about 10 nm to about 100 nm.

9. The method of claim 1 , further comprising a step of annealing the nanoparticle by heating the nanoparticle to a temperature of about 80-90° C. for about 1-15 minutes.

10. The method of claim 1 , wherein the nanoparticle emits white fluorescence upon excitation at about 254 nm or at about 405 nm, and wherein the white fluorescence emission has chromaticity coordinates in which x is about 0.30 to about 0.40 and y is about 0.30 to about 0.40.

11. The method of claim 1 , further comprising a step of forming a metal nanocluster in the protein, either before or after the crosslinking step, wherein the metal is gold.

12. The method of claim 1 , further comprising labeling the nanoparticle with a biological compound.

13. The method of claim 12 , wherein the biological compound is selected from the group consisting of taurine and biotin.

14. The method of claim 1 , wherein the nanoparticle has a particle size of about 10 nm to about 100 nm.

15. A method of synthesizing a white-emitting protein fluorescent nanoparticle, the method comprising:
labeling a protein by covalent linkage with at least three different fluorescent dye reagents; and
crosslinking the protein with a crosslinking agent,
to thereby form the white-emitting protein fluorescent nanoparticle,
wherein the protein is labeled with the fluorescent dyes either before or after the crosslinking step,
wherein the three fluorescent dye reagents are selected from the group consisting of: 1-pyrenebutanoic acid N-succinimidyl ester; 7-methoxycoumarin-3-carboxylic acid N-succinimidyl ester; 7-diethylaminocoumarin-3-carboxylic acid N-succinimidyl ester; fluorescein isothiocyanate; tetramethylrhodamine-5-(and-6)-isothiocyanate; and 5(6)-carboxy-X-rhodamine N-succinimidyl ester.

16. A method of synthesizing a white-emitting protein fluorescent nanoparticle, the method comprising:
labeling a protein by covalent linkage with at least three different fluorescent dye reagents; and
crosslinking the protein with a crosslinking agent,
to thereby form the white-emitting protein fluorescent nanoparticle,
wherein the protein is labeled with the fluorescent dyes either before or after the crosslinking step,
wherein the protein is bovine serum albumin, and the protein is labeled with three or four different fluorescent dye reagents selected from 7-methoxycoumarin-3-carboxylic acid N-succinimidyl ester, 7-diethylaminocoumarin-3-carboxylic acid N-succinimidyl ester, fluorescein isothiocyanate, and 5(6)-carboxy-X-rhodamine N-succinimidyl ester.

Assignees

• Univ Connecticut

Inventors

• Kumar Challa Vijaya
• Stromer Bobbi Shanyelle

Classifications

• G01N2333/765

  Serum albumin, e.g. HSA · CPC title

• G01N33/54346

  Nanoparticles · CPC title

• G01N33/84

  involving inorganic compounds or pH · CPC title

• G01N33/587 Primary

  Nanoparticles · CPC title

• G01N33/533

  with fluorescent label · CPC title