Signal amplification in solution-based plasmonic specific-binding partner assays

Abstract

The present invention relates to analyte detection devices and methods of using such devices to detect minute quantities of a target analyte in a sample. In particular, the invention provides a method of detecting a target analyte in a sample comprising mixing the sample with a first detection conjugate and a second detection conjugate in solution, wherein the first and second detection conjugates comprise metallic nanostructures coupled to binding partners that are capable of specifically binding to the target analyte if present in the sample to form a complex between the first detection conjugate, the analyte, and the second detection conjugate, wherein a change in an optical signal upon complex formation indicates the presence of the target analyte in the sample. Methods of preparing nanostructures and nanoalloys, as well as nanostructures and nanoalloys conjugated to binding partners, are also described.

What is claimed is: 
     
         1 . A method of detecting a target analyte in a sample comprising:
 (a) mixing the sample with a first detection conjugate and a second detection conjugate, wherein the first and second detection conjugates comprise composite metallic nanostructures coupled to binding partners that are capable of specifically binding to the target analyte if present in the sample to form a complex between the first detection conjugate, the analyte, and the second detection conjugate;   (b) exposing the complex to a light source at a wavelength range within the ultraviolet-visible-infrared spectrum; and   (c) measuring an optical signal from the complex, wherein a change in the optical signal indicates the presence of the target analyte in the sample.   
     
     
         2 . The method of  claim 1 , wherein the optical signal is reflectance, an absorbance spectrum, scattering spectrum, or an emission spectrum. 
     
     
         3 . The method of  claim 1 , wherein the change in the optical signal comprises a spectral peak wavelength shift and/or a total spectral wavelength shift. 
     
     
         4 . The method of  claim 3 , wherein the total spectral wavelength shift is a difference spectrum. 
     
     
         5 . The method of  claim 1 , wherein the presence of nanogram quantities of the target analyte is detected. 
     
     
         6 . The method of  claim 1 , wherein the presence of picogram quantities of the target analyte is detected. 
     
     
         7 . The method of  claim 1 , wherein the presence of femtogram quantities of the target analyte is detected. 
     
     
         8 . The method of  claim 1 , wherein step (a) is performed in a spectrophotometric cuvette, an analytical rotor, a microwell plate, a clinical analyzer, a flow chamber, on the tip of an optical fiber, or in a transparent gel. 
     
     
         9 . The method of  claim 1 , wherein the composite metallic nanostructures comprise at least two metals selected from gold, silver, copper, platinum, palladium, cadmium, iron, nickel, and zinc. 
     
     
         10 . The method of  claim 1 , wherein each of the composite metallic nanostructures comprises a core of a first metal and a coating of a second metal. 
     
     
         11 . The method of  claim 9 , wherein each of the composite metallic nanostructures comprises a gold coating and a silver core. 
     
     
         12 . The method of  claim 9 , wherein each of the composite metallic nanostructures comprises a silver coating and a gold core. 
     
     
         13 . The method of  claim 1 , wherein each of the composite metallic nanostructures is an alloy of a first metal and a second metal. 
     
     
         14 . The method of  claim 1 , wherein the composite metallic nanostructures have a geometry selected from spherical nanoparticles, pyramidal nanoparticles, hexagonal nanoparticles, nanotubes, nanostars, nanoshells, nanorods, nanoislands, nanodots, nanowires, or combinations thereof. 
     
     
         15 . The method of  claim 1 , wherein the binding partner is a biological macromolecule. 
     
     
         16 . The method of  claim 15 , wherein the biological macromolecule is selected from an antibody or a fragment thereof, an antigen, a receptor, a ligand, a polynucleotide, an aptamer, a polypeptide, a polysaccharide, a lipopolysaccharide, a glycopeptide, a lipoprotein, or a nucleoprotein. 
     
     
         17 . The method of  claim 16 , wherein the biological macromolecule is an antibody. 
     
     
         18 . The method of  claim 16 , wherein the biological macromolecule is an antigen. 
     
     
         19 . The method of  claim 1 , wherein the first detection conjugate and the second detection conjugate comprise binding partners that are antibodies. 
     
     
         20 . The method of  claim 19 , wherein the antibodies bind different epitopes on the target analyte. 
     
     
         21 . The method of  claim 1 , wherein the target analyte is selected from a protein, enzyme, antigen, antibody, peptide, nucleic acid, hormone, glycoprotein, polysaccharide, toxin, virus, virus particle, drug molecule, hapten, and a chemical. 
     
     
         22 . The method of  claim 1 , wherein the target analyte is a pathogenic antigen or antibody to a pathogenic antigen. 
     
     
         23 . The method of  claim 22 , wherein the pathogenic antigen is a viral antigen. 
     
     
         24 . The method of  claim 23 , wherein the viral antigen is from a virus selected from feline leukemia virus, canine parvovirus, foot and mouth virus, influenza virus, hepatitis a virus, hepatitis b, hepatitis c virus, HIV virus, human papilloma virus, Epstein Barr virus, and rabies virus. 
     
     
         25 . The method of  claim 22 , wherein the pathogenic antigen is a bacterial antigen. 
     
     
         26 . The method of  claim 25 , wherein the bacterial antigen is selected from  Ehrlichia, Borrelia, Anaplasma, Salmonella, Bacillus , and  Rickettsia.    
     
     
         27 . The method of  claim 26 , wherein is the bacterial antigen is selected from  Ehrlichia canis, Ehrlichia chafeensis, Ehrlichia ewingii, Borrelia burgdorferi, Anaplasma platys, Anaplasma phagocytophilum, Salmonella enterica, Bacillus anthracis , and  Rickettsia rickettsii.    
     
     
         28 . The method of  claim 22 , wherein the pathogenic antigen is a fungal antigen or a parasitic antigen. 
     
     
         29 . The method of  claim 28 , wherein the fungal antigen or parasitic antigen is selected from canine heartworm,  Giardia lamblia, plasmodium falciparum, African trypanosomiasis , and  Trypanosoma brucei.    
     
     
         30 . The method of  claim 1 , wherein step (a) of mixing occurs in the presence of a polymeric material selected from polyethylene glycol, polyvinylpyrrolidone, polyallylamine, polyethyleneimine, polylysine, polyacrylic acid, polyvinylalcohol, and polyaspartic acid. 
     
     
         31 . The method of  claim 30 , wherein the polymeric material is polyethylene glycol. 
     
     
         32 . The method of  claim 1 , wherein step (a) of mixing occurs in the presence of a polysaccharide. 
     
     
         33 . The method of  claim 32 , wherein the polysaccharide is selected from maltodextrin, corn syrup, and polyglucose. 
     
     
         34 . The method of  claim 33 , wherein the polysaccharide is maltodextrin. 
     
     
         35 . The method of  claim 34 , wherein the final concentration of maltodextrin in the reaction mixture is about 2% to about 20% wt/vol. 
     
     
         36 . The method of  claim 35 , wherein the final concentration of maltodextrin in the reaction mixture is about 5% to about 10% wt/vol. 
     
     
         37 . The method of  claim 1 , wherein step (a) of mixing occurs in the presence of a blocking agent. 
     
     
         38 . The method of  claim 37 , wherein the blocking agent is selected from bovine serum albumin, casein, gelatin, ovalbumin, and gamma-globulins. 
     
     
         39 . The method of  claim 38 , wherein the blocking agent is bovine serum albumin. 
     
     
         40 . The method of  claim 39 , wherein the final concentration of bovine serum albumin in the reaction mixture is about 1% to about 5% wt/vol. 
     
     
         41 . An analyte detection device comprising:
 a first detection conjugate, wherein the first detection conjugate comprises a metallic nanostructure coupled to a binding partner that is capable of specifically binding to the target analyte if present in the sample; and   a second detection conjugate, wherein the second detection conjugate comprises a metallic nanostructure coupled to a binding partner that is capable of specifically binding to the target analyte if present in the sample,   wherein the metallic nanostructure in the first detection conjugate and/or the second detection conjugate is a composite metallic nanostructure.   
     
     
         42 . The analyte detection device of  claim 41 , wherein the analyte detection device is a spectrophotometric cuvette, an analytical rotor, a microwell plate, or a flow chamber. 
     
     
         43 . The analyte detection device of  claim 42 , wherein the analyte detection device is an analytical rotor. 
     
     
         44 . The analyte detection device of  claim 43 , wherein the analytical rotor contains one or more reaction chambers in which the first detection conjugate and the second detection conjugate is located. 
     
     
         45 . The analyte detection device of  claim 41 , wherein the first detection conjugate and/or the second detection conjugate is lyophilized. 
     
     
         46 . The analyte detection device of  claim 41 , wherein the device is configured to receive a test sample. 
     
     
         47 . The analyte detection device of  claim 41 , wherein the device is configured to expose a complex of the first detection conjugate, the analyte, and the second detection conjugate to a light source at a wavelength range within the ultraviolet-visible-infrared spectrum. 
     
     
         48 . The analyte detection device of  claim 47 , wherein the device is further configured to measure an optical signal from the complex, wherein a change in the optical signal indicates the presence of the target analyte in the sample.