Functionalized encapsulated fluorescent nanocrystals

• US 7,244,413 B2
• Filed: 12/12/2005
• Issued: 07/17/2007
• Est. Priority Date: 02/18/2000
• Status: Expired due to Term

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Barbera-Guillem, Emilio
• Primary Examiner(s): ZEMEL, IRINA SOPJIA
• Application Number:
  US11/301,050
• Publication Number:
  US 20060099147A1
• Time in Patent Office: 582 Days
• Field of Search: 424/9.1, 424/9.32, 424/9.321, 424/9.34, 424/9.36, 424/9.6, 428/402.24, 428/403, 428/404, 427/213.3, 427/215, 427/220
• US Class Current: 424/9.6
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/161 Vesicles, e.g. liposome
• CPC Class Codes: G01N 33/586 Liposomes, microcapsules or cells
• CPC Class Codes: G01N 33/588 with semiconductor nanocrystal label, e.g. quantum dots
• CPC Class Codes: Y10T 428/2989 Microcapsule with solid core [includes liposome]
• CPC Class Codes: Y10T 428/2991 Coated
• CPC Class Codes: Y10T 428/2993 Silicic or refractory material containing [e.g., tungsten oxide, glass, cement, etc.]

Abstract

Provided are a functionalized, encapsulated fluorescent nanocrystal comprising a liposome having encapsulated therein one or more fluorescent nanocrystals; use of the functionalized, encapsulated fluorescent nanocrystals in detection systems; and a method of producing functionalized, encapsulated fluorescent nanocrystals. A method of using the functionalized encapsulated fluorescent nanocrystals having affinity molecule bound thereto comprises contacting the functionalized encapsulated fluorescent nanocrystals with a sample so that complexes are formed between the functionalized encapsulated fluorescent nanocrystals and substrate for which the affinity molecule has binding specificity, if the substrate is present; exposing the complexes in the detection system to an excitation light source, and detecting a fluorescence peak emitted from the complexes, if present.

Claims

1. An encapsulated fluorescent nanocrystal comprising

a liposome;

one or more fluorescent nanocrystals encapsulated by the liposome, the fluorescent nanocrystal being comprised of a core semiconductor material and a shell semiconductor material;

wherein an outer surface of the liposome comprise surface groups, the surface groups being selected from the group consisting of one or more reactive functionalities, one or more affinity molecules, and a combination thereof.

2. The encapsulated fluorescent nanocrystal of claim 1, wherein the liposome further comprises a component for substitution selected from the group consisting of a membrane stabilizer, an isotonic agent, a pH adjusting agent, an aggregation minimizer, an affinity molecule, an amino acid, and a combination thereof.

3. The encapsulated fluorescent nanocrystal of claim 1, wherein the liposome comprises a membrane stabilizer selected from the group consisting of one or more sterols, one or more fatty acids, one or more amino acids, and a combination thereof.

4. The encapsulated fluorescent nanocrystal of claim 1, wherein the liposome comprises one or more phospholipids and one or more sterols.

5. The encapsulated fluorescent nanocrystal of claim 1, wherein the surface groups comprise a reactive functionality comprising free amino groups.

6. The encapsulated fluorescent nanocrystal of claim 1, wherein the surface groups comprise a reactive functionality comprising thiol-reactive groups.

7. The encapsulated fluorescent nanocrystal of claim 1, wherein the surface groups comprise a reactive functionality comprising free thiol groups.

8. The encapsulated fluorescent nanocrystal of claim 1, wherein the surface groups comprise a reactive functionality comprising free carboxyl groups.

9. The encapsulated fluorescent nanocrystal of claim 1, wherein the surface groups comprise an affinity molecule comprising a monoclonal antibody.

10. The encapsulated fluorescent nanocrystal of claim 1, wherein the surface groups comprise an affinity molecule comprising a nucleobase.

11. The encapsulated fluorescent nanocrystal of claim 1, wherein the surface groups comprise an affinity molecule comprising a nucleic acid molecule.

12. The encapsulated fluorescent nanocrystal of claim 1, wherein the core semiconductor material comprises at least one of a Group II–

VI semiconductor material, Group III–

V semiconductor material, a Group IV semiconductor material, or a combination thereof.

13. The encapsulated fluorescent nanocrystal of claim 1, wherein the shell semiconductor material comprises at least one of a Group II–

VI semiconductor material, Group III–

V semiconductor material, a Group IV semiconductor material, or a combination thereof.

14. The encapsulated fluorescent nanocrystal of claim 1, wherein the core semiconductor material is CdSe, and the shell semiconductor material is ZnS.

15. A method of using encapsulated fluorescent nanocrystals in a detection system, wherein the encapsulated fluorescent nanocrystal comprises a liposome, one or more fluorescent nanocrystals encapsulated by the liposome, the fluorescent nanocrystal being comprised of a core semiconductor material and a shell semiconductor material, wherein an outer surface of the liposome comprise surface groups, the surface groups comprising an affinity molecule, the method comprising:

contacting the encapsulated fluorescent nanocrystals with a sample being analyzed for the presence or absence of a substrate for which the affinity molecule

has binding specificity, wherein if the substrate is present in the sample, complexes are formed comprising the encapsulated fluorescent nanocrystals bound to the substrate;

exposing the complexes, if formed, in the detection system to an excitation light source suitable for exciting the functionalized encapsulated fluorescent nanocrystals to emit a fluorescence peak; and

detecting the fluorescence peak emitted by the complexes, if present, by a detection means for detecting the fluorescence peak;

wherein the detection of a fluorescence peak is indicative of the presence of the substrate.

16. The method of claim 15, wherein the presence of the substrate is detected, and further comprises quantitating the amount of substrate present by measuring the intensity of the fluorescence peak emitted.

17. The method of claim 15, wherein fluorescence is detected, the method further comprising contacting the functionalized, encapsulated fluorescent nanocrystals in the detection system with a lipolytic agent in an effective amount to disrupt liposome portions of the encapsulated fluorescent nanocrystals in releasing fluorescent nanocrystals;

and removing the released fluorescent nanocrystals from the detection system so as to quench the fluorescence.

18. A method of making encapsulated fluorescent nanocrystals, the method comprising:

mixing fluorescent nanocrystals with a lipid mixture to form a dried lipid mixture film;

contacting the dried lipid mixture film with an aqueous solution; and

mixing the dried lipid mixture film with the aqueous solution to form encapsulated fluorescent nanocrystals.

19. The method of claim 18, wherein the fluorescent nanocrystals and lipid mixture are mixed in an organic solvent, and the organic solvent is evaporated to form the dried lipid mixture.

20. The method of claim 18, wherein the dried lipid mixture film is formed further comprising:

evaporating an organic solvent containing fluorescent nanocrystals to form a dried preparation of fluorescent nanocrystals;

evaporating an organic solvent containing the lipid mixture to form a dried preparation of lipid mixture; and

mixing the dried preparation of fluorescent nanocrystals and the dried preparation of lipid mixture.

21. The method of claim 18, wherein the dried lipid mixture film further comprises a membrane stabilizer, an isotonic agent, a pH adjusting agent, an aggregation minimizer, an affinity molecule, an amino acid, or combinations thereof.

22. The method of claim 19, wherein the aqueous solution further comprises a membrane stabilizer, an isotonic agent, a pH adjusting agent, an aggregation minimizer, an affinity molecule, an amino acid, or combinations thereof.