SYNTHESIS OF FLUORESCENT NOBLE METAL NANOPARTICLES

• US 20130183665A1
• Filed: 09/23/2011
• Published: 07/18/2013
• Est. Priority Date: 09/24/2010
• Status: Active Grant

• Current Assignee: The Governing Council of the University of Toronto (University of Toronto)
• Original Assignee: The Governing Council of the University of Toronto (University of Toronto)
• Inventors: Chan, Warren, Perrault, Steven, Chou, Leo
• Granted Patent: US 9,395,371 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: A61K 33/242 Gold; Compounds thereof
• CPC Class Codes: B22F 1/054 Nanosized particles
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C22C 5/02 Alloys based on gold
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/587 Nanoparticles

Abstract

A process for the production of fluorescent nanoparticles selected from noble metal, silica or polymer nanoparticles which comprises: 1. A process for the production of fluorescent nanoparticles selected from noble metal or silica nanoparticles which comprises: (1) providing a platform of nanoparticles; (2) covering the surfaces of the nanoparticles to saturation with thiol-terminated polymers by one of the following methods: 1. mixing the nanoparticles with methoxy-(polyethylene glycol)-thiol and biotin-(polyethylene glycol)-thiol; 2. mixing the nanoparticles with fluorescently-labeled methoxy-(polyethylene glycol)-thiol and/or biotin-(polyethylene glycol)-thiol 3. coordinating thiol and biotin thiol to the surfaces of the nanoparticles by a non-covalent bond; and 4. directly conjugating methoxy-thiol and biotin-thiol to the surfaces of the nanoparticles, so that the polymers bind to the surfaces of the nanoparticles as a brush layer via thiol particle coordination of the thiol ends so that the biotin or methoxy ends are free; and (3) homogeneously mixing the resulting biotin nanoparticles with fluorescent avidin or a derivative thereof in proportions such that the final concentration is 1 biotin molecule for every 10 to 1000 avidin molecules in the fluorescent multi-coloured nanoparticle-avidin complexes, each being capable of having a different targeting molecule, and which may be mixed with biotin related targets, and the fluorescent labeled avidin or a derivative thereof being spaced away from the particle surface, thus reducing or removing the potential quenching of the dye.

Claims

1. A process for the production of fluorescent nanoparticles selected from noble metal or silica nanoparticles which comprises:

(1) providing a platform of nanoparticles;

(2) covering the surfaces of the nanoparticles to saturation with thiol-terminated polymers by one of the following methods;

1. mixing the nanoparticles with methoxy-(polyethylene glycol)-thiol and biotin-(polyethylene glycol)-thiol;

2. mixing the nanoparticles with fluorescently-labeled methoxy-(polyethylene glycol)-thiol and/or biotin-(polyethylene glycol)-thiol

3. coordinating thiol and biotin thiol to the surfaces of the nanoparticles by a non-covalent bond; and

4. directly conjugating methoxy-thiol and biotin-thiol to the surfaces of the nanoparticles,

so that the polymers bind to the surfaces of the nanoparticles as a brush layer via thiol particle coordination of the thiol ends so that the biotin or methoxy ends are free; and

(3) homogeneously mixing the resulting biotin nanoparticles with fluorescent avidin or a derivative thereof in proportions such that the final concentration is 1 biotin molecule for every 10 to 1000 avidin molecules in the fluorescent multi-coloured nanoparticle-avidin complexes, each being capable of having a different targeting molecule, and which may be mixed with biotin related targets, and the fluorescent labeled avidin or a derivative thereof being spaced away from the particle surface, thus reducing or removing the potential quenching of the dye.

2. The process as claimed in claim 1 wherein the fluorophores are selected from the group comprising Cy5 and FITC and are coated onto the surfaces of the nanoparticles via a polyethylene-thiol spacer.

3. The process of claim 1 wherein the distance between the fluorophores to the Au surface is equivalent to from about 4 to about 15 nm.

4. The process as claimed in claim 1 wherein the nanoparticles are noble metal nanoparticles.

5. The process as claimed in claim 1 wherein the target molecules are selected from antibodies, peptides and aptamers.

10. A probe comprising the fluorescent multi-coloured noble metal nanoparticle-avidin complexes as claimed in claim 5 and at least one biotin related target or a probe comprising the fluorescent multi-coloured gold nanoparticle-avidin complexes as claimed in claim 6 and at least one biotin related target.

12. An assay kit of fluorescent, functionalized noble metal nanoparticles comprising the following separate components:

Solution A comprising an aqueous solution of nanoparticles;

Solution B comprising a conjugation buffer;

Solution C comprising streptavidin-fluorophore conjugate stock solutions in a conjugation buffer, each with a different fluorophore; and

instructions for assembling the components in accordance with the process as claimed in claim 1 for the production of fluorescent nanoparticles.

13. The assay kit of claim 12 for use in in vitro cell labelling, in vitro diagnostic assay labelling, or in vivo tracking or tumor targeting.

14. The assay kit of claim 12 wherein the nanoparticles are gold nanoparticles.

6. A process for the production of fluorescent gold nanoparticles which comprises:

(1) providing a platform of gold nanoparticles;

(2) covering the surfaces of the gold nanoparticles to saturation with thiol-terminated polymers by one of the following methods;

1. mixing the gold nanoparticles with methoxy-(polyethylene glycol)-thiol and biotin-(polyethylene glycol)-thiol;

2. coordinating thiol and biotin thiol to the surfaces of the gold nanoparticles by a non-covalent bond; and

3. directly conjugating methoxy-thiol and biotin-thiol to the surfaces of the gold nanoparticles,

so that the polymers bind to the surfaces of the gold nanoparticles as a brush layer via thiol particle coordination of the thiol ends so that the biotin or methoxy ends are free; and

(3) homogeneously mixing the resulting biotin gold nanoparticles with fluorescent avidin or a derivative thereof in proportions such that the final concentration is 1 biotin molecule for every 10 to 1000 avidin molecules in the fluorescent multi-coloured gold nanoparticle-avidin complexes, each being capable of having a different targeting molecule, and which may be mixed with biotin related targets, and the fluorescent labeled avidin or a derivative thereof being spaced away from the gold nanoparticle surface, thus reducing or removing the potential quenching of the fluorescent dye.

7. Fluorescent metallic or non-metallic nanoparticle-avidin complexes comprising a noble metal cluster core, a poly-(ethylene glycol) (PEG) outer brush layer, and a biotin linker presented on the outside of a proportion of the outer brush PEG layer that allows attachment of at least one fluorescent dye by biotin-avidin or derivative of avidin linkage.

8. The fluorescent gold nanoparticle-avidin complexes of claim 7, wherein the noble metal cluster core is a gold cluster core.

9. Fluorescent gold nanoparticles created by complexing a gold cluster core with a PEG outer brush layer labeled with a fluorescent dye molecule.

11. (canceled)