FLUORESCENCE ENHANCING PLASMONIC NANOSCOPIC GOLD FILMS AND ASSAYS BASED THEREON

• US 20130172207A1
• Filed: 12/27/2012
• Published: 07/04/2013
• Est. Priority Date: 12/28/2011
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Dai, Hongjie, Tabakman, Scott M., Hong, Guosong, Zhang, Bo
• Granted Patent: US 9,823,246 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G01N 21/658 enhancement Raman, e.g. surface plasmons
• CPC Class Codes: G01N 2610/00 Assays involving self-assembled monolayers [SAMs]
• CPC Class Codes: G01N 33/553 Metal or metal coated
• CPC Class Codes: Y10T 428/24851 Intermediate layer is discontinuous or differential
• CPC Class Codes: Y10T 428/24909 Free metal or mineral containing
• CPC Class Codes: Y10T 428/24917 including metal layer

Abstract

Disclosed are nanostructured gold films which may be produced by solution-phase depositions of gold ions onto a variety of surfaces. The resulting plasmonic gold films are used for enhanced spectroscopic-based immunoassays in multiplexed microarray format with detection mechanisms based on either surface-enhanced Raman scattering or near-infrared fluorescence enhancement. The preparation of the films and subsequent modifications of the gold film surfaces afford increased sensitivity for various microarrays. The films are discontinuous, forming gold “islands.” Sensitivity, size, shape, and density of the nanoscopic gold islands comprising the discontinuous nanostructured gold film are controlled to enhance the intensity of Raman scattering and fluorescence in the near-infrared, allowing for improved measurements in clinical diagnostic or biomedical research applications.

Claims

1. A microarray comprising:

(a) a substrate;

(b) a discontinuous gold film applied to said substrate, said discontinuous gold film having isolated island areas of between about 100 nm² and 250,000 nm² in area and configured to enhance plasmonic near-infrared fluorescence; and

(c) an array of biological molecules, for use as capture agents specifically binding to an analyte, disposed in discrete locations on the discontinuous gold film and coupled to the discontinuous gold film, whereby near-infrared fluorescence emission caused by an analyte captured by a capture agent is enhanced by the discontinuous gold film.

2. The microarray of claim 1 wherein the isolated island areas are separated by gaps of between 5 and 100 nm.

3. The microarray of claim 1 wherein the isolated island areas are between 10,000 nm² and 25,000 nm² in area.

4. The microarray of claim 1 wherein the isolated island areas are selected from the group consisting of (a) separated gold portions of a solution phase deposition;

(b) separated gold islands etched on the film;

(c) discrete gold particles assembled on the substrate;

(d) gold nanorods assembled on the substrate; and

(e) gold nanoplates assembled on the substrate.

5. The microarray of claim 1 further comprising a self-assembled monolayer between the discontinuous gold film and the biological molecules.

6. The microarray of claim 5 further comprising a branched PEG layer between the discontinuous gold film, the self-assembled monolayer and the biological molecules.

7. The microarray of claim 1 further comprising an avidin or streptavidin layer between the discontinuous gold film, and the biological molecules.

8. The microarray of claim 1 wherein the biological molecules are one of proteins, peptides, antigens, antibodies, nucleic acids, polysaccharides, carbohydrates or biomolecules in whole cells.

9. The microarray of claim 8 wherein the biological molecules are proteins that are cytokines.

10. The microarray of claim 1 wherein the gold film is over the array of biological molecules.

11. The microarray of claim 1 wherein the gold film is under the array of biological molecules.

12. The microarray of claim 1 further comprising, in the discrete locations, a self-assembled monolayer on the discontinuous gold film, a hydrophilic polymer linked to a self-assembled monolayer on the discontinuous gold film, wherein the biological molecules are linked to the hydrophilic polymer.

13. A method for preparing a microarray, comprising:

(a) applying to a substrate a solution containing gold ions;

(b) precipitating the gold ions from solution onto the substrate using a basic solution;

(c) reducing the gold ions precipitated onto the substrate in step (b) to produce on the substrate Au(0) seed particles;

(d) adding gold ions from solution to the gold seeds from step (c) together with a reducing agent to grow isolated island areas in a discontinuous film; and

(e) applying to the discontinuous gold film an array of biological molecules for use as capture agents specifically binding to an analyte, disposed as different molecular species in discrete locations on the discontinuous gold film and coupled to the discontinuous gold film, whereby near-infrared fluorescence emission caused by an analyte captured by a capture agent is enhanced by the discontinuous gold film.

14. The method of claim 13 wherein the Au(0) seed particles are between 0.1 nm² and 100 nm² in area and between about 10 nm and 100 nm apart.

15. The method of claim 13 wherein the isolated island areas are between 100 nm² and 250,000 nm² in size.

16. The method of claim 13 wherein the gaps between isolated island areas are between about 1 nm to 1000 nm.

17. The method of claim 16 wherein the gaps are about 10 nm.

18. The method of claim 13 where the isolated island areas produce plasmonic modes in the 500-2000 nm NIR range.

19. The method of claim 13 further comprising the step of modifying the gold film for attachment of the biological molecule by applying to the gold film a material selected from the group consisting of thiol, mercaptan, poly-L lysine, dextran, amino dextran, carboxy-methyl dextran and polyethylene ethylene glycol.

20. The method of claim 19 wherein the material is branched polyethylene glycol.

21. The method of claim 13 further comprising the step of coating the gold film with a layer of an avidin material.

22. The method of claim 21 comprising the step of applying biotinylated biological molecules to the avidin material.

23. The method of claim 13 further comprising the step of applying a self-assembled thiol containing monolayer directly on the gold film, applying a layer of polyethylene glycol to the self-assembled monolayer via covalent reactions with the terminal groups of the SAM, and applying the analyte capture agents to the terminal groups of polyethylene glycol layer.

24. The method of claim 13 wherein the isolated island areas have at least one of the following properties:

(a) about 100 nm² and 250,000 nm² in area, (b) a gap distance between about 1 and 1000 nm, and (c) heights of 5-500 nm.

25. The method of claim 24 wherein the isolated island areas have heights of between 30 nm and 100 nm.

26. The method of claim 13 wherein reducing is carried out using one of ascorbic acid, hydrazine, hydroxylamine, ammonium borohydrate, formic acid and a hydrogen gas atmosphere.

27. The method of claim 13 wherein the substrate is one of glass, polyvinyl chloride, polydimethyl siloxane, quartz, silicon, or silicon dioxide.

28. A method of detecting one or more analytes in a sample, comprising:

(a) providing a microarray having

(i.) a substrate;

(ii.) a discontinuous gold film applied to said substrate, said gold film having isolated island areas of between about 100 nm² and 250,000 nm² in area, configured to enhance plasmonic near-infrared fluorescence; and

(iii.) an array of biological molecules, for use as capture agents specifically binding to an analyte, disposed as different molecular species in discrete locations on the discontinuous gold film and coupled to the discontinuous gold film, whereby near-infrared fluorescence emission caused by an analyte captured by a capture agent is enhanced by the discontinuous gold film;

(b) applying to said microarray said sample and a near-infrared fluorphore label for said one or more analytes; and

(c) detecting fluorescence emission from fluorophore label bound to one or more analytes.

29. The method of claim 28 wherein the label emits in the 500-1000 nm range.

30. The method of claim 28 wherein the label emits in the including the near-infrared (NIR) range of 650-900 nm.

31. The method of claim 28 wherein the label is one of IRDye 800, Cy7, or Cy5.

32. The method of claim 28 wherein the step of applying a label comprises applying multiple fluorophores with non-overlapping fluorescence in the 500-1000 nm range to detect different analytes.

33. The method of claim 28 wherein the biological molecules are purified proteins.

34. The method of claim 28 wherein the biological molecules are antibodies.

35. The method of claim 34 wherein the antibodies are antibodies to cytokines.