MULTICOLOR MICROWAVE-ACCELERATED METAL-ENHANCED FLUORESCENCE (M-MAMEF)

• US 20120238035A1
• Filed: 03/15/2012
• Published: 09/20/2012
• Est. Priority Date: 03/18/2011
• Status: Active Grant

• Current Assignee: GEDDES, CHRIS D.
• Original Assignee: GEDDES, CHRIS D.
• Inventors: GEDDES, CHRIS D.
• Granted Patent: US 8,735,175 B2
• Time in Patent Office: Days
• US Class Current: 436/501
• 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: G01N 2021/6432 Quenching
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 21/6428 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 Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G01N 21/6489 Photoluminescence of semiconductors
• CPC Class Codes: G01N 21/76 Chemiluminescence; Bioluminescence
• CPC Class Codes: G01N 21/763 Bioluminescence
• CPC Class Codes: G01N 33/54346 Nanoparticles
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/553 Metal or metal coated

Abstract

The present invention relates to the use of multiple different light emitting molecules that emit different and detectable emission signals to provide systems and methods to detect different target products in a single assay sample, wherein the different light emitting molecules are positioned an optimal distance from metallic particles thereby enhancing emissions. Preferably, the systems and methods further comprise use of either microwave or sonic energy to increase binding reactions, timing of such reactions within the assay sample and reduce background non-specific biological absorption.

Claims

1. A method of decreasing the detection time of a metal-enhanced fluorescence assay used for detecting a single nucleotide sequence or multiple and different target nucleotides, the method comprising:

applying a multiplicity of metallic particles to a substrate surface used in the assay system;

connecting at least two different capture nucleotides to the metallic particles, wherein each of the capture nucleotides has binding affinity for a different target nucleotide;

introducing a solution suspected of including the different target nucleotides;

introducing at least two different detector nucleotides, wherein each of the detector nucleotides has binding affinity for a different target nucleotide and wherein each of the detector nucleotides includes a different fluorescence molecule, wherein the fluorescence molecule is positioned from about 5 nm to about 30 nm from the metallic particles;

applying microwave or sonic energy to the assay system for a time period sufficient to increase binding reactions between the capture nucleotides and/or detector nucleotides with target nucleotides;

applying electromagnetic energy at a frequency to excite the fluorescence molecules, using either one or multiphoton excitation; and

measuring any fluorescence signals.

2. The method of claim 1, wherein the metallic material used for fabricating the metallic particles is selected from silver, gold, copper, zinc, indium nickel, iron, palladium, aluminum, nickel, rhodium, platinum, oxides and mixtures thereof.

3. The method of claim 1, wherein the metallic particles are metallic islands, colloids, or nanostructures of any geometric shape, such as spherical, triangular, elliptical, oblong, rod shape, hexagonal or multifaceted.

4. The method of claim 1, wherein the surface substrate is selected from the group consisting of glass, quartz, polymeric material, semiconductor chips, indium tin oxide, paper, cellulose, cotton, nylon, silk, sapphire, diamond, ruby, metal oxides, continuous thin metal firms and dielectric materials.

5. The method of claim 1, wherein the substrate is transparent or non-transparent.

6. A method for detecting different targeted DNAs from the same target pathogen or different target pathogens in a sample, the method comprising:

providing a system comprising;

immobilized metallic nanostructures positioned on a surface substrate, wherein the immobilized metallic structures have attached thereto at least one capture nucleotide, wherein the capture nucleotide has binding affinity for known DNA sequences from the same target pathogen or different target pathogens in a sample; and

free capture DNA sequence probes that are complementary to the known DNA sequences, wherein the free capture DNA sequences are different and in an amount sufficient to bind to different sequences of the same target pathogen or to bind to sequences of different target pathogens, and wherein the free capture DNA sequence probes have attached thereto an excitable energy emitting molecule, wherein the free capture DNA sequence probes comprise excitable energy emitting molecules that are specific for the different target pathogens or different sequences of the same target pathogen suspected of being in the sample, wherein the excitable energy emitting molecules emit energy in the UV to IR range;

contacting the sample with the immobilized capture DNA sequence probes, wherein the different DNA sequences of the target pathogen binds to the corresponding immobilized capture DNA sequence probes or the DNA sequences of the different target pathogens binds to the corresponding immobilized capture DNA sequence probes;

contacting the bound DNA sequences with the free capture DNA sequence probes, wherein binding of free capture DNA sequence probes to the DNA sequences causes the excitable energy emitting molecule to be positioned a sufficient distance from the immobilized metallic material to enhance energy emission;

applying to the system microwave or sonic energy in an amount sufficient to increase the speed of the binding reactions; and

irradiating the system with electromagnetic energy in a range from UV to IR to induce emissions by the excitable energy emitting molecule positioned a predetermined distance from the metallic material, wherein the irradiating can be conducted before, during or after the applying of either microwave or ultrasound energy.

7. The method of claim 6, wherein the excitable energy emitting molecule is an intrinsic fluorophore, extrinsic fluorophore, fluorescent dye, chromophore, phosphorus compound, luminophore, carbon compound or allotropes, carbon nanotubes, carbon nanodots, silicon luminescent compounds, semiconductor quantum dot, multiatom gold luminescent cluster, multiatom silver atom luminescent cluster or a bioluminescent molecule that emits a detectable signal in response to a chemical reaction.

8. A kit for detecting different target molecules in a sample, the kit comprising a container comprising a layer of immobilized metal particles deposited on a substrate fabricated of a polymeric or quartz material, wherein at least two different immobilized probes are connected to the metal particles and wherein each of the immobilized probe has affinity for different target molecule;

at least two different excitable molecules that emit light upon excitation, wherein each has affinity for a different target molecule, wherein the binding of the target molecule to the immobilized probe and an excitable molecule having affinity for the target molecules causes the excitable molecule to be positioned a sufficient distance from the immobilized metal particles to enhance emission once the excitable molecule is excited with electromagnetic energy in a range from UV to IR; and

a source of microwave or ultrasonic energy to increase binding reactions within the system and a source of electromagnetic energy to excite the excitable molecules.

9. A method of metal-enhanced emission sensing, comprising:

providing a substrate surface having metallic particles immobilized thereon;

introducing a solution containing a multiplicity of receptor biomolecules for disposing on or near the metallic particles;

introducing ligands, wherein each ligand has binding affinity for binding with one of the receptor biomolecules, wherein the ligands are different and bind with the corresponding receptor biomolecules having affinity therewith, wherein an excitable light emitting molecule is attached to each ligand and provides an indication of the binding of the ligand to the specific receptor biomolecule; and

measuring the different emission signals from the excitable light emitting molecules to determine different receptor biomolecules in the solution.

10. The method of claim 9, wherein the biomolecule receptors are proteins, peptides or nucleotide sequences.

11. The method of claim 9, wherein the metallic particles are fabricated silver, gold, copper, zinc, nickel, iron, palladium, aluminum, indium, nickel, platinum, or mixtures thereof.

12. The method of claim 9, wherein the excitable light emitting molecule is positioned from about 5 nm to about 50 nm from the metallic particles.

13. The method of claim 9, wherein the excitable light emitting molecules are fluorophores, chromophores, luminophores, or carbon nanodots.

14. The method of claim 9, wherein the substrate surface is glass, quartz, plastics semiconductors, paper, cellulose, cotton, nylon, silk, sapphire, diamond, ruby, or a dielectric material.

15. The method of claim 14, wherein the substrate is transparent or non-transparent and excitation can be delivered from top, side or bottom of the substrate.

16. The method of claim 9, wherein the receptor biomolecules is a capture DNA immobilized on the metallic particles.

17. The method of claim 9, wherein the metallic particles are in the form of metallic islands, colloids, or nanostructures.

18. The method of claim 17, wherein the nanostructures have a geometric shape selected from spherical, triangular, elliptical, rod shape, hexagonal or multifaceted.