Multicolor microwave-accelerated metal-enhanced fluorescence (M-MAMEF)

• US 8,735,175 B2
• Filed: 03/15/2012
• Issued: 05/27/2014
• Est. Priority Date: 03/18/2011
• Status: Expired due to Fees

• Current Assignee: Chris D. Geddes
• Original Assignee: Chris D. Geddes
• Inventors: Geddes, Chris D.
• Primary Examiner(s): Haq, Shafiqul
• Assistant Examiner(s): Hollinden, Gary E
• Application Number:
  US13/420,702
• Publication Number:
  US 20120238035A1
• Time in Patent Office: 803 Days
• Field of Search: None
• US Class Current: 436/525
• 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 from two to seven different target nucleotides, the method comprising:

applying a multiplicity of metallic particles to a glass substrate surface used in the assay system, wherein the metallic particles are silver or a combination of silver and gold or a combination of silver and aluminum;

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

introducing a solution suspected of including from two to seven different target nucleotides;

introducing from two to seven different detector nucleotides, wherein each of the detector nucleotides has binding affinity for a different target nucleotide and wherein each of the two to seven detector nucleotides includes a different fluorescent molecule, wherein the different fluorescent molecule is positioned from about 6 nm to about 30 nm from the metallic particles, and wherein the metallic particles are positioned a distance from each other to avoid inter-molecular energy transfer between fluorescent molecules upon excitation;

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

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

detecting different fluorescence signals by either visual discrimination of emissions having emission wavelengths of perceptibly different colors or using a variety of filters and diffraction gratings for independent emission detection.

2. The method of claim 1, wherein the substrate is transparent.

3. A method for detecting from two to seven different targeted DNAs from different target pathogens in a sample, the method comprising:

providing a system comprising;

immobilized metallic nanoparticles positioned on a glass surface substrate, wherein the immobilized metallic nanoparticles are silver, a combination of silver and gold or a combination of silver and aluminum, wherein the immobilized metallic nanoparticles have attached thereto from two to seven different capture nucleotides, wherein the two to seven different capture nucleotides have binding affinity for known DNA sequences from two to seven different target pathogens in a sample;

two to seven different free capture DNA sequence probes that are complementary to the known DNA sequences of the two to seven different targeted DNA, wherein the two to seven different free capture DNA sequences are in an amount sufficient to bind to sequences of the two to seven different target pathogens, and wherein each of the two to seven different 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 two to seven different target pathogens 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 from two to seven different immobilized capture DNA sequence probes, wherein the DNA sequences of the two to seven different target pathogens bind 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 distance from about 6 nm to about 30 nm from the immobilized metallic nanoparticles to enhance energy emission, and wherein the immobilized metallic nanoparticles are positioned a distance from each other to avoid inter-molecular energy transfer between excitable energy emitting molecules upon excitation;

applying to the system microwave in an amount sufficient to increase the speed of the binding reactions;

irradiating the system with electromagnetic energy in a range from UV to IR to induce emissions by the excitable energy emitting molecules positioned a predetermined distance from the immobilized metallic nanoparticles, wherein the irradiating can be conducted before, during or after the applying of microwave energy; and

detecting different emissions by the excitable energy emitting molecules by either visual discrimination of emissions having emission wavelengths of perceptibly different colors or using a variety of filters and diffraction gratings for independent emission detection.

4. The method of claim 3, wherein the excitable energy emitting molecules is selected from a group consisting of intrinsic fluorophores, extrinsic fluorophores, fluorescent dyes, phosphorus compounds, and carbon nanodots.

5. A method of metal-enhanced emission sensing using different excitable energy emitting molecules for identifying from two to seven ligands in a testing sample, comprising:

providing a glass substrate surface having immobilized metallic nanoparticles positioned thereon, wherein the immobilized metallic nanoparticles are silver or a combination of silver and gold or a combination of silver and aluminum, wherein the immobilized metallic nanoparticles are position from about 6 to 9 nm from each other to avoid inter-molecular energy transfer between excitable energy emitting molecules upon excitation;

connecting two to seven different receptor biomolecules to the metallic nanoparticles;

introducing two to seven ligands, wherein each ligand has binding affinity for binding with one of the two to seven different receptor biomolecules, wherein the two to seven ligands are different and bind with the corresponding receptor biomolecules having affinity therewith, wherein two to seven different excitable light emitting molecules are provided and one is attached to each of the two to seven binding molecules, wherein the binding molecules are different from each other and having specific binding affinity for a specific ligand and provides an indication of the binding of the ligand to the specific receptor biomolecule when positioned from about 6 nm to about 30 nm from the immobilized metallic nanoparticles;

irradiating the different excitable light emitting molecules with electronmagnetic energy frequencies that excite the different excitable light emitting molecules; and

detecting the different emission signals from the excitable light emitting molecules by either visual discrimination of emissions having emission wavelengths of perceptibly different colors or using a variety of filters and diffraction gratings for independent emission detection.

6. The method of claim 5, wherein the biomolecule receptors are proteins, peptides or nucleotide sequences.

7. The method of claim 5, wherein the excitable light emitting molecules are fluorophores, chromophores, luminophores, or carbon nanodots.

8. The method of claim 5, wherein the glass substrate is transparent and excitation can be delivered from top, side or bottom of the substrate.

9. The method of claim 5, wherein the receptor biomolecules comprise capture DNA immobilized on the metallic particles.

10. The method of claim 5, wherein the metallic nanoparticles are in the form of metallic islands.