Solution-based plasmon-specific binding partner assays and metal nanostructures

Abstract

The present invention relates to nanostructure-bonding partner conjugates, as well as reaction mixtures, analyte detection devices, and methods for fabricating and using conjugates. [Solution] In particular, the present invention provides a method for detecting a target analyte in a sample, comprising the step of mixing the sample with a first detection conjugate and a second detection conjugate in a solution, wherein the first and second detection conjugates include metal nanostructures linked to binding partners that can specifically bind to the target analyte when it is present in the sample, forming a complex between the first detection conjugate and the analyte and the second detection conjugate, and the change in optical signal during complex formation indicates the presence of the target analyte in the sample.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 451,932, filed on 30 January 2017, which is incorporated herein by reference in its entirety.

[0002] Field of Invention The present invention relates to a system and method for detecting a target analyte in a sample. In particular, the present invention provides a localized surface plasmon resonance-based analyte detection system that can detect small amounts of a target analyte in a sample. [Background technology]

[0003] Background of the Invention Current immunoassays and biomolecular binding assays typically require multiple steps and high-performance equipment to perform the assay. The lack of sensitivity and complexity associated with performing such heterogeneous assays arise from the specific need to separate labeled specific binding partners from unlabeled specific binding partners.

[0004] Attempts have been made to develop assays based on the localized surface plasmon resonance (LSPR) properties of precious metal nanoparticles (Tokel et al., Chem Rev., Vol. 114: 5728-5752, 2014 (Non-Patent Literature 1)). LSPR is a collective oscillation of electrons in nanometer-sized structures induced by incident light. Metal nanoparticles exhibit a strong electromagnetic response to refractive index changes in their immediate vicinity, and therefore, the shift in the resonance frequency of the nanoparticles can be measured as an indicator of molecular bonding to the nanoparticle surface. While metal nanoparticles, particularly gold nanoparticles, have been used in diagnostic assays to detect bonding events, such assays generally suffer from low sensitivity and cannot be used to quantitatively monitor the kinetics of continuous bonding events.

[0005] Therefore, improved assay methods are needed that use a homogeneous format while providing increased sensitivity. Assays utilizing standard experimental techniques such as spectroscopy are also considered desirable. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Tokel et al., Chem Rev., Vol. 114: 5728-5752, 2014 [Overview of the project]

[0007] This application describes the use of localized surface plasmon resonance (LSPR) techniques for performing assays involving specific binding partners, including, but not limited to, ligands, receptors, transcription factors, binding DNA elements, antigens, and antibodies. More specifically, this application relates to processes and materials for achieving significant amplification in such assays using nanostructure-binding partner conjugates. In several aspects, this disclosure provides compositions and methods for achieving highly sensitive detection of molecules using LSPR techniques and for minimizing nonspecific binding (NSB) levels in the provided assays.

[0008] In various embodiments described herein, this application relates to a nanostructure-binding partner conjugate, wherein the nanostructure is a metallic nanostructure comprising a plurality of spikes. In some embodiments, the nanostructure is a metallic nanostructure having an average diameter of at least 50 nm. In further embodiments, the nanostructure is a metallic nanostructure having an average diameter of about 50 nm to about 120 nm. In some embodiments, this disclosure provides the use of such metallic nanostructure-binding partner conjugates in solution for qualitatively or quantitatively determining the binding of a specific binding partner. In some embodiments, this disclosure provides a method for producing the conjugates described herein.

[0009] In one aspect, the present disclosure provides a method and composition for detecting a target analyte in a sample, the method comprising the steps of mixing the sample in solution with a first detection conjugate and a second detection conjugate, wherein the first and second detection conjugates include nanostructures linked to binding partners that can specifically bind to the target analyte in the sample if present, thereby forming a complex between the first detection conjugate and the analyte and the second detection conjugate. In some embodiments, the nanostructures are anisotropic nanostructures comprising a plurality of protrusions on a spherical core, the average diameter of the nanostructures from tip to tip being at least about 50 nm. In further embodiments, the average diameter of the nanostructures is about 70 nm or about 90 nm. In some embodiments, the nanostructures are spherical nanostructures. In further embodiments, the method further comprises the step of exposing the complex to a light source in the wavelength range within the ultraviolet-visible-infrared spectrum. In yet another embodiment, the method comprises the step of measuring an optical signal from the complex, wherein a change in the optical signal indicates the presence of a target analyte in the sample.

[0010] In some embodiments, three or more detection conjugates are used. For example, a third, fourth, fifth, or more detection conjugates are added. In some embodiments, each of the detection conjugates can bind to the same target analyte to form a complex. In some embodiments, each of the detection conjugates binds to non-overlapping epitopes on the target analyte. In some embodiments, some or all of the conjugates are anisotropic.

[0011] In some embodiments, the mixing step is carried out in the presence of 3-((3-collamidopropyl)dimethylammino)-1-propanesulfonate (CHAPS). In some embodiments, CHAPS is present at a concentration of about 0.1% w / v to about 0.5% w / v. In further embodiments, CHAPS is present at a concentration of about 0.2% w / v. Thus, in some embodiments, the solution provided herein contains CHAPS.

[0012] In some embodiments, the mixing step is carried out in the presence of a polymer material selected from polyethylene glycol (PEG), polyvinylpyrrolidone, methylcellulose, dextran, polyallylamine, polyethyleneimine, polylysine, polyacrylic acid, polyglutamic acid, polyvinyl alcohol, and polyaspartic acid. Thus, in some embodiments, the solution provided herein comprises one or more of PEG, polyvinylpyrrolidone, methylcellulose, dextran, polyallylamine, polyethyleneimine, polylysine, polyacrylic acid, polyglutamic acid, polyvinyl alcohol, and / or polyaspartic acid. In some embodiments, the polymer material is PEG. In some embodiments, PEG is present at a concentration of about 0.05% to about 5% w / v, or about 0.1% to about 3%. In some embodiments, PEG has a molecular weight of 1,000 to 300,000, or 2,000 to 250,000, or 3,000 to 200,000.

[0013] In some embodiments, the mixing step is carried out in the presence of a viscosity enhancer. Thus, in some embodiments, the solutions provided herein contain a viscosity enhancer. In further embodiments, the viscosity enhancer is selected from trehalose, maltodextrin, sucrose, sorbitol, mannitol, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), cyclodextrin, randomly alkylated cyclodextrin, methylcellulose, trehalose, sucrose, sorbitol, mannitol, and ficol, dextran, or any combination thereof. In certain embodiments, the mixing step is carried out in the presence of dextran at concentrations ranging from about 0.05% to about 5%, depending on the molecular weight. For example, in some embodiments, dextran is present at concentrations of about 0.05%, about 0.1%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%.

[0014] In some embodiments, the mixing step is carried out in the presence of gelatin. Therefore, in some embodiments, the solution provided herein contains gelatin. In some embodiments, the gelatin is present at a concentration between about 0.1% and about 3%.

[0015] In some embodiments, the solutions and reaction mixtures provided herein include at least one binding partner-nanostructure conjugate, CHAPS buffer, PEG, one or more Hofmeister series salts, EDTA, polymer-based blocking agents such as Biolipidure®, BSA, gelatin, or any combination thereof. In some embodiments, the Hofmeister series salt is magnesium chloride. In other embodiments, the Hofmeister series salt is calcium chloride. In some embodiments, the reaction mixture includes multiple salts, such as Hofmeister series salts. In some embodiments, the solutions and reaction mixtures provided herein include MgCl2 or NaSCN at concentrations of about 10 mM to about 250 mM, or about 100 mM. In some embodiments, the solutions and reaction mixtures provided herein include a citrate of a divalent cation, such as Mg2+ citrate or Ca2+ citrate. In some embodiments, the solution and reaction mixture contain a thiocyanate, manganese, cobalt, nickel, ethylenediaminetetraacetic acid (EDTA), and / or ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). In some embodiments, EDTA and / or EGTA are present in the solution at concentrations of about 5 mM to about 100 mM.

[0016] In some embodiments, the nanostructures used in the methods and compositions provided herein include a plurality of protrusions, and the average diameter from tip to tip of the nanostructure is about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, or about 120 nm. In some embodiments, the nanostructure is a metallic nanostructure. In some embodiments, the nanostructure is a gold metallic nanostructure. In some embodiments, the nanostructure provided herein is a spherical nanostructure. In some embodiments, the average diameter of the spherical nanometer is about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, or about 120 nm.

[0017] In some embodiments, the Disclosure provides a method for detecting a target analyte in a sample, comprising the steps of: (a) mixing the sample in solution with a first detection conjugate, a second detection conjugate, CHAPS, bovine serum albumin (BSA), one or more polymer materials, one or more viscosity enhancers, a salt, and optionally a chelating agent, wherein the first and second detection conjugates comprise nanostructures linked to binding partners that can specifically bind to the target analyte in the sample if it is present, thereby forming a complex between the first detection conjugate and the analyte and the second detection conjugate; (b) exposing the complex to a light source in the 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 a target analyte in the sample.

[0018] In some embodiments, the polymer material is selected from the group consisting of PEG, polyvinylpyrrolidone, gelatin, methylcellulose, dextran, polyallylamine, polyethyleneimine, polylysine, polyacrylic acid, polyvinyl alcohol, and polyaspartic acid. In some embodiments, the viscosity enhancer is selected from the group consisting of trehalose, maltodextrin, sucrose, sorbitol, mannitol, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), cyclodextrin, methylcellulose, dextran, and Ficol. In some embodiments, the salt is selected from the group consisting of NaCl, MgCl2, CaCl2, and NaSCN. In some embodiments, the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA) and ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

[0019] In some embodiments, the solutions and reaction mixtures provided herein include Biolipidure® reagents. In some embodiments, the Biolipidure® reagents are Biolipidure® 205, 206, 1002, 1201, 1202, or combinations thereof. In some embodiments, the nanostructures are selected from the group consisting of spherical nanoparticles and nanoparticles containing multiple protrusions. Accordingly, in some embodiments, the disclosure provides methods, solutions, and reaction mixtures comprising a first detection conjugate, a second detection conjugate, CHAPS, BSA, gelatin, PEG, EDTA, MgCl2, Biolipidure® reagents, or any combination thereof.

[0020] In some embodiments, the optical signal is a reflectance, absorbance spectrum, scattering spectrum, or emission spectrum. In some embodiments, the change in the optical signal includes a spectral peak wavelength shift and / or an overall spectral profile shift. In some embodiments, the overall spectral profile shift is a difference spectrum. In some embodiments, the methods provided herein provide for the detection of amounts of target analytes in nanograms, picograms, or femtograms.

[0021] In some embodiments, the methods provided herein are performed in a spectrophotometric cuvette, in an analytical rotor, in a microwell plate, in a clinical analyzer, in a flow chamber, on the tip of an optical fiber, or in a transparent gel.

[0022] In one aspect, the present disclosure provides a reaction mixture comprising at least one binding partner-nanostructure conjugate, wherein the nanostructure comprises a plurality of protrusions and the average diameter of the nanostructure is at least about 50 nm, or at least about 70 nm, or at least about 90 nm, or at least about 120 nm. In another aspect, the present disclosure provides a reaction mixture comprising at least one binding partner-nanostructure conjugate, wherein the nanostructure is a spherical nanostructure. In multiple embodiments, the reaction mixture further comprises an amphoteric surfactant. In some embodiments, the amphoteric surfactant is selected from the group consisting of 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS) and sulfobetaine surfactants. In some embodiments, CHAPS is present at a concentration of about 0.1% to about 1%. In a further embodiment, CHAPS is present at a concentration of about 0.5%.

[0023] In some embodiments, the binding partner is a biopolymer. In further embodiments, the biopolymer is selected from an antibody or fragment thereof, antigen, receptor, ligand, polynucleotide, aptamer, polypeptide, polysaccharide, lipopolysaccharide, glycopeptide, lipoprotein, or nucleoprotein. In some embodiments, the methods and compositions provided herein include a first detection conjugate and a second detection conjugate, wherein one of the binding partners of the detection conjugate is an antibody. In further embodiments, both the first and second detection conjugates include a binding partner that is an antibody. In some embodiments, the antibodies conjugated to the first and second conjugates bind to different epitopes on the same target analyte. In some embodiments, the first and second antibodies, or the first and second conjugates, bind to two different non-overlapping epitopes on the target analyte. In other embodiments, the first and second antibodies, or the first and second conjugates, bind to two different antigens. In some embodiments, the two different antigens are two interacting molecules. In some embodiments, the interacting molecules are two biopolymers including, without limitation, a receptor and its ligand (e.g., a protein hormone and its binding receptor), a DNA-binding transcription factor and another transcription factor and / or DNA, etc.

[0024] In some embodiments, the target analyte is selected from proteins, enzymes, antigens, antibodies, peptides, nucleic acids, hormones, glycoproteins, polysaccharides, toxins, viruses, viral particles, drug molecules, haptens, and chemical substances. In further embodiments, the target analyte is a pathogenic antigen or an antibody against a pathogenic antigen. In further embodiments, the pathogenic antigen is a viral antigen. In further embodiments, the viral antigen is derived from a virus selected from feline leukemia virus, canine parvovirus, foot-and-mouth disease virus, influenza virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, HIV virus, human papillomavirus, Epstein-Barr virus, and rabies virus. In other embodiments, the pathogenic antigen is a bacterial antigen. In a further embodiment, the bacterial antigen is selected from the genera Ehrlichia, Borrelia, Anaplasma, Salmonella, Bacillus, and Rickettsia. In a further embodiment, the bacterial antigen is selected from Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia ewingii, Borrelia burgdorferi, Anaplasma platys, Anaplasma phagocytophilum, Salmonella enterica, Bacillus anthracis, and Rickettsia rickettsii.

[0025] In other embodiments, the pathogenic antigen is a fungal or parasitic antigen. In further embodiments, the fungal or parasitic antigen is selected from canine heartworm, Giardia lamblia, Plasmodium falciparum, African trypanosomiasis, and Trypanosoma brucei.

[0026] In some embodiments, the mixing of the sample provided herein with the first and second detection conjugates is carried out in the presence of a blocking agent. In further embodiments, the blocking agent is selected from bovine serum albumin (BSA), casein, gelatin, ovalbumin, and gamma globulin. In some embodiments, the blocking agent is BSA present at a concentration of about 1% to about 5% w / v.

[0027] In one aspect, the Disclosure provides a method for preparing a conjugate comprising a binding partner and an anisotropic metal nanostructure, suitable for detecting changes in an optical signal based on the presence of a target analyte, wherein the anisotropic metal nanostructure comprises a plurality of protrusions (spikes) and the diameter of the metal nanostructure is at least about 50 nm. In another aspect, the Disclosure provides a method for preparing a conjugate comprising a binding partner and a metal nanostructure, suitable for detecting changes in an optical signal based on the presence of a target analyte, wherein the metal nanostructure is a spherical nanostructure. In several embodiments, the method comprises the steps of: (c) mixing a solution containing a metal nanostructure with a solution containing a binding partner to form a binding partner-nanostructure conjugate; (d) blocking the conjugate with or without the presence of a viscosity enhancer and / or one or more Hofmeister series salts and / or EDTA and / or EGTA, using a blocking agent provided herein (e.g., BSA and / or gelatin and / or PEG and / or Biolipidure® reagent); (c) centrifuging the conjugate; and (d) resuspending the conjugate in a diluent containing a buffer such as phosphate-buffered saline (PBS), Tris-buffered saline (TBS), or borate buffer, a blocking agent provided herein (e.g., BSA and / or gelatin and / or PEG), and CHAPS. In some embodiments, the binding partner is an antibody. In further embodiments, the antibody is an antibody containing a hydrophobic region.

[0028] In some embodiments, the metal nanostructure and / or solution containing the binding partner further comprises a viscosity enhancer. In some embodiments, the viscosity enhancer is selected from trehalose, maltodextrin, dextran, sucrose, sorbitol, mannitol, polyvinylpyrrolidone (PVP), and polyvinyl alcohol (PVA). In some embodiments, the viscosity enhancer is dextran. In some embodiments, the viscosity enhancer is methylcellulose.

[0029] In some embodiments, the centrifugation step of the method for preparing the conjugate provided herein includes centrifugation at about 2000 g or more. In further embodiments, the method includes centrifugation at about 5000 g or more. In further embodiments, the method includes centrifugation at about 10,000 g or more. In further embodiments, the method includes centrifugation at about 50,000 g or more. In further embodiments, the method includes centrifugation at about 75,000 g or more. In further embodiments, the method includes centrifugation at about 100,000 g or more.

[0030] In some embodiments, the disclosure provides a freeze-drying step after resuspending the conjugate. In further embodiments, the freeze-drying step includes distributing the conjugate in liquid nitrogen and freeze-drying it using vacuum and temperature cycling. [Invention 1001] A method for detecting a target analyte in a sample, including the following steps: (a) A step of mixing a sample in solution with a first detection conjugate and a second detection conjugate, wherein the first and second detection conjugates include nanostructures linked to binding partners that can specifically bind to a target analyte if present in the sample to form a complex between the first detection conjugate, the analyte, and the second detection conjugate, the nanostructures including a plurality of protrusions, and the average diameter from tip to tip of the nanostructures being at least about 50 nm; (b) Exposing the composite to a light source in the wavelength range within the ultraviolet-visible-infrared spectrum; and (c) A step of measuring an optical signal from the composite, wherein a change in the optical signal indicates the presence of the target analyte in the sample. [Invention 1002] The method of the present invention 1001, wherein the average diameter of the nanostructure is approximately 70 nm. [Invention 1003] The method of the present invention 1001, wherein the average diameter of the nanostructure is approximately 90 nm. [Invention 1004] The method of the present invention 1001, wherein the mixing step (a) is carried out in the presence of 3-((3-collamidopropyl)dimethylammino)-1-propanesulfonate (CHAPS). [Invention 1005] The method of the present invention 1004, wherein CHAPS is present at a concentration of approximately 0.1% w / v to approximately 0.5% w / v. [Invention 1006] The method of the present invention 1004, wherein CHAPS is present in the solution at a concentration of approximately 0.2% w / v. [Invention 1007] The method of the present invention 1001, wherein the mixing step (a) is carried out in the presence of a polymer material selected from polyethylene glycol (PEG), polyvinylpyrrolidone, gelatin, cellulose, or a combination thereof. [Invention 1008] The method of the present invention 1007, wherein the polymer material is selected from the group consisting of methylcellulose, dextran, polyallylamine, polyethyleneimine, polylysine, polyacrylic acid, polyvinyl alcohol, and polyaspartic acid. [Invention 1009] The method of the present invention 1007, wherein the polymer material is PEG, and the PEG is present at a concentration of about 0.1% to about 5% w / v. [Invention 1010] The method of the present invention 1001, wherein the solution further contains a viscosity enhancer. [Invention 1011] The method of the present invention 1010, wherein the viscosity enhancer is selected from the group consisting of trehalose, maltodextrin, sucrose, sorbitol, mannitol, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), cyclodextrin, methylcellulose, dextran, and ficol. [Invention 1012] The method of the present invention 1001, wherein the solution further comprises a salt selected from MgCl2 and NaSCN. [Invention 1013] The method of the present invention 1012, wherein MgCl2 or NaSCN is present in the solution at a concentration of about 10 mM to about 250 mM. [Invention 1014] The method of the present invention 1012, wherein MgCl2 or NaSCN is present in the solution at a concentration of approximately 100 mM. [Invention 1015] The method of the present invention 1001, wherein the solution further comprises ethylenediaminetetraacetic acid (EDTA) or ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). [Invention 1016] The method of the present invention 1015, wherein EDTA or EGTA is present in the solution at a concentration of approximately 5 mM to approximately 100 mM. [Invention 1017] The method of the present invention 1001, wherein the solution further comprises Biolipidure® reagent. [Invention 1018] The method of the present invention 1017, wherein the Biolipidure® reagent is selected from the group consisting of Biolipidure® reagents 205, 206, 1002, 1201, and 1202. [Invention 1019] The method of the present invention 1001, wherein the optical signal is a reflectance spectrum, an absorbance spectrum, a scattering spectrum, or an emission spectrum. [Invention 1020] The method of the present invention 1001, wherein the change in the optical signal includes a spectral peak wavelength shift and / or a full spectral profile shift. [Invention 1021] The method of the present invention 1020, wherein the entire spectral profile shift is a difference spectrum. [Invention 1022] The method of the present invention 1001, which detects the presence of a target analyte in nanogram quantities. [Invention 1023] The method of the present invention 1001, which detects the presence of a picogram amount of target analyte. [Invention 1024] The method of the present invention 1001, wherein the presence of a target analyte in a femtogram quantity is detected. [Invention 1025] The method of the present invention 1001, wherein step (a) is carried out in a spectrophotometric cuvette, in an analytical rotor, in a microwell plate, in a clinical analyzer, in a flow chamber, on the tip of an optical fiber, or in a transparent gel. [Invention 1026] The method of the present invention 1001, wherein the metal nanostructure is a gold metal nanostructure. [Invention 1027] The method of the present invention 1001, wherein the binding partner is a biopolymer. [Invention 1028] The method of the present invention 1027, wherein the biopolymer is selected from antibodies or fragments thereof, antigens, receptors, ligands, polynucleotides, aptamers, polypeptides, polysaccharides, lipopolysaccharides, glycopeptides, lipoproteins, or nucleoproteins. [Invention 1029] The method of the present invention 1027, wherein the biopolymer is an antibody. [Invention 1030] The method of the present invention 1027, wherein the biopolymer is an antigen. [Invention 1031] The method of the present invention 1001, wherein the first detection conjugate and the second detection conjugate each include a binding partner which is an antibody. [Invention 1032] The method of the present invention 1031, wherein the antibody binds to different epitopes on the target analyte. [Invention 1033] The method of the present invention 1001, wherein the target analyte is selected from proteins, enzymes, antigens, antibodies, peptides, nucleic acids, hormones, glycoproteins, polysaccharides, toxins, viruses, viral particles, drug molecules, haptens, and chemical substances. [Invention 1034] The method of the present invention 1001, wherein the target analyte is a pathogenic antigen or an antibody against a pathogenic antigen. [Invention 1035] The method of the present invention 1034, wherein the pathogenic antigen is a viral antigen. [Invention 1036] The method of the present invention 1035, wherein the viral antigen is derived from a virus selected from feline leukemia virus, canine parvovirus, foot-and-mouth disease virus, influenza virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, HIV virus, human papillomavirus, Epstein-Barr virus, and rabies virus. [Invention 1037] The method of the present invention 1034, wherein the pathogenic antigen is a bacterial antigen. [Invention 1038] The method of the present invention 1037, wherein the bacterial antigen is selected from the genera Ehrlichia, Borrelia, Anaplasma, Salmonella, Bacillus, and Rickettsia. [Invention 1039] The method of the present invention 1037, wherein the bacterial antigen is selected from Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia ewingii, Borrelia burgdorferi, Anaplasma platys, Anaplasma phagocytophilum, Salmonella enterica, Bacillus anthracis, and Rickettsia rickettsii. [Invention 1040] The method of the present invention 1034, wherein the pathogenic antigen is a fungal antigen or a parasitic antigen. [Invention 1041] The method of the present invention 1040, wherein the fungal or parasitic antigen is selected from canine heartworm, Giardia lamblia, Plasmodium falciparum, African trypanosomiasis, and Trypanosoma brucei. [Invention 1042] The method of the present invention 1001, wherein the mixing step (a) is carried out in the presence of a blocking agent. [Invention 1043] The method of the present invention 1042, wherein the blocking agent is selected from bovine serum albumin (BSA), casein, gelatin, ovalbumin, and gamma globulin. [Invention 1044] The method of the present invention 1043, wherein the blocking agent is BSA present at a concentration of approximately 1% to approximately 5% w / v. [Invention 1045] A method for detecting a target analyte in a sample, including the following steps: (a) A step of mixing a sample in solution with a first detection conjugate, a second detection conjugate, CHAPS, bovine serum albumin (BSA), one or more polymer materials, one or more viscosity enhancers, a salt, and optionally a chelating agent, wherein the first and second detection conjugates include nanostructures linked to binding partners that can specifically bind to the target analyte if present in the sample to form a complex between the first detection conjugate and the analyte and the second detection conjugate; (b) Exposing the composite to a light source in the wavelength range within the ultraviolet-visible-infrared spectrum; and (c) A step of measuring an optical signal from the composite, wherein a change in the optical signal indicates the presence of the target analyte in the sample. [Invention 1046] The method of the present invention 1045, wherein the polymer material is selected from the group consisting of PEG, polyvinylpyrrolidone, gelatin, methylcellulose, dextran, polyallylamine, polyethyleneimine, polylysine, polyacrylic acid, polyvinyl alcohol, and polyaspartic acid. [Invention 1047] The method of the present invention 1045, wherein the viscosity enhancer is selected from the group consisting of trehalose, maltodextrin, sucrose, sorbitol, mannitol, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), cyclodextrin, methylcellulose, dextran, and ficol. [Invention 1048] The method of the present invention 1045, wherein the salt is selected from the group consisting of NaCl, MgCl2, CaCl2, and NaSCN. [Invention 1049] The method of the present invention 1045, wherein the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA) and ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). [Invention 1050] The method of the present invention 1045, wherein the solution in step (a) further comprises Biolipidure® reagent. [Invention 1051] The method of the present invention 1045, wherein the nanostructure is selected from the group consisting of spherical nanoparticles and nanoparticles containing multiple protrusions. [Invention 1052] A reaction mixture comprising at least one binding partner-nanostructure conjugate and an amphoteric surfactant, wherein the nanostructure comprises a plurality of protrusions and the average diameter of the nanostructure is at least about 50 nm. [Invention 1053] The reaction mixture of the present invention 1052, wherein the amphoteric surfactant is selected from the group consisting of 3-((3-collamidopropyl)dimethylammino)-1-propanesulfonate (CHAPS) and sulfobetaine surfactants. [Invention 1054] The reaction mixture of the present invention 1053, wherein CHAPS is present at a concentration of approximately 0.1% to approximately 1%. [Invention 1055] A reaction mixture according to Invention 1054, in which CHAPS is present at a concentration of approximately 0.5%. [Invention 1056] The reaction mixture of the present invention 1052, wherein the average diameter of the nanostructure is approximately 70 nm. [Invention 1057] The reaction mixture of the present invention 1052, wherein the average diameter of the nanostructure is approximately 90 nm. [Invention 1058] The reaction mixture of the present invention 1052 further comprises a sample containing a target analyte. [Invention 1059] The reaction mixture of the present invention 1058, wherein the target analyte is selected from proteins, enzymes, antigens, antibodies, peptides, nucleic acids, hormones, glycoproteins, polysaccharides, toxins, viruses, viral particles, drug molecules, haptens, and chemical substances. [Invention 1060] The reaction mixture of the present invention 1052, wherein the binding partner is a biopolymer. [Invention 1061] The reaction mixture of the present invention 1060, wherein the biopolymer is selected from antibodies or fragments thereof, antigens, receptors, ligands, polynucleotides, aptamers, polypeptides, polysaccharides, lipopolysaccharides, glycopeptides, lipoproteins, or nucleoproteins. [Invention 1062] The reaction mixture of the present invention 1060, wherein the biopolymer is an antibody. [Invention 1063] The reaction mixture of the present invention 1052, wherein the metal nanostructure is a gold metal nanostructure. [Invention 1064] The reaction mixture of the present invention 1052, comprising a first detection conjugate and a second detection conjugate, wherein the first and second detection conjugates comprise binding partners that are first and second antibodies. [Invention 1065] The reaction mixture of the present invention 1064, wherein the first and second antibodies bind to different and non-overlapping epitopes on the target analyte. [Invention 1066] A method for preparing a conjugate comprising a binding partner and a metal nanostructure, suitable for detecting changes in optical signal based on the presence of a target analyte, wherein the metal nanostructure comprises a plurality of protrusions, the diameter of the metal nanostructure is at least about 50 nm, and the method comprises the following steps: (a) A step of mixing a solution containing the metal nanostructure with a solution containing the binding partner to form a binding partner-nanostructure conjugate; (b) Blocking the conjugate with BSA, PEG, Biolipidure® reagent, or a combination thereof; (c) the step of centrifuging the conjugate; and (d) Resuspending the conjugate in a diluent containing a buffer selected from the group consisting of PBS, TBS, and borate; a blocking agent selected from the group consisting of BSA, PEG, Biolipidure® reagent, or a combination thereof; and CHAPS. [Invention 1067] The method of the present invention 1066, wherein the binding partner is an antibody. [Invention 1068] The method of the present invention 1067, wherein the antibody is an antibody containing a hydrophobic region. [Invention 1069] The method of the present invention 1066, wherein the solution comprises a viscosity enhancer selected from trehalose, maltodextrin, sucrose, sorbitol, mannitol, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), cyclodextrin, methylcellulose, dextran, and Ficol. [Invention 1070] The method of the present invention 1066, wherein step (c) includes centrifugation at approximately 2,000 g or more. [Invention 1071] The method of the present invention 1066, wherein the average diameter of the nanostructure is approximately 70 nm. [Invention 1072] The method of the present invention 1066, wherein the average diameter of the nanostructure is approximately 90 nm. [Invention 1073] The method of the present invention 1066, further comprising a freeze-drying step after step (d). [Invention 1074] The method of the present invention 1073, wherein the freeze-drying step includes distributing the conjugate in liquid nitrogen and freeze-drying using vacuum and temperature cycling. [Invention 1075] The method of the present invention 1066, wherein the solution further comprises EDTA or EGTA. [Invention 1076] The method of the present invention 1066, wherein the solution further comprises MgCl2 or NaSCN. [Invention 1077] The method of the present invention 1066, wherein the solution further comprises EDTA and MgCl2. [Brief explanation of the drawing]

[0031] [Figure 1] The principle of the LSPR immunoassay described herein is illustrated. Metal nanoparticles containing multiple spikes exhibit an optical spectrum of their own. Subtle changes on the surface of the nanoparticles due to the initial primary bond and subsequent secondary bond cause progressive changes in the properties of light interacting with the nanoparticle-binding partner conjugate. Such changes can be recorded by a suitable spectrometer and can provide qualitative and quantitative information. [Figure 2] This shows the titration of anti-TSH antibody C1 onto a nanostructure containing multiple protrusions to form an antibody-nanostructure conjugate. [Figure 3] This shows the titration of anti-TSH antibody C6 onto a nanostructure containing multiple protrusions to form an antibody-nanostructure conjugate. [Figure 4]This shows large-scale (100 ml nanostructure) titrations of antibodies C1 and C6 onto nanostructures containing multiple protrusions to form C1 antibody-nanostructure conjugates or C6 antibody-nanostructure conjugates. The inset shows strips with stripes of protein A immersed in conjugate solutions with or without BSA blocking. [Figure 5] The peak shifts for non-conjugate nanostructures (573.8 nm) and C1 nanostructures (shifted to 585.3 nm) or C6 nanostructures (shifted to 585.9 nm) are shown. [Figure 6] This shows the peak shifts of non-conjugate nanostructures, C1 antibody-nanostructures, and C6 antibody-nanostructures, as well as the peak shifts of the C1 antibody-nanostructures and C6 antibody-nanostructures after BSA blocking. [Figure 7] The image shows a lateral flow strip with stripes of protein A (0.5 mg / mL) immersed in a solution containing a conjugate nanostructure generated using an absorption protocol, or a conjugate nanostructure generated using an absorption protocol and blocked with BSA. [Figure 8] The spectral shifts of nanostructure conjugates generated using a thiol-mediated conjugation protocol are shown for (i) 50 nm nanostructures before conjugation; (ii) 50 nm nanostructures conjugated with C1 antibody; (iii) 50 nm nanostructures conjugated with C6 antibody; (iv) C1 conjugated nanostructures after BSA blocking; and (v) C6 conjugated nanostructures after BSA blocking. [Figure 9] The image shows a lateral flow strip with Protein A (0.5 mg / mL) stripes immersed in a solution containing a conjugate nanostructure generated using a thiol-mediated protocol, or a conjugate nanostructure generated using a thiol-mediated protocol and blocked with BSA. [Figure 10]Figure 10A shows the change in composite λmax for C1 and C6 conjugates generated using a 50 nm nanostructure and adsorption protocol. Figure 10B shows the change in composite λmax for C1 and C6 conjugates generated using a 50 nm nanostructure and thiol-mediated conjugation protocol. [Figure 11] The effect of the presence of a reaction accelerator on the spectral shift of conjugates prepared by the adsorption protocol (left panel; 0.25% PEG) and the thiol-mediated protocol (right panel; 1% PEG) is shown. [Figure 12] The time-course dose-response curves and kinetics of a covalently bonded conjugate in the presence of 0.1% PEG and 0.5% methylcellulose are shown in the presence of increasing amounts of antigen (TSH). [Figure 13] The reaction curves obtained for various ratios of C1 and C6 antibodies in the conjugate (C1 0 / C6 40 ratio, top left panel; C1 40 / C6 0 ratio, top right panel; C1 30 / C6 10 ratio, middle left panel; C1 20 / C6 20 ratio, middle right panel; C1 10 / C6 30 ratio, bottom panel) are shown, either in the absence of hTSH (0 ng) or in the presence of 0.25 ng. [Figure 14A] This shows the peak shift for the conjugate in the presence of increasing amounts of TSH and 0.5% PEG. [Figure 14B] This shows the peak shift for the conjugate in the presence of increasing amounts of TSH and 1.0% PEG. [Figure 15] Figure 15A (upper panel) shows the conjugation of anti-hTSH antibodies C1 and C6 to 70 nm and 90 nm nanostructures, and their reactivity with protein A lateral flow strips. Figure 15B (lower panel) shows that the protein A line on nitrocellulose reacted as expected before and after blocking with BSA. [Figure 16] This shows the spectral shift of a conjugate containing nanostructures with a diameter of approximately 70 nm. [Figure 17] This shows the spectral shift of a conjugate containing nanostructures with a diameter of approximately 90 nm. [Figure 18] This shows the detection of TSH measured by peak shift between a 50 nm nanostructure containing multiple protrusions and a nanorod. [Figure 19] This shows the net peak shift of a CHAPS-treated conjugate containing a nanostructure with multiple protrusions, conjugated to an antibody at pH 6.0. [Figure 20] Images of Biolipidure® polymer substrates are shown, which have a polar charged head and a tail with varying properties derived from hydrophobic, anionic, cationic, and / or hydrogen bond donor groups. The images are from the website of NOF, the distributor of Biolipidure® reagents. [Figure 21] This shows the wavelength shift over time of anti-TSH coated nanoparticles blocked with a range of Biolipidure® reagents and BSA. Compared to the BSA conjugate, blocking with 1002, 1201, 1202, 205, and 206 all enhance the sensitivity of the conjugate at 10 minutes compared to the standard BSA conjugate. [Figure 22A] Figures 22A and 22B show the nonspecific adsorption of conjugates blocked with Biolipidure® reagents 1002, 1003, 1201, 1202, 205, 206, and BSA (Figure 22A). Due to a large wavelength shift originating from 1003, Figure 22B shows the nonspecific adsorption of only 1002, 1201, 1202, 205, 206, and BSA. [Figure 22B] Figures 22A and 22B show the nonspecific adsorption of conjugates blocked with Biolipidure® reagents 1002, 1003, 1201, 1202, 205, 206, and BSA (Figure 22A). Due to a large wavelength shift originating from 1003, Figure 22B shows the nonspecific adsorption of only 1002, 1201, 1202, 205, 206, and BSA. [Figure 23]This shows the wavelength shift of nanoparticle conjugates in canine serum, compared to BSA blocking reagents, with four Biolipidure® blocking reagents that demonstrate the greatest effect in both improving sensitivity and reducing nonspecific wavelength shift in serum. [Figure 24] The figure shows improved wavelength shift in response to 1 ng / mL, which was diluted 1 / 20 for a final concentration of 50 pg / mL cTSH antigen in TBS BSA buffer. This figure compares the positive response of an 80 nm sphere blocked with BSA versus Biolipidure® 1002, and a 90 nm nanourchin blocked with BSA versus Biolipidure® 1002. In both cases, the Biolipidure® blocking reagent improves the wavelength shift in response to the antigen. The assay conditions for the results provided in Figure 24 included: pH 7.7, 50 mM Tris, 150 mM NaCl, 1% BSA. [Figure 25] A schematic diagram of the Hofmeister series of salts is provided (Zhang Y, Cremer PS, "Interactions between macromolecules and ions: The Hofmeister series" Curr Opin Chem Biol. 2006 Dec;10(6):658-63). [Figure 26] This study demonstrates the effect of the presence of MgCl2 on the level of nonspecific binding in assays using nanosphere cTSH conjugates. Kilo and Mister are two different normal canine serum samples. [Figure 27] This shows the effect of the presence of MgCl2, NaCl, or NaSCN on nonspecific binding. [Figure 28] The results of the LSPR peak shift at 5 minutes in studies testing the presence of MgCl2, NaCl, or NaSCN are shown. [Figure 29] This demonstrates the effect of Mg on nonspecific binding in LSPR assays. [Figure 30]This study demonstrates the effect of EDTA on nonspecific binding in LSPR assays. [Modes for carrying out the invention]

[0032] Detailed description of the invention The present invention is partly based on the discovery that significant amplification in LSPR-based assays can be achieved with anisotropic metal nanostructure-labeling partners. Accordingly, the present invention provides an analyte detection method utilizing multiple detection conjugates comprising anisotropic metal nanostructures linked to biomolecules. The metal nanostructures provided herein are, in some embodiments, multi-branched anisotropic nanoparticles having multiple protrusions on their surface. Nanostructures having multiple protrusions on their surface, as provided herein, also have a diameter of at least about 50 nm. In some embodiments, the metal nanostructures provided herein are spherical or non-spherical metal nanostructures.

[0033] The present invention overcomes the problems of current immunoassays, ligand-receptor binding assays, nucleic acid-protein binding assays, or other specific binding partner assays, which generally require multiple steps and high-performance equipment to carry out such steps. The lack of sensitivity and complexity associated with carrying out such heterogeneous assays arise from the specific need to separate labeled specific binding partners from unlabeled specific binding partners. The present invention overcomes such limitations by carrying out all steps involved in the assay in a homogeneous form, where separation of reacted and unreacted assay components is unnecessary, because the binding event alters the LSPR properties, which are measured in real time by any spectroscopic technique used by those skilled in the art in spectroscopy. The separation-free one-pot assay of the present invention utilizes the relevant effects to provide refractive index sensing, plasmon coupling, and amplification of the final LSPR-modulated signal. Moreover, the methods and metal nanostructures provided enable improved detection. Surprisingly, the methods and metal nanostructures provided enable improved detection and minimized nonspecific binding (referred to herein as "NSB"). This disclosure provides an anisotropic nanostructure-antibody conjugate and a method for producing it, which provides unexpectedly high sensitivity for the detection of analytes. This disclosure provides an anisotropic nanostructure-antibody conjugate and a method for fabricating the same, which provides unexpectedly high sensitivity for analyte detection while offering unexpectedly low levels of NSB. In one aspect, the nanostructure comprises multiple protrusions. Since it has been well known in the art that rod-shaped metal nanoparticles are sensors with better reflectivity compared to circular, spherical, or other nanostructures, the high sensitivity detection achieved with such star-shaped nanostructures is unexpected in some embodiments. Furthermore, given that reports in the literature suggest better sensitivity with nanorods than with the star-shaped structures described herein, the superior effect of the anisotropic nanostructure having a spherical core and multiple spikes provided herein is remarkable.In other embodiments, the nanostructures are star-shaped or spherical, and the sensitivity and low levels of NSB provided herein are achieved by the conjugation methods provided herein.

[0034] As will be apparent to those skilled in the art, the present invention may be applied to the detection of various antigenic analytes, for example, those related to infectious diseases in both humans and animals, such as antigens associated with infectious diseases and antibodies produced in response thereto. The techniques described herein may also be used to perform assays involving specific binding partners, such as ligands and receptors, as well as transcription factors and their associated DNA-binding elements, beyond the detection of antigens and antibodies. Furthermore, RNA-RNA, RNA-DNA, DNA-DNA, or protein-nucleic acid interactions may be detected using appropriate conjugates of anisotropic metal nanoparticles and specific binding partners.

[0035] As provided herein, the present invention describes the use of metal nanoparticles in solution (as opposed to adhesion to a surface via chemical or physical deposition) for qualitatively or quantitatively determining the binding of specific binding partners. Changes in the properties of light interacting with regions containing unbound and bound partners attached to the metal nanoparticles can be measured, thereby enabling the measurement of both qualitative and quantitative interactions between specific binding partners using a suitable detector.

[0036] In a first aspect, the present application provides a method for detecting a target analyte in a sample. In some embodiments, the method includes the step of mixing a sample with a plurality of detection conjugates comprising anisotropic metal nanostructures linked to binding partners. In some embodiments, the nanostructures comprise a plurality of protrusions or spikes. In some embodiments, the nanostructures comprising protrusions have a diameter of at least 50 nm. In other embodiments, the nanostructures are spherical. In some embodiments, the nanostructures are gold nanostructures. In one embodiment, the method comprises a first detection conjugate and a second detection conjugate, the first and second detection conjugates comprising metal nanostructures linked to binding partners that can specifically bind to the target analyte if present in the sample, thereby forming a complex between the first detection conjugate and the analyte and the second detection conjugate. For example, the conjugates comprise a first binding partner and a second binding partner, each binding to a different epitope on the same target analyte. In a further embodiment, the method includes the steps of: exposing the complex to a light source in the wavelength range within the ultraviolet-visible-infrared spectrum; and measuring an optical signal from the complex, wherein a change in the optical signal indicates the presence of a target analyte in the sample. In an exemplary embodiment, the metal nanostructure in the first detection conjugate and / or the second detection conjugate is a metal nanostructure of gold. In another exemplary embodiment, the mixing step is carried out in the presence of a polymer material selected from polyethylene glycol (PEG), polyvinylpyrrolidone, polyallylamine, polyethyleneimine, polylysine, polyacrylic acid, polyvinyl alcohol, polyglutamic acid, and polyaspartic acid. In a preferred embodiment, the polymer material is PEG. In yet another exemplary embodiment, the mixing step is carried out in the presence of a polysaccharide or other viscosity enhancer. In some embodiments, the viscosity enhancer is selected from trehalose, maltodextrin, sucrose, sorbitol, mannitol, polyvinylpyrrolidone (PVP), or polyvinyl alcohol (PVA).In some embodiments, the polysaccharide is selected from maltodextrin, trehalose, sucrose, corn syrup, and polyglucose. In preferred embodiments, the polysaccharide is maltodextrin or trehalose. In yet another exemplary embodiment, the mixing step is carried out in the presence of a blocking agent. In some embodiments, the blocking agent is selected from bovine serum albumin (BSA), casein, gelatin, ovalbumin, and gamma globulin. In preferred embodiments, the blocking agent is BSA.

[0037] In some embodiments, the Disclosure provides methods and compositions comprising blocking agents that have been previously used in assays such as lateral flow assays but have not been previously used or are not intended for use in LSPR assays. For example, in some embodiments, the Disclosure provides methods and compositions for LSPR assays described herein, in which one or more Biolipidure® reagents are used as blocking agents. Surprisingly, despite the fact that the effect on wavelength shift in LSPR assays cannot be predicted based on the use of such agents in non-LSPR assays (e.g., lateral flow assays), the inventors have found that Biolipidure® reagents provide excellent effects in LSPR assays provided herein. Biolipidure® reagents are polymer agents exhibiting one or more of the following characteristics: enhanced sensitivity and accuracy of detection; suppression of nonspecific adsorption; stabilization of antibodies and enzymes; and elimination of lot-to-lot variability. Biolipidure® reagents do not require biohazard handling and are used in several embodiments by preparing a buffer solution containing Biolipidure® (e.g., about 0.1 wt%, about 0.25 wt%, about 0.5 wt%, about 0.75 wt%, about 1 wt%, about 1.25 wt%, about 1.5 wt%, about 2 wt%, about 5 wt%, or more) and by dissolving the sample to be tested in the buffer. In certain embodiments, Biolipidure® reagents are used at a concentration of 1 wt%.

[0038] In the various embodiments described herein, the methods of the present invention can be configured in the form of a sandwich assay, a direct assay, an indirect assay, and competitive and secondary labeling.

[0039] In some embodiments, the detection method is a sandwich assay. In such embodiments, the detection conjugate comprises an anisotropic metal nanostructure, as provided herein, linked to a binding partner that can specifically bind to the target analyte if present in the sample. For example, in one embodiment, the method in sandwich assay form comprises a first detection conjugate and a second detection conjugate, the first and second detection conjugates comprising a spherical metal nanostructure and / or a metal nanostructure having a plurality of protrusions, the nanostructure being linked to a binding partner that can specifically bind to the target analyte if present in the sample to form a complex between the first detection conjugate and the analyte and the second detection conjugate. In an exemplary embodiment, the metal nanostructure in the first detection conjugate and / or the second detection conjugate is an anisotropic gold metal nanostructure. The complex is exposed to a light source and the optical signal is measured, the change in the optical signal indicating the presence of the analyte in the sample. As an example, when a sample containing a target analyte is mixed with first and second detection conjugates, the target analyte binds to its binding partner in the detection conjugate, forming a complex between the first detection conjugate, the analyte, and the second detection conjugate. This complex formation results in very close proximity of metal nanostructures in the detection conjugates, i.e., plasmon-plasmon coupling. The amount of light absorbed, scattered, or transmitted by the metal nanostructures is affected by the proximity of the metal nanostructures in the complex, thus producing an enhanced shift in the peak absorption wavelength, which indicates the presence of the target analyte in the sample.

[0040] In another embodiment, the detection method is a competitive assay. In such an embodiment, the first detection conjugate contains a metal nanostructure conjugated to the target analyte of interest. As in a sandwich assay, the second detection conjugate can specifically bind to the target analyte. In this type of assay, the first detection conjugate will initially bind to the second detection conjugate. When a sample containing the target analyte is mixed with these first conjugates, any unlabeled or free target analyte in the sample will compete with the first detection conjugate for binding to the second detection conjugate. The change in the optical signal in this type of assay is due to the replacement of the metal nanostructure in the first detection conjugate by the second detection conjugate, which will proportionally reduce the wavelength shift at the peak absorption wavelength.

[0041] As described above, the method of the present invention may utilize multiple detection conjugates. Depending on the assay configuration, the detection conjugate includes a spherical metal nanostructure or a metal nanostructure having multiple protrusions and linked to a binding partner that can specifically bind to a target analyte or another detection conjugate. For example, in an embodiment in which the method is configured in a sandwich assay format, the detection conjugate includes a metal nanostructure linked to or conjugated to a binding partner that can specifically bind to a target analyte. In other embodiments in which the method is configured in a direct competitive assay format, at least one of the detection conjugates includes a metal nanostructure linked to or conjugated to a target analyte.

[0042] In some embodiments, the detection conjugate includes a binding partner that can specifically bind to the target analyte. As used herein, “specific binding” means high affinity, for example, at least 10 -6This refers to binding to a target molecule at affinity M. In some embodiments, the binding partner is a biomacromolecule, including haptens and other small molecules, drugs, hormones, non-limiting antibodies or fragments thereof (e.g., Fv, Fab, F(ab)2, single-stranded, CDR, etc.), antigens, receptors, ligands, polynucleotides, aptamers, polypeptides, polysaccharides, lipopolysaccharides, glycopeptides, lipoproteins, or nucleoproteins. In certain embodiments, the binding partner is an antibody. In other embodiments, the binding partner is an antigen.

[0043] In some embodiments, the detection conjugates, for example, a first detection conjugate and a second detection conjugate, include a binding partner that is a molecule of the same type but preferably binds to the target analyte at a distinct position from the other. For example, both the first and second detection conjugates may be antibodies that recognize the target analyte, but the epitope to which the first detection conjugate binds to the target analyte is distinct from and, ideally, does not overlap with the epitope to which the second detection conjugate binds to the target analyte. Thus, in certain embodiments, the first detection conjugate includes an antibody that recognizes a first epitope of the target analyte, and the second detection conjugate includes a different antibody that recognizes a second epitope of the target analyte. In various embodiments described herein, the first detection conjugate may include a monoclonal antibody that recognizes a first epitope of the target analyte. In a further embodiment, the second detection conjugate may include a monoclonal antibody that recognizes a second epitope of the target analyte, which is distinct from and, ideally, does not overlap with the epitope recognized by the first detection conjugate. Alternatively, the first detection conjugate and / or the second detection conjugate may include a polyclonal antibody. For example, the first detection conjugate may include a polyclonal antibody, while the second detection conjugate includes a monoclonal antibody. In some embodiments, the first detection conjugate includes a polyclonal antibody, and the second detection conjugate includes a polyclonal antibody.

[0044] The metal nanostructures in the detection conjugate may consist of noble metals or composites thereof. In some embodiments, the metal nanostructures in the detection conjugate may consist of transition metals or composites thereof. In some embodiments, the metal nanostructures in the detection conjugate may contain alkali metals or lanthanides in combination with noble metals or transition metals. In certain embodiments, the metal nanostructures in the detection conjugate include metals selected from gold, silver, copper, platinum, palladium, ruthenium, rhodium, osmium, iridium, titanium, chromium, cadmium, zinc, iron, cobalt, nickel, and composites thereof. In one embodiment, the metal nanostructure is a gold nanostructure. In another embodiment, the metal nanostructure is a silver nanostructure. In yet another embodiment, the metal nanostructure in the detection conjugate is a composite metal nanostructure. "Composite metal nanostructure" means a nanostructure containing at least two noble metals, transition metals, alkali metals, or lanthanides. Two or more metals may be mixed together, as in an alloy, or two or more metals may be present in separate parts of the nanostructure. For example, one metal may form the core of the nanostructure, while a second metal forms the shell or coating of the nanostructure. In some embodiments, the composite metal nanostructure comprises at least two metals selected from gold, silver, copper, platinum, palladium, ruthenium, rhodium, osmium, iridium, titanium, chromium, cadmium, zinc, iron, cobalt, and nickel. In other embodiments, the composite metal nanostructure comprises at least two metals selected from gold, silver, copper, platinum, palladium, cadmium, iron, nickel, and zinc. In one particular embodiment, the composite metal nanostructure comprises gold and silver. In another embodiment, the composite metal nanostructure comprises gold and copper. In yet another embodiment, the composite metal nanostructure comprises silver and copper. In some embodiments, the composite metal nanostructure used in the method of the present invention comprises a core of a first metal and a coating of a second metal. For example, the composite metal nanostructure may comprise a silver core and a gold coating. In other embodiments, the composite metal nanostructure comprises a copper core and a gold coating.In another embodiment, the core is silver and the coating is copper. In some embodiments, each of the composite metal nanostructures comprises a dielectric core (e.g., silicon dioxide, gold sulfide, titanium dioxide, silica, and polystyrene), a first coating of the first metal, and a second coating of the second metal. In some embodiments, the core containing the first metal is dissolved after a coating process with the second metal to generate a hollow structure composed of the second metal. As an example, coating a silver core with gold nanoparticles generates a gold shell around the silver core, which is then dissolved or decomposed, resulting in the formation of a hollow nano-gold shell structure.

[0045] The nanostructures disclosed herein include, in some embodiments, multiple protrusions, such as spikes or conical protrusions. Thus, the nanostructures provided herein are highly branched nanoparticles. In some embodiments, the surface of the inner core of the nanostructure is essentially covered by protrusions. The diameter of the nanostructures enumerated herein includes the protrusions; that is, the enumerated diameter is from tip to tip of the protrusions covering the nanostructure.

[0046] The average diameter of the nanostructures provided herein, having multiple protrusions or spikes on a single surface, is about 50 nm to about 120 nm. The average diameter of the nanostructure includes the protrusions thereon. Therefore, the average diameter is described herein as the diameter from tip to tip in some embodiments. In some embodiments, the average diameter is about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, or greater. In some embodiments, the average diameter is about 70 nm. In other embodiments, the average diameter is about 90 nm. In some embodiments, the nanostructure comprises a mixture of average diameters from about 50 nm to about 90 nm. The average diameter of the spherical nanostructures provided on a single surface is about 50 nm to about 120 nm. In some embodiments, the average diameter of the spherical nanostructures is approximately 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, or greater.

[0047] In another aspect, this disclosure provides a reaction mixture comprising a binding partner-nanostructure conjugate disclosed herein. In further embodiments, the reaction mixture comprises one or more capping reagents and / or one or more zwitterionic surfactants. In some embodiments, the capping agent is a zwitterionic surfactant. For example, in some embodiments, the reaction mixture comprises CHAPS. Surprisingly, the inventors have found that the presence of CHAPS, the capping agent, and the zwitterionic surfactant makes it possible to effectively conjugate the nanostructure provided herein, which comprises multiple surface protrusions, to a binding partner such as an antibody. In addition, the presence of CHAPS enables faster centrifugation and shorter centrifugation times. For example, the inventors have found that in the presence of CHAPS, centrifugation rates that would normally be considered to cause the collapse of anisotropic nanoparticle antibody conjugates (e.g., greater than 15,000 g, i.e., about 40,000 g) can be used to centrifuge the nanostructure provided herein. Thus, the presence of CHAPS enables more efficient generation of the conjugate. Furthermore, the presence of CHAPS enables easy resuspension of antibody conjugates after centrifugation. In particular, conjugates containing hydrophobic antibodies that would otherwise be impossible to resuspend after centrifugation are readily resuspended in the presence of CHAPS. Moreover, the presence of the CHAPS surfactant helps to prevent nonspecific size / shape changes that would lead to aggregation. Particles, such as the nanostructures provided herein having multiple spikes, may come out of the solution. Surprisingly, the inventors have found that even if the particles have come out of the solution completely, they can be rescued by adding CHAPS.In some embodiments, the reaction mixture comprises a binding partner-nanostructure conjugate, and the mixture comprises one or more capping agents or amphoteric surfactants selected from the sulfobetaine series, Triton series (x-100) surfactants; Tween series (Tween 20) surfactants; cationic surfactant series such as CTAB; and anionic surfactants such as SDS.

[0048] Methods for conjugating molecules to metal nanostructures disclosed herein are also provided. Such methods include conjugation chemistry, e.g., those involving 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), sulfo-NHS coupling, hydrophobic bonding, or thioether chemistry. In some embodiments, the binding partner or target analyte can be linked to the metal nanostructure through various chemical functionalities, including thiols, amines, dithiols, acrylic phosphoramidites, azides, or alkynes. In some embodiments, the molecule can be linked to the metal nanostructure indirectly through a larger carrier molecule or protein. Such indirect linkage is particularly useful when the molecule is small, e.g., hormones, drugs, and other small molecules less than 10 kD. Preferably, the carrier protein cannot specifically interact with the target analyte. In some embodiments, protein A or protein G or protein A / G may be conjugated or linked to the nanoparticles.

[0049] In some embodiments, a method for conjugating a molecule to a metal nanostructure provided herein comprises the steps of: (a) mixing a solution containing the metal nanostructure with a solution containing a binding partner to form a binding partner-nanostructure conjugate; (c) blocking the conjugate with BSA; (d) centrifuging the conjugate; and (b) resuspending the conjugate in a diluent containing a buffer such as PBS, a blocking agent such as BSA, and CHAPS. In some embodiments, the binding partner is an antibody. In further embodiments, the antibody is an antibody containing a hydrophobic region. In some embodiments, the metal nanostructure is adjusted to a basic pH before titration of the antibody to the nanostructure. For example, in some embodiments, the nanostructure pH is adjusted to a pH of about 8, about 8.5, about 8.8, or about 9.2. However, in some embodiments, the inventors have surprisingly found that the pH of a solution containing nanostructures can be adjusted to a neutral or acidic pH (e.g., about 5.5, about 6, about 6.5, or about 7), and that it is possible to successfully form conjugates capable of highly sensitive antigen detection. In some embodiments, the neutral or acidic pH resulted in conjugates that were difficult to resuspend in standard conjugate diluents containing BSA and phosphate buffer. Surprisingly, such insoluble conjugates dissolved rapidly in a buffer containing CHAPS.

[0050] In some embodiments, one or more metals used in the first detection conjugate may be the same as one or more metals used to fabricate the metal nanostructures in the second detection conjugate. For example, in one embodiment, the first detection conjugate contains gold nanostructures, and the second detection conjugate contains gold nanostructures. In other embodiments, the metal used in the first detection conjugate is different from one or more metals used to fabricate the metal nanostructures in the second detection conjugate.

[0051] In some embodiments, the reaction environment can be prepared with appropriate buffers, ionic strength, and other reaction accelerators. In a preferred embodiment, the reaction environment includes polyethylene glycol (PEG), which can enhance the intensity of the LSPR signal and the rate at which the signal is generated, as described herein. Other similar polymer materials, non-limited to polyvinylpyrrolidone, polyallylamine, polyethyleneimine, polylysine, polyacrylic acid, polyvinyl alcohol, and polyaspartic acid, may also be used.

[0052] The present invention also provides an analyte detection apparatus for utilizing the methods described herein to detect a target analyte in a sample. Suitable analyte detection apparatus may, but not limited to, a spectrophotometric cuvette, an analytical rotor, a microwell plate, or a flow chamber. As will be understood by those skilled in the art, a fiber optic tip or a transparent gel may also be used to carry out the detection methods disclosed herein.

[0053] In certain embodiments, all components of the analyte detection apparatus described herein are contained within a centrifuge rotor or disk. For example, the rotor or disk may contain one or more reaction chambers in which a plurality of detection conjugates are arranged. In some embodiments, the detection conjugates exist in the form of lyophilized compositions, e.g., lyophilized beads or pellets. In some embodiments, the analyte detection apparatus comprises a rotor or disk having one or more chambers, each reaction chamber comprising a plurality of detection conjugates (e.g., a first detection conjugate and a second detection conjugate), the detection conjugates being first and second specific binding partners linked to metal nanoparticles. Such an apparatus provides a one-step analyte detection assay, thereby bringing a test sample into contact with the rotor or disk, and the application of centrifugal force to the rotor or disk delivers the test sample to the reaction chamber, where the sample mixes with the first and second detection conjugates. In embodiments in which the rotor or disk contains two or more reaction chambers, the detection conjugates can be selected so that a variety of analytes can be detected in each reaction chamber. These rotor-type detection devices can be configured in a sandwich assay format, a direct competition format, or both formats if the rotor includes multiple reaction chambers.

[0054] In some embodiments, direct competitive assays or sandwich assays may be performed in a centrifuge rotor, for example, the rotors described in U.S. Patents 5,061,381, 5,122,284, 5,186,844, 5,304,348, 5,457,053, and 5,693,233. In some embodiments, the disclosure provides a multiplex assay that enables separate detection via multiple cuvettes, for example, by using a disk or rotor that enables multiplex analysis.

[0055] In some embodiments, nanoparticle conjugates of two paired monoclonal antibodies or nanoparticle conjugates of a polyclonal antibody mixture conjugating to two or more epitopes are added as lyophilized beads. Solution-phase LSPR assays work with both monoclonal and polyclonal antibodies. In some embodiments, the disclosure provides antibody pairs that enable highly sensitive detection in LSPR assays. For example, in some embodiments, the antibody pair is anti-TSH antibody clones C1 and C6, each conjugating to a different epitope of TSH. In other embodiments, the antibody pair is anti-TSH antibody close to C1 and 5409. In some embodiments, the best signal-to-noise ratio is obtained with gold conjugates prepared from anti-TSH close to 5405 and 5409.

[0056] The present invention also includes a kit comprising an analyte detection apparatus of the present invention as disclosed herein. In one embodiment, the kit comprises a plurality of detection conjugates (e.g., a first detection conjugate and a second detection conjugate), the detection conjugate being a specific binding partner linked to a metal nanostructure provided herein. In some embodiments, one or more of the detection conjugates may be lyophilized, for example, in the form of pellets or beads. In one embodiment, all of the detection conjugates are lyophilized. In further embodiments, the kit may comprise one or more additional reagents. In some embodiments, one or more of the additional reagents are provided in a lyophilized form. In some embodiments, the kit may comprise a blocking agent, a sugar, a polymer reaction promoter, sodium chloride, and / or a combination thereof. A “blocking agent” is an agent that prevents the association of proteins present in the sample with the detectable agent and / or analyte. The blocking agent is typically a protein itself and may not be limited to bovine serum albumin (BSA), casein, gelatin, ovalbumin, gamma globulin, and IgG from non-immunized animals. In some embodiments, the sugar is a polysaccharide. In one embodiment, the polysaccharide is selected from maltodextrin, corn syrup, and polyglucose. In a preferred embodiment, the polysaccharide is maltodextrin. In another embodiment, the sugar is trehalose. In some embodiments, the reagent kit may contain maltodextrin and trehalose. In some embodiments, the polymer reaction promoter is PEG.

[0057] The kit of the present invention may also include instructions for using an apparatus for detecting analytes in a test sample, an apparatus or tool for collecting biological samples, and / or an extraction buffer for obtaining samples from solid materials such as soil, food, and biological tissue.

[0058] As described herein, the test sample may be any type of liquid sample, including a biological sample or an extract prepared from an environmental or food sample. In one particular embodiment, the test sample is a biological sample. Biological samples include, but are not limited to, whole blood, plasma, serum, saliva, urine, pleural fluid, sweat, bile, cerebrospinal fluid, fecal matter, vaginal fluid, sperm, eyepiece fluid, mucus, synovial fluid, ascites, amniotic fluid, biopsy tissue, saliva, and cell lysates. Biological samples may be obtained from human or animal subjects suspected of having a disease condition such as cancer, infectious diseases (e.g., viral, bacterial, parasitic, or fungal infections), cardiovascular disease, metabolic disease, or autoimmune disease. Biological samples may also be obtained from healthy subjects (e.g., humans or animals) undergoing routine health checkups.

[0059] In some embodiments of the method, the test sample is mixed with a first detection conjugate, and the mixture is then brought into contact with a second detection conjugate. In certain embodiments, the sample, the first detection conjugate, and the second detection conjugate are brought into contact simultaneously. For example, contact between the sample and both reagents may be performed simultaneously in a rotor-type detection apparatus as described herein.

[0060] As described above, this application relates in some embodiments to the use of metal nanostructures conjugated to a binding partner, the nanostructure having multiple protrusions, such as spikes or conical protrusions, and having an average diameter of about 50 nm or more. Nanorods were expected to provide superior results because they are known to be better sensors of refractive index changes, but the inventors have surprisingly found that the sensitivity of solution-based assays is significantly enhanced with protrusion-filled nanostructures compared to the use of nanorods (having a smooth rod-shaped surface). In fact, the inventors have surprisingly found that in solution-based assays disclosed herein, metal nanostructure conjugates containing nanostructures with multiple protrusions exhibited robust antigen detection, whereas nanorod conjugates did not. The inventors have further found that larger nanostructures containing protrusions exhibit better detection sensitivity compared to smaller nanostructures having the same protrusion characteristics. For example, in some embodiments, if the average diameter of the nanostructures used in the assay is increased from about 50 nm to about 70 nm, the detection sensitivity increases. In further embodiments, if the average diameter of the nanostructures used in the assay is increased from about 70 nm to about 90 nm, the detection sensitivity increases even further.

[0061] In one embodiment, the solution contains polysaccharides at a final concentration of about 2% to about 20% wt / vol. In another embodiment, the solution contains polysaccharides at a final concentration of about 4% to about 15% wt / vol. In yet another embodiment, the solution contains polysaccharides at a final concentration of about 5% to about 10% wt / vol. In an exemplary embodiment, the solution contains polysaccharides at a final concentration of about 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or higher, encompassing all values ​​in between. In a particular embodiment, trehalose may be used in the analytical rotor to prevent sedimentation of the detection conjugate. In certain embodiments, the trehalose concentration includes all values ​​between them, approximately 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or higher. In various embodiments described herein, the sensitivity of the assay may be improved when a polysaccharide, such as sucrose, trehalose, maltodextrin, sorbitol, mannitol, or Ficol, is added to the solution, compared to assays performed in solutions containing alternative sugars. In an exemplary embodiment, the polysaccharide is maltodextrin. In another exemplary embodiment, the polysaccharide is trehalose. In yet another exemplary embodiment, the polysaccharide is dextran.

[0062] In one embodiment, the solution contains a blocking agent at a final concentration of about 0.1% to about 20% wt / vol. In another embodiment, the solution contains a blocking agent at a final concentration of about 0.5% to about 10% wt / vol. In yet another embodiment, the solution contains a blocking agent at a final concentration of about 1% to about 5% wt / vol. In an exemplary embodiment, the solution contains a blocking agent at a final concentration of about 1%, 2%, 3%, 4%, or 5%, encompassing all values ​​in between. In various embodiments described herein, the sensitivity of the assay may be improved when a blocking agent is added to the solution compared to an assay performed in the absence of the blocking agent. In some embodiments, the blocking agent is selected from bovine serum albumin, casein, gelatin, ovalbumin, and γ-globulin. In an exemplary embodiment, the blocking agent is bovine serum albumin (BSA).

[0063] In some embodiments, the solution comprises one or more of maltodextrin, trehalose, PEG, a blocking agent (e.g., BSA), and / or sodium chloride. In exemplary embodiments, one or more of the solution components, e.g., maltodextrin, may be provided as lyophilized beads or pellets that are suspended upon addition of a liquid, e.g., water, saline solution, or liquid biological sample. For example, one or more of the solution components may be provided in a spectrophotometric cuvette or analytical rotor reaction chamber as beads suspended in the solution after the addition of the liquid.

[0064] In an additional embodiment, the LSPR signal may be substantially amplified by mixing the first and second detection conjugates with the analyte in the presence of a polymer reaction promoter selected from polyethylene glycol, polyvinylpyrrolidone, polyallylamine, polyethyleneimine, polylysine, polyacrylic acid, polyvinyl alcohol, and polyaspartic acid. In an exemplary embodiment, the polymer material is polyethylene glycol (PEG). In one embodiment, the reaction mixture contains the polymer material, e.g., PEG, at a final concentration of about 0.1 mg / mL to about 200 mg / mL. In another embodiment, the reaction mixture contains the polymer material, e.g., PEG, at a final concentration of about 0.2 mg / mL to about 100 mg / mL. In yet another embodiment, the reaction mixture contains the polymer material, e.g., PEG, at a final concentration of about 0.5 mg / mL to about 10 mg / mL. In yet another embodiment, the reaction mixture contains the polymer material, e.g., PEG, at a final concentration of about 2 mg / mL to about 8 mg / mL. In exemplary embodiments, the reaction mixture contains a polymer material, such as PEG, at a final concentration of about 2, 3, 4, 5, 6, 7, or 8 mg / mL, encompassing all values ​​in between. In some embodiments, PEGs of various molecular weights may be used; for example, a smaller amount of higher molecular weight PEG can be used for a substantial effect. In some embodiments, the PEG concentration required for assay enhancement varies with the molecular weight of the polymer.

[0065] The detection method of the present invention may be used to determine the qualitative or quantitative amount of a target analyte. Such a method is particularly useful for determining an appropriate amount of a target analyte in a sample and can be used in particular to diagnose a particular medical condition or to evaluate the efficacy of a drug therapy. In one embodiment, the amount of a target analyte can be determined by establishing a standard curve for a particular analyte by measuring the change in optical signal from metal nanoparticles as described herein for a sample having a known amount of the target analyte; determining the change in optical signal for a test sample; and comparing the change in optical signal for the test sample to a value obtained from the standard curve. In some embodiments, determining the amount of a complex between a first reagent and a second reagent involves comparing the absorbance ratio and / or reaction rate from a test sample to the absorbance ratio and / or reaction rate from one sample having a known amount of the complex, thereby determining the amount of the complex in the test sample. The quantitative value obtained from the test sample may be compared to a predetermined threshold, which indicates either an abnormal or normal level of the target analyte.

[0066] The detection method of the present invention provides a highly sensitive technique for detecting trace amounts of target analytes in a sample. In some embodiments, amplification of a surface plasmon resonance-based signal can be achieved with a gold nanostructure conjugate so that nanogram amounts of the target analyte can be detected in the sample. Thus, in one embodiment of the method, the presence of nanogram amounts of the target analyte is detected. In some embodiments, a plasmon resonance-based signal derived from a detection conjugate containing gold nanoparticles can be amplified using a composite metal nanostructure detection conjugate. The use of gold-coated silver nanostructures conjugated to an analyte-specific antibody may enable the detection of picogram amounts of the target analyte. Thus, in some embodiments of the method, the presence of picogram amounts of the target analyte is detected. In other embodiments of the method, the presence of femtogram amounts of the target analyte is detected. Higher sensitivity may be obtained by changing the composition and / or shape of the composite metal nanostructure.

[0067] When incident light is applied to a metallic nanostructure, conduction band electrons in the metal collectively vibrate at the same frequency as the incident electromagnetic wave. As a result of these resonant vibrations, the nanostructure strongly absorbs and scatters light in a specific wavelength range. For metallic nanostructures containing noble metals or transition metals, this wavelength range is within the ultraviolet-visible-infrared spectrum, depending on the specific composition of the nanostructure. Therefore, a light source for applying electromagnetic energy suitable for use in the method of the present invention can include any source capable of applying wavelengths within the ultraviolet-visible spectrum or the ultraviolet-visible-infrared spectrum, including arc lamps and lasers. In some embodiments, the light source may be equipped with a monochromator so that light of a specific wavelength can be applied.

[0068] The optical properties of metal nanostructures depend on their size, shape, and composition. For example, solid gold nanoparticles have absorption peak wavelengths (λ) ranging from approximately 515 nm to approximately 560 nm, depending on the particle size. max ) has. Gold spherical nanoparticles with a diameter of 30 nm absorb maximum at approximately 520 nm, λ max The wavelength shifts to longer wavelengths as the particle diameter increases. Silver and copper particles are λ in the ultraviolet / blue or red region (e.g., about 350 nm to about 500 nm). max The increase in particle diameter results in a longer wavelength of λ. max This causes a shift in the lateral λ. max1 and vertical λ max2 Alloys of different metals typically exhibit absorption peaks in the intermediate range between the absorption peaks of the constituent metals. For example, nanostructures containing a 50 / 50 alloy of gold and silver exhibit absorption peaks at approximately 480 nm λ. maxThe LSPR signal exhibits this property, and increasing the amount of gold causes a shift in the absorption peak to longer wavelengths. The sensitivity of the LSPR signal to changes in the local medium refractive index can be modified by changing the shape or geometric arrangement of the nanostructure. For example, non-spherical particles (e.g., nanoprisms, nanorods, nanoshells, etc.) have increased LSPR sensitivity to changes in refractive index compared to spheres. In some embodiments, optical properties (e.g., absorption / scattering at specific wavelengths) are tuned to specific applications by varying the size, shape, or composition of the metallic nanostructure used in the detection conjugate.

[0069] The interaction between incident light and metal nanostructures can be monitored as reflected or transmitted light. The amount of incident light absorbed or scattered can be measured as an absorption spectrum in the reflection mode or an absorption spectrum in the transmission mode. In some embodiments, the optical signals measured from metal nanostructures can be light reflection, absorbance spectra, scattering spectra, and / or emission spectra.

[0070] Plasmon coupling between metal nanostructures in a detection conjugate, resulting from the formation of a complex between the binding partner and the target analyte, leads to changes in the localized surface plasmon resonance spectrum of the metal nanostructure. For example, such changes may include increased optical attenuation, increased optical reflection, and / or increased scattering and / or emission signals. In some embodiments, changes in the optical signal indicating the presence of the target analyte in a sample include shifts, increases, or decreases in light scattering, or a combination of these features. In certain embodiments, the change in the optical signal indicating the presence of the target analyte in a sample is a spectral peak wavelength shift. In certain other embodiments, the change in the optical signal indicating the presence of the target analyte in a sample is a wavelength shift at a position other than the peak. For example, changes in the optical signal indicating the presence of the target analyte in a sample may be a midpoint spectral wavelength shift, a spectral wavelength shift at the base of the wavelength, or a whole spectral wavelength shift such as a difference spectrum. In one embodiment, a wavelength shift at an optical spectral peak may be a redshift (e.g., a shift to longer wavelengths) within a spectral window of 200 nm to 1200 nm. In another embodiment, the wavelength shift in the optical spectral peak may be a blue shift (e.g., a shift to shorter wavelengths) within a spectral window of 200 nm to 1200 nm. The change in the optical signal can be measured at a specific point in time after a predetermined reaction period. Additionally or alternatively, the change in the optical signal over the reaction period (e.g., rate determination) may be measured. Both types of measurements can be used for either qualitative or quantitative analysis of the target analyte.

[0071] Various means for measuring optical signals at different wavelengths and obtaining attenuation, scattering, or emission spectra are known in the art. Any spectrophotometric or photometric instrument is suitable for use in the disclosed methods. Some non-limiting examples include plate readers, Cobas Fara analyzers, and Piccolo xpress® and Vetscan analyzers (Abaxis, Inc., Union City, CA), fiber optic readers (e.g., LightPath® S4 (LamdaGen, Menlo Park, CA)), SPR instruments (e.g., Biacore instruments available from GE Healthcare), and centrifuge analyzers from Olympus, Hitachi, and others.

[0072] The present invention also includes an assay complex comprising (i) a first detection conjugate having a plurality of protrusions provided herein, linked to a binding partner, (ii) a target analyte, and (iii) a second detection conjugate having a plurality of protrusions provided herein, linked to a binding partner, wherein the binding partner in the first detection conjugate binds to a first epitope on the target analyte, and the binding partner in the second detection conjugate binds to a second epitope on the target analyte, thereby forming a complex comprising the first detection conjugate, the target analyte, and the second detection conjugate. In some embodiments, the assay complex is contained in a cuvette adapted for use in a centrifuge rotor. In other embodiments, the assay complex is contained in a reaction chamber in a centrifuge rotor or disk.

[0073] Any type of target analyte can be detected using the methods, apparatus, and assay complexes of the present invention, particularly those significant in the diagnosis of disease. Target analytes can, indefinitely, include proteins, enzymes, antigens, antibodies, peptides, nucleic acids (RNA, DNA, mRNA, miRNA), hormones, glycoproteins, polysaccharides, toxins, viruses, viral particles, drug molecules, haptens, or chemical substances. In some embodiments, the target analyte is a marker or antigen associated with infectious diseases in humans and / or animals. In other embodiments, the target analyte is a marker or antigen associated with a specific physiological or pathological condition.

[0074] In certain embodiments, the target analyte is a pathogenic antigen or an antibody against a pathogenic antigen. For example, pathogenic antigens can be viral antigens (e.g., feline leukemia virus, canine parvovirus, foot-and-mouth disease virus, influenza virus, hepatitis A, B, and C viruses, HIV virus, human papillomavirus, Epstein-Barr virus, rabies virus, etc.), bacterial antigens (e.g., Ehrlichia, Borrelia, Anaplasma, Salmonella, Bacillus, Rickettsia, etc.), fungal antigens, or parasitic antigens (e.g., canine heartworm, Giardia lamblia, Plasmodium falciparum, African trypanosomiasis, Trypanosoma brusey, etc.). In specific embodiments, the bacterial antigen may be derived from Ehrlichia canis, Ehrlichia shafensis, Ehrlichia ewingi, Borrelia burgdorferi, Anaplasma platis, Anaplasma phagocytophyllum, Salmonella enterica, Bacillus anthracis, and Rickettsia rickettii. In other embodiments, the target analyte is a disease-related antigen or an antibody against a disease-related antigen. Disease-related antigens include, but are not limited to, cancer-related antigens or markers (e.g., PSA, AFP, CA125, CA15-3, CA19-9, CEA, NY-ESO-1, MUC1, GM3, GD2, ERBB2, etc.), cardiovascular disease-related antigens or markers (e.g., troponin, C-reactive protein, brain sodium excretion-increasing peptide, CKMB, fatty acid-binding protein, etc.), metabolism-related antigens or markers (e.g., thyroid-stimulating hormone, thyroxine, leptin, insulin), or autoimmune disease-related antigens or markers (e.g., autoantibodies). In certain embodiments, the target analyte is an inflammatory antigen or marker (e.g., C-reactive protein, MRP14, MRP8, 25F9, etc.). In other embodiments, the target analyte is a pregnancy-related antigen or marker (e.g., fetal antigen, human chorionic gonadotropin).

[0075] In some embodiments, the Disclosure provides methods for synthesizing the nanostructures provided herein. In certain embodiments, silver / gold nanoparticles are synthesized in a single vessel by adding predetermined amounts of the following reagents in succession and with thorough mixing: (1) a surfactant (e.g., ionic [anionic, cationic, or amphoteric], or nonionic) or a capping agent, e.g., 3-((3-collamidopropyl)dimethylammino)-1-propanesulfonate (CHAPS), SDS, Tween, Triton, or any sulfobetaine surfactant; (2) gold chloride; (3) water; (4) silver nitrate; (5) trisodium citrate; and finally (6) ascorbic acid to initiate the formation of nanoparticles. In other embodiments, nanoparticles are synthesized in a single vessel by adding predetermined amounts of the following in the following order: (1) a surfactant or capping agent, e.g., CHAPS, SDS, Tween, Triton, CTAB, or any sulfobetaine surfactant; (2) gold chloride; (3) silver nitrate; (4) trisodium citrate; (5) water; and (6) a reducing agent. In some embodiments, the reducing agent consists of CHAPS, ascorbic acid, trisodium citrate, and water. In further embodiments, the reducing agent consists of about 200 mg of CHAPS, about 4 g of ascorbic acid, about 117.6 mg of trisodium citrate, and about 15.68 g of water. In some embodiments, approximately 1 mL of aqueous 1% (wt / wt) CHAPS is continuously mixed with approximately 0.25 mL of 0.1 M gold chloride, approximately 0.5 mL of 0.02 M silver nitrate, approximately 0.05 mL of 1 M trisodium citrate, approximately 6.2 mL of water, and approximately 2 mL of a reducing agent. By varying the concentrations of various active ingredients such as metal salts, capping agents, and reducing agents, and the pH of the solution, nanoparticles of various particle types (e.g., nanospheres, nanostars, or nanorods) and various compositions are obtained as a result.

[0076] In some embodiments, nanostars are formed by mixing, in order, water, cetyltrimethylammonium bromide (CTAB), gold chloride, ascorbic acid, and pre-formed gold nanosphere seeds. In a further embodiment, about 0.825 mL of water, about 0.1 mL of 20% CTAB, about 0.025 mL of 0.1 M gold chloride, about 0.05 mL of 1 M ascorbic acid, and about 0.05 mL of gold nanosphere seeds are mixed in that order. The age of the seeds and the ratio of seeds to metal ions affect the geometric arrangement and, therefore, the optical spectrum of the nanoparticles. Nanostars of gold only are made by reducing gold chloride with a reducing agent consisting of about 200 mg of CHAPS, about 4 g of ascorbic acid, about 117.6 mg of trisodium citrate, and about 15.68 g of water. The size of the nanostars formed is determined by the concentration of gold chloride. Gold nanostars prepared by this method can be purified by centrifugation and stored in water at 2-8°C.

[0077] The formation of nanomaterials using the methods provided herein is essentially completed within minutes, but may be allowed to reach equilibrium overnight. The synthesis of nanoparticles can be monitored by spectroscopy and confirmed by scanning or transmission electron microscopy.

[0078] In some embodiments, the size and therefore optical properties can be altered by changing the concentrations of surfactants or capping agents, ascorbic acid, trisodium citrate, gold chloride, and / or silver nitrate. The size of the synthesized nanostars increases with increasing silver content up to a certain point, and then decreases. These changes are reflected in the LSPR peak of the synthesized nanostars as a red shift in the peak with increasing silver / gold ratio, and then begin to blue shift at a gold:silver molar ratio of 5:2. The final concentration of the selected surfactant in the reaction mixture can vary from 0.05 to 5%, with smaller particles prevailing at higher concentrations of surfactant. Increasing the concentration of ascorbic acid results in smaller nanostars, and the final concentration of ascorbic acid varies from 0.05 to 0.2 M. Similarly, increasing the concentration of trisodium citrate from 10 mM to 100 mM reduces the nanostar size.

[0079] In some embodiments, gold-silver nanoalloys can be synthesized under alkaline reducing conditions by mixing CTAB (e.g., CTAB dissolved in alcohol) with gold chloride and silver nitrate. In some embodiments, nanoalloy formation can be induced, in order, by mixing water, CTAB, gold chloride (0.5 mM to 5 mM), silver nitrate (20% to 80% of gold), ascorbic acid (10 mM to 200 mM), or a reducing agent containing ascorbic acid, trisodium citrate, and CHAPS, as well as NaOH (50% to 200% of ascorbic acid). In further embodiments, the nanoalloy is formed by mixing about 0.825 ml of water, about 0.1 ml of 20% CTAB prepared in isopropanol, about 0.025 ml of 0.1 M gold chloride, about 0.005 to 0.025 ml of 0.1 M silver nitrate, about 0.05 ml of 1 M ascorbic acid, and about 0.05 ml of 1 M NaOH. The concentration of CTAB can vary from 0.05 M to 0.2 M, with lower concentrations favoring the synthesis of nanostars with higher content. Acidic pH is favorable for nanorod formation, and higher aspect ratios are obtained by decreasing pH.

[0080] The present invention is further illustrated by the following additional embodiments, which should not be construed as limiting. Those skilled in the art will recognize that, in light of this disclosure, many modifications can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention, and similar or comparable results can still be obtained.

[0081] All patent and non-patent documents referenced throughout this disclosure are incorporated herein by reference in their entirety for all purposes. [Examples]

[0082] Example 1. Titration of antibody clones for conjugation to nanostructures We conducted research to identify protocols for conjugating nanostructures with multiple spikes to exemplary antibodies. First, titration experiments were performed by titrating anti-TSH clone C1 to the nanostructures to determine the amount of antibody required for optimal conjugation.

[0083] The reaction was set up in a 1 ml cuvette placed on a Lambda950 spectrophotometer. 1 ml of nanostructure was placed in the cuvette after pH adjustment to 9.2 with 10 microliters of 0.5 M borate (pH 9.2). The spectrum was recorded, which showed λmax at 573.8 nm. Binding of antibody C1 was determined by observing the peak shift upon successive addition of 2 μg of antibody. Equivalence was reached at approximately 6–8 micrograms of antibody per OD unit. Finally, 10 microliters of 20% BSA was added to block nonspecific binding events. This resulted in an additional 2 nm shift in λmax. The results are shown in Figure 2.

[0084] Similarly, titration experiments were performed to determine the amount of antibody required for conjugation of the C6 antibody (specific for a different epitope of TSH). As for the C1 antibody, the reaction was set up in a 1 ml cuvette placed in a Lambda950 spectrophotometer. 1 ml of the nanostructure with multiple spikes was placed in the cuvette after adjusting the pH to 9.2 with 10 microliters of 0.5 M borate (pH 9.2). The spectrum was recorded, which showed Lmax(λ max ) at 573.7 nm. Binding of antibody C6 was determined by observing peak shifts upon successive addition of 2 μg of the antibody. The equivalence point was reached with approximately 6 micrograms of antibody per OD unit. Finally, 10 microliters of 20% BSA was added. This resulted in an additional 1.4 nm shift at λmax. The results are provided in Figure 3.

[0085] Next, to determine the scalability of conjugation, large-scale (100 ml) conjugations of the C1 and C6 antibodies to the nanostructure were performed. The results are provided in Figure 4. In this preparation, approximately 0.5 mg of C1 and C6 were separately added with rapid stirring to two containers each containing 100 ml of the nanostructure solution pre-adjusted to pH 9.2. The resulting conjugate solutions were tested for antibody binding before and after blocking with BSA. Strips with stripes of 0.5 mg / ml protein A were immersed in the conjugate solution diluted with phosphate-buffered BSA and Tween solution. The resulting lines (1 and 6) showed that C1 or C6 had bound to the nanostructure and the antibody remained bound after BSA blocking (1B and 6B).

[0086] The C1 and C6 conjugates were recovered by centrifugation, washed once with conjugate diluent, and recentrifuged. The precipitate containing the conjugates readily dissolved in a conjugate storage solution containing PBS / BSA / CHAPS. Diluents of the final conjugates were prepared in water and compared. The original non-conjugate nanostructures had a peak at 573.8 nm, which was redshifted to 585–586 nm in the blocked conjugates. The shift is shown in Figure 5. The final conjugate solution can be stored at 2–8C until use.

[0087] Example 2. Conjugation of nanostructures to antibodies using an adsorption conjugation protocol. To generate nanostructure conjugates, nanostructures with multiple protrusions and an average diameter of 50 nm (λmax - 1.0 OD / mL at 575 nm) were adjusted to pH 8.8 with 0.1 M borate. C1 or C6 antibody (approximately 33 picomoles per OD nanostructure) was titrated and mixed thoroughly for 15 minutes. 2 mg of BSA per ml was added and mixed for a further 15 minutes. The nanostructure / C1 or nanostructure / C6 mixture was centrifuged at 15,000 g for 10 minutes; the supernatant was removed; and the conjugate was resuspended in conjugate diluent CG-1P containing PBS / BSA and CHAPS. CHAPS was particularly important for the resuspending of the hydrophobic C6 antibody. Furthermore, the CHAPS surfactant helped to prevent nonspecific size / shape changes that would lead to aggregation.

[0088] Figure 6 shows the spectral shifts for conjugates generated using the adsorption protocol: (i) 50 nm nanostructure before conjugation; (ii) 50 nm nanostructure conjugated with C1 antibody; (iii) 50 nm nanostructure conjugated with C6 antibody; (iv) C1 conjugated nanostructure after blocking with BSA; and (v) C6 conjugated nanostructure after blocking with BSA. The study shows that antibody binding caused a 4–5 nm redshift, and an additional 1 nm shift was induced when the conjugate was blocked with BSA.

[0089] To confirm that antibody molecules bound to the nanostructure, lateral flow strips with protein A (0.5 mg / mL) stripes were immersed in a solution containing the conjugated nanostructure or the conjugated nanostructure blocked with BSA. Figure 7 shows that conjugates were formed and that BSA blocking did not destroy the conjugates.

[0090] Example 3. Conjugation of nanostructures to antibodies using a thiol-mediated conjugation protocol. To generate nanostructure conjugates using a thiol-mediated conjugation protocol, nanostructures with multiple protrusions and an average diameter of 50 nm (λmax - 1.0 OD / mL at 575 nm) were adjusted to pH 8.8 with 0.1 M borate. TCEP-reduced C1 or C6 antibody (approximately 33 picomoles per OD nanostructure) was titrated and thoroughly mixed for 15 minutes. 2 mg of BSA per ml was added and mixed for a further 15 minutes. The nanostructure / C1 or nanostructure / C6 mixture was centrifuged at 15,000 g for 10 minutes; the supernatant was removed; and the conjugate was resuspended in conjugate diluent CG-1P containing PBS / BSA and CHAPS. CHAPS was particularly important for the resuspending of the hydrophobic C6 antibody. Furthermore, the CHAPS surfactant helped prevent nonspecific size / shape changes that would lead to aggregation.

[0091] Figure 8 shows the spectral shifts for conjugates generated using the thiol-mediated conjugation protocol for (i) 50 nm nanostructures before conjugation; (ii) 50 nm nanostructures conjugated with C1 antibody; (iii) 50 nm nanostructures conjugated with C6 antibody; (iv) C1 conjugated nanostructures after blocking with BSA; and (v) C6 conjugated nanostructures after blocking with BSA. The study shows that antibody binding caused a 4–5 nm redshift, and an additional 1 nm shift was induced when the conjugates were blocked with BSA.

[0092] To confirm that antibody molecules bound to the nanostructure, lateral flow strips with protein A (0.5 mg / mL) stripes were immersed in a solution containing the conjugate nanostructure or the conjugate nanostructure blocked with BSA. Figure 9 shows that conjugates were formed and that BSA blocking did not destroy the conjugates.

[0093] Example 4. High-sensitivity antigen detection using antibody-conjugate nanostructures The ability of conjugates to detect the presence of antibodies was tested. Changes in the combined λmax of C1 and C6 adsorbed conjugates in the absence and presence of TSH were observed. Anti-TSH clones C1 and C6 were purchased from Arista Biologicals. These antibodies target the β-fragment of TSH. To obtain a baseline using a Nicoya® lifesciences OpenSPR spectrophotometer, clones C1 and C6 conjugated to 50 nm nanostructures were mixed together in PBS / BSA buffer and scanned for changes in λmax for 250 seconds. TSH was then added to 5 ng / ml, and λmax was monitored for an additional 450 seconds. Figure 10A shows the combined λmax of C1 and C6 conjugates generated using the 50 nm nanostructure and adsorption protocol. maxThe changes are shown in Figure 10B. The composite λ of C1 and C6 conjugates generated using a 50 nm nanostructure and a thiol-mediated conjugation protocol. max This shows the change.

[0094] Next, the effect of the presence of reaction accelerators on the spectral shifts of conjugates prepared by the adsorption protocol and the thiol-mediated protocol was evaluated. The results showed that various concentrations of reaction accelerators elicited diverse responses in the adsorption protocol conjugate versus the thiol-mediated covalent conjugate. For example, lower molecular weight and lower concentrations of PEG produced similar responses from the adsorption versus covalent conjugate (Figure 11).

[0095] Figure 12 shows the time-course dose-response curves and kinetics of covalent conjugates in the presence of increasing amounts of antigen (TSH) in the presence of 0.1% PEG and 0.5% methylcellulose. Figure 12 shows the peak-shift dose-response of anti-TSH C1 and anti-TSH C6 covalent conjugates in the presence of 0.1% polyethylene glycol and 0.5% methylcellulose.

[0096] Next, studies were conducted to determine the effect of the ratio of the conjugates used (e.g., C1 nanostructures and C6 nanostructures). Figure 13 shows the results obtained from C1 or C6 alone, or from the two antibodies in various ratios. Reaction curves were obtained in the absence of hTSH (0 ng) or in the presence of 0.25 ng. In this particular assay, clone C1 was essentially unreactive on its own, but made a significant contribution once C6 was introduced. While we do not wish to be constrained by theory, assays in some embodiments may depend on the formation of 3D nanoparticle assemblies, and appropriate changes in ionic strength, pH, and surfactant may provide additional enhancement in sensitivity.

[0097] In another study, two conjugates (15 μl of C1 and 25 μl of C6) were mixed with a PBS / BSA / PEG 8K solution containing 0–2 ng of TSH. The final concentration of PEG in the reaction mixture was 0.5% (Figure 14A) or 1.0% (Figure 14B). Components of the reaction mixture (0.8 ml) were placed in disposable cuvettes to record the change in λmax measured using a Nicoya OpenSPR® spectrometer. The curves obtained in these experiments were then fitted using regression analysis. max The changes were calculated from regression analysis over a 10-minute period and plotted to generate curves, as shown in Figures 14A and 14B. An increase in PEG concentration from 0.5% (Figure 14A) to 1% (Figure 14B) nearly doubled the sensitivity.

[0098] Example 5. Larger nanostructures having multiple spikes and an average diameter of 70 nm or 90 nm Conjugation and detection were tested using nanostructures with multiple spikes and average diameters of 70 nm or 90 nm (including the spikes). Figure 15A (upper panel) shows the conjugation of anti-hTSH antibodies C1 and C6 to 70 nm and 90 nm nanostructures, as well as their reactivity with protein A lateral flow strips. Figure 15B (lower panel) shows that the protein A lines on nitrocellulose reacted as expected before and after blocking with BSA. The unconjugated 70 nm and 90 nm nanostructures showed λmax of 609.5 and 641.9, respectively. The attachment of C1 and C6 and subsequent blocking with BSA caused a shift of up to 8 nm in λmax. The table below shows the quantitative data from Figure 15A.

[0099] (Table 1) Lmax of C1 and C6 conjugates at 70 nm and 90 nm in the presence or absence of BSA blocking TIFF2026076214000002.tif93128

[0100] The sensitivity of nanostructures of various diameters was compared. The spectral shift of conjugates containing nanostructures with a diameter of approximately 70 nm was improved compared to that of conjugates with a diameter of approximately 90 nm. Notably, further increasing the diameter of the nanostructures up to approximately 90 nm resulted in a further increase in sensitivity, as increasing the diameter to 90 nm produced nanostructure-conjugates that could produce a net spectral shift greater than 15 nm (Figures 16 and 17). In addition, the study showed that increasing the diameter of nanostructures with multiple spikes resulted in increased detection sensitivity. It was also observed that increasing PEG improved sensitivity up to a certain PEG concentration, but very high concentrations had a counterproductive effect.

[0101] Example 6. Activity of nanostructures with multiple protrusions versus nanorods in a solution-based assay. To compare the detection sensitivity of the nanostructure conjugates provided herein, which include multiple protrusions, with that of nanorod conjugates containing the same antibody, we conducted a study.

[0102] Nanorods (45.5 ± 6.3 nm in length and 17.4 ± 1.2 nm in width) were obtained from Nanocomposix and conjugated to antibody clones 1 and 6 using an adsorption conjugation protocol. 50 nm nanostructures containing multiple protrusions were also conjugated to antibody clones 1 and 6 using passive adsorption in borate buffer at pH 9.2, as described above. The results of the study are provided in Figure 18. The ability of the nanorod conjugates to detect TSH was tested in buffers containing PBS, BSA, and 1% PEG 20000, with or without the addition of 10 ng of TSH. Figure 18 shows no net change in λmax, calculated by subtracting the value obtained with TSH from the value obtained without TSH. λmax changes were recorded using Nicoya Lifesciences' OpenSPR®. Nanostructures with multiple protrusions were similarly tested, but the reaction rate was too fast at a TSH concentration of 10 ng / ml, resulting in a TSH amount of only 0.5 ng. Notably, the nanostructures with multiple protrusions exhibited robust TSH detection when measured by peak shift analysis, whereas the nanorods failed to detect TSH. This result was unexpected, at least since nanorods are believed to be excellent sensors for refractive index changes. However, in the solution-based assay of the present invention, nanostructures with multiple protrusions, rather than nanorods, exhibit superior performance.

[0103] Example 7. A low pH conjugation protocol produces a conjugate that exhibits high sensitivity detection. We conducted a study to evaluate the ability of conjugates formed using nanostructures containing multiple protrusions conjugated to antibodies at lower pH levels.

[0104] Anti-TSH antibody clones C1 and C6 were conjugated to gold nanostars prepared by a single-container seed-free method. The nanostars were diluted with distilled water to OD=1. The pH was approximately 6.0. Clones 1 and 6 were added separately to the nanostar solution to obtain 5 μg of antibody per 1 OD nanostar solution. After incubation for 15 minutes, the conjugates were blocked with 2 mg BSA per 1 ml of conjugate. The conjugates were then separated from the reaction by centrifugation at 25000 g for 15 minutes. If the pellet was loose, centrifugation may be repeated by adding 10 mM phosphate-buffered BSA (1%). The final precipitate was dissolved in the following CHAPS-containing buffers: PBS (1×), BSA (1%), and CHAPS (2%). Dissolution was facilitated by sonication for up to 30 seconds.

[0105] The ability of these conjugates to detect TSH was tested by diluting the final C1 (3%) and C6 (1%) conjugates with PBS / BSA (1% BSA in PBS) to an OD of approximately 0.5, and by recording the peak shift over time in the presence or absence of 1 ng / ml TSH using an OpenSPR® spectrometer. The results are shown in Figure 19, which shows the net shift plotted against reaction time (the net shift is calculated by subtracting the shift observed in the absence of any TSH from the shift caused by 1 ng / ml TSH). The study demonstrated that the conjugates exhibited robust detection activity.

[0106] Example 8. Simultaneous reduction of nonspecific adsorption of serum proteins and increased sensitivity of immunoassays. Biolipidure® reagents are synthetic polymer reagents containing a phosphorylcholine (PC) polar group, as well as a polymer tail containing hydrophobic, anionic, cationic, and / or hydrogen bond donor groups (Figure 20). These reagents are also known as 2-methacryloyloxyethyl phosphorylcholine polymer (MPC) and are incorporated into polymer biomaterials due to their properties of resistance to nonspecific protein adsorption, cell adhesion, and blood coagulation. Some of the features of these reagents include enhanced sensitivity and accuracy, suppression of nonspecific adsorption, stabilization of antibodies and enzymes, and reduced lot-to-lot variability, without the hassle of handling biohazards.

[0107] For many applications, Biolipidure® reagents are added to the final working solution to achieve the desired product results. To prevent nonspecific adsorption in immunoassays, Biolipidure® reagents can be applied by coating microplates, coating magnetic beads, and adding them to antibodies present in solution. In some embodiments, Biolipidure® reagents can be used by preparing buffer solutions with 1 wt% Biolipidure® reagent; dissolving samples (e.g., mucus) in buffer; and loading diluted samples onto immunochromatographic sample pads.

[0108] Remarkably, Biolipidure® reagents, when added to the surface of gold nanoparticles during the blocking phase of passive IgG adsorption, can both reduce nonspecific adsorption of serum proteins and enhance assay sensitivity. Briefly, gold nanospheres were coated with mouse IgG for 15 minutes, and then with one of Biolipidure® reagents 205, 206, 1002, 1003, 1201, 1202, or BSA for 15 minutes. The antibody-gold conjugates were washed three times for pre-test storage and suspended in conjugate diluents. To test the sensitivity of Biolipidure® conjugates to the antigen compared to BSA conjugates, the conjugates were tested with 500 pg / mL of antigen over a 10-minute course in Tris-buffered saline / bovine serum albumin (TBS BSA), a buffer solution. Surprisingly, the 5 / 6 Biolipidure® blocking conjugate exhibits a 2-3x enhancement compared to the standard BSA blocking conjugate. The results are shown in Figure 21.

[0109] Even more surprisingly, when tested for nonspecific protein adsorption in serum samples, blocking with Biolipidure® demonstrated a reduction in NSB compared to standard BSA conjugates. All Biolipidure® conjugates were tested with the same canine serum samples verified to have low / normal TSH levels. The serum samples were diluted 1 / 20 in TBS / BSA, and their wavelength shifts were monitored over time. Biolipidure® reagent 1003 showed a large 10 nm wavelength shift in response to the addition of serum to the nanoparticle conjugate, as shown in Figure 22A. This shift was approximately 3–4 times larger than all other conjugates and was therefore removed to clearly observe the wavelength shifts of the other conjugates (Figure 22B). The wavelength shift of the BSA-blocking IgG conjugate over a 10-minute time course in response to the addition of canine serum was approximately 3.5 nm. Biolipidure® conjugate 1202 showed a wavelength shift similar to that of the BSA-blocking conjugate. Conjugates blocked with 205, 206, 1002, and 1201 all showed a decrease in wavelength shift in the presence of canine serum.

[0110] Due to the variability of Biolipidure® reagents, they exhibit diverse responses to canine serum and enhanced sensitivity in sandwich immunoassays. Biolipidure® reagents 205, 206, 1002, and 1201 all demonstrate the ability to both reduce wavelength shift in canine serum and improve antigen-responsive LSPR shift in this homogeneous sandwich immunoassay. Biolipidure® reagent 1202 was able to improve the response to the antigen, while the wavelength shift in response to the addition of canine serum to the sample was comparable to that of the BSA conjugate.

[0111] The increased sensitivity of antigen detection and the reduction in nonspecific wavelength shift in canine serum were not limited to spherical nanoparticles. When Biolipidure® reagent was added to the surface of IgG conjugates, the sensitivity of antibody conjugates to the antigen increased, and the wavelength shift in response to serum addition was significantly reduced compared to BSA-blocking conjugates (Figure 23). For 1 ng / mL antigen diluted 20-fold to 50 pg / mL canine antigen, there was a significant increase in the response of 90 nm nanourtin (i.e., anisotropic nanoparticles with multiple protrusions on the surface) conjugates blocked with 2x Biolipidure® reagent 1002 (Figure 24). These conjugates were also much more sensitive than spherical gold conjugates blocked with Biolipidure® 1002.

[0112] The results of this study provided a remarkable method for increasing sensitivity while reducing nonspecific binding in assays. Changing the blocking agent during the gold nanoparticle-antibody passive conjugation procedure from BSA to several Biolipidure® reagents resulted in a significant increase in the wavelength shift for the antigen under buffer conditions, followed by a decrease in the wavelength shift in response to the addition of serum to the conjugate, which indicated a possible reduction in nonspecific adsorption derived from serum components.

[0113] Example 9. Salt combinations and EDTA reduce nonspecific binding. To determine the reasons for nonspecific binding in LSPR assays and to identify means of reducing nonspecific binding, we conducted a study. Nonspecific binding may be due to electrostatic or hydrophobic interactions between the gold conjugate and large serum proteins. The initial study focused on NaCl. Based on Hofmeister's theory, experiments were conducted to determine whether MgCl2 or NaSCN could be beneficial in inhibiting protein aggregation. In addition, chaotropic salts may have an effect on the colloidal stability of the gold conjugate. A schematic diagram of salts in the Hofmeister series is provided in Figure 25.

[0114] The initial study using the chaotropic salt MgCl2 showed promising results. In the absence of the salt, normal canine serum samples exhibited significant nonspecific binding. With 50–100 mM MgCl2, the nonspecific binding signal was drastically reduced (Figure 26). The results of the LSPR peak shift at 5 minutes are shown in Table 2 below. Nonspecific binding and the 5 ng / ml LSPR signal were disproportionately reduced in the presence of MgCl2.

[0115] (Table 2) LSPR peak shift in the presence or absence of MgCl2 TIFF2026076214000003.tif37162

[0116] In another study, the effects of other chaotropic salts on 80 nm nanosphere cTSH conjugates were evaluated. MgCl2, NaCl, and NaSCN were tested. Both MgCl2 and NaSCN showed a reduction in nonspecific binding (Figure 27). The LSPR peak shift results after 5 minutes are provided in Figure 28 and Table 3 below.

[0117] (Table 3) LSPR peak shift in the presence of NaCl, MgCl2, or NaSCN TIFF2026076214000004.tif31166

[0118] In another study, the beneficial effect of Mg(II) on nonspecific binding in LSPR-based canine TSH assays was confirmed using very small pooled samples (cTSH <100 pg / ml in TBS buffer) and large samples (10 ng / ml in TBS buffer). In the presence of 0 mM MgCl2, the wavelength shifts of the very small and large cTSH samples were the same (Figure 29, upper left panel). With increasing concentrations of MgCl2 (1 mM, 10 mM, or 100 mM, upper right, lower left, and lower right panels of Figure 29, respectively), the wavelength shift in the very small sample was reduced. Therefore, the study confirmed that Mg(II) substantially reduced nonspecific binding when present at 100 mM (Figure 29). To understand the mechanism of the Mg(II) effect on the LSPR signal, a second experiment using EDTA (chelating agent) was performed. Surprisingly, EDTA showed an effect similar to that of Mg(II) (Figure 30). Even more surprisingly, EDTA and Mg(II) did not appear to cancel each other out. This was particularly surprising, as EDTA is expected to chelate Mg(II) and render it ineffective. Thus, the study demonstrated that combinations of Mg(II), optionally other Hofmeister series salts, and EDTA or ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) completely controlled nonspecific binding in LSPR-based assays. These surprising results were achieved in studies using either spherical nanostructures or nanoparticles with multiple protrusions on their surface.

[0119] It is understood that the disclosed invention is not limited to the specific methodologies, protocols, and materials described herein, as these may vary. It is also understood that the technical terms used herein are for the purpose of describing specific aspects and are not intended to limit the scope of the invention, which is limited solely by the appended claims.

[0120] Those skilled in the art will be able to recognize, or confirm by routine experimental methods, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the appended claims.

[0121] Unless otherwise defined, all technical and scientific terms herein have the same meaning as those commonly understood by those skilled in the art in which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the present invention, but preferred methods and materials are described herein. All publications, patents, and patent publications cited herein are incorporated herein by reference in their entirety for all purposes.

[0122] The publications discussed herein are provided solely for disclosures prior to the filing date of this application. Nothing herein should be construed as an acknowledgment that the present invention is not granted prior rights to such publications by prior art.

Claims

1. A method for detecting a target analyte in a sample, including the following steps: (a) A step of mixing a sample in solution with a first detection conjugate and a second detection conjugate, wherein the first and second detection conjugates include nanostructures linked to binding partners that can specifically bind to a target analyte if present in the sample to form a complex between the first detection conjugate, the analyte, and the second detection conjugate, the nanostructures including a plurality of protrusions, and the average diameter of the nanostructures from tip to tip is at least about 50 nm; (b) Exposing the composite to a light source in the wavelength range within the ultraviolet-visible-infrared spectrum; and (c) A step of measuring an optical signal from the composite, wherein a change in the optical signal indicates the presence of the target analyte in the sample.

2. The method according to claim 1, wherein the average diameter of the nanostructure is about 70 nm.

3. The method according to claim 1, wherein the average diameter of the nanostructure is about 90 nm.

4. The method according to claim 1, wherein the mixing step (a) is carried out in the presence of 3-((3-collamidopropyl)dimethylammino)-1-propanesulfonate (CHAPS).

5. The method according to claim 4, wherein CHAPS is present at a concentration of approximately 0.1% w / v to approximately 0.5% w / v.

6. The method according to claim 4, wherein CHAPS is present in the solution at a concentration of approximately 0.2% w / v.

7. The method according to claim 1, wherein the mixing step (a) is carried out in the presence of a polymer material selected from polyethylene glycol (PEG), polyvinylpyrrolidone, gelatin, cellulose, or a combination thereof.

8. The method according to claim 7, wherein the polymer material is selected from the group consisting of methylcellulose, dextran, polyallylamine, polyethyleneimine, polylysine, polyacrylic acid, polyvinyl alcohol, and polyaspartic acid.

9. The method according to claim 7, wherein the polymer material is PEG, and the PEG is present in a concentration of about 0.1% to about 5% w / v.

10. The method according to claim 1, wherein the solution further comprises a viscosity enhancer.

11. The method according to claim 10, wherein the viscosity enhancer is selected from the group consisting of trehalose, maltodextrin, sucrose, sorbitol, mannitol, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), cyclodextrin, methylcellulose, dextran, and ficol.

12. The aforementioned solution is MgCl 2 The method according to claim 1, further comprising a salt selected from NaSCN.

13. MgCl 2 Alternatively, the method according to claim 12, wherein NaSCN is present in the solution at a concentration of about 10 mM to about 250 mM.

14. MgCl 2 The method according to claim 12, wherein NaSCN is present in the solution at a concentration of about 100 mM.

15. The method according to claim 1, wherein the solution further comprises ethylenediaminetetraacetic acid (EDTA) or ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

16. The method according to claim 15, wherein EDTA or EGTA is present in the solution at a concentration of about 5 mM to about 100 mM.

17. The method according to claim 1, wherein the solution further comprises Biolipidure® reagent.

18. The method according to claim 17, wherein the Biolipidure® reagent is selected from the group consisting of Biolipidure® reagents 205, 206, 1002, 1201, and 1202.

19. The method according to claim 1, wherein the optical signal is a reflectance spectrum, an absorbance spectrum, a scattering spectrum, or an emission spectrum.

20. The method according to claim 1, wherein the change in the optical signal includes a spectral peak wavelength shift and / or a full spectral profile shift.

21. The method according to claim 20, wherein the entire spectral profile shift is a difference spectrum.

22. The method according to claim 1, wherein the presence of a nanogram amount of target analyte is detected.

23. The method according to claim 1, wherein the presence of a picogram amount of the target analyte is detected.

24. The method according to claim 1, wherein the presence of a femtogram of the target analyte is detected.

25. The method according to claim 1, wherein step (a) is carried out in a spectrophotometric cuvette, in an analytical rotor, in a microwell plate, in a clinical analyzer, in a flow chamber, on the tip of an optical fiber, or in a transparent gel.

26. The method according to claim 1, wherein the metal nanostructure is a gold metal nanostructure.

27. The method according to claim 1, wherein the binding partner is a biopolymer.

28. The method according to claim 27, wherein the biopolymer is selected from antibodies or fragments thereof, antigens, receptors, ligands, polynucleotides, aptamers, polypeptides, polysaccharides, lipopolysaccharides, glycopeptides, lipoproteins, or nucleoproteins.

29. The method according to claim 27, wherein the biopolymer is an antibody.

30. The method according to claim 27, wherein the biopolymer is an antigen.

31. The method according to claim 1, wherein the first detection conjugate and the second detection conjugate include a binding partner which is an antibody.

32. The method according to claim 31, wherein the antibody binds to different epitopes on the target analyte.

33. The method according to claim 1, wherein the target analyte is selected from proteins, enzymes, antigens, antibodies, peptides, nucleic acids, hormones, glycoproteins, polysaccharides, toxins, viruses, viral particles, drug molecules, haptens, and chemical substances.

34. The method according to claim 1, wherein the target analyte is a pathogenic antigen or an antibody against a pathogenic antigen.

35. The method according to claim 34, wherein the pathogenic antigen is a viral antigen.

36. The method according to claim 35, wherein the viral antigen is derived from a virus selected from feline leukemia virus, canine parvovirus, foot-and-mouth disease virus, influenza virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, HIV virus, human papillomavirus, Epstein-Barr virus, and rabies virus.

37. The method according to claim 34, wherein the pathogenic antigen is a bacterial antigen.

38. The method according to claim 37, wherein the bacterial antigen is selected from the genera Ehrlichia, Borrelia, Anaplasma, Salmonella, Bacillus, and Rickettsia.

39. The method according to claim 37, wherein the bacterial antigen is selected from Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia ewingii, Borrelia burgdorferi, Anaplasma platys, Anaplasma phagocytophilum, Salmonella enterica, Bacillus anthracis, and Rickettsia rickettsii.

40. The method according to claim 34, wherein the pathogenic antigen is a fungal antigen or a parasitic antigen.

41. The method according to claim 40, wherein the fungal or parasitic antigen is selected from canine heartworm, Giardia lamblia, Plasmodium falciparum, African trypanosomiasis, and Trypanosoma brucei.

42. The method according to claim 1, wherein the mixing step (a) is carried out in the presence of a blocking agent.

43. The method according to claim 42, wherein the blocking agent is selected from bovine serum albumin (BSA), casein, gelatin, ovalbumin, and gamma globulin.

44. The method according to claim 43, wherein the blocking agent is BSA present at a concentration of about 1% to about 5% w / v.

45. A method for detecting a target analyte in a sample, including the following steps: (a) A step of mixing a sample in solution with a first detection conjugate, a second detection conjugate, CHAPS, bovine serum albumin (BSA), one or more polymer materials, one or more viscosity enhancers, a salt, and optionally a chelating agent, wherein the first and second detection conjugates include nanostructures linked to binding partners that can specifically bind to the target analyte if present in the sample to form a complex between the first detection conjugate and the analyte and the second detection conjugate; (b) Exposing the composite to a light source in the wavelength range within the ultraviolet-visible-infrared spectrum; and (c) A step of measuring an optical signal from the composite, wherein a change in the optical signal indicates the presence of the target analyte in the sample.

46. The method according to claim 45, wherein the polymer material is selected from the group consisting of PEG, polyvinylpyrrolidone, gelatin, methylcellulose, dextran, polyallylamine, polyethyleneimine, polylysine, polyacrylic acid, polyvinyl alcohol, and polyaspartic acid.

47. The method according to claim 45, wherein the viscosity enhancer is selected from the group consisting of trehalose, maltodextrin, sucrose, sorbitol, mannitol, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), cyclodextrin, methylcellulose, dextran, and ficol.

48. The method according to claim 45, wherein the salt is selected from the group consisting of NaCl, MgCl2, CaCl2, and NaSCN.

49. The method according to claim 45, wherein the chelating agent is selected from the group consisting of ethylenediaminetetraacetic acid (EDTA) and ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

50. The method of claim 45, wherein the solution of step (a) further comprises Biolipidure® reagent.

51. The method according to claim 45, wherein the nanostructure is selected from the group consisting of spherical nanoparticles and nanoparticles having a plurality of protrusions.

52. A reaction mixture comprising at least one binding partner-nanostructure conjugate and an amphoteric surfactant, wherein the nanostructure comprises a plurality of protrusions and the average diameter of the nanostructure is at least about 50 nm.

53. The reaction mixture according to claim 52, wherein the amphoteric surfactant is selected from the group consisting of 3-((3-collamidopropyl)dimethylammino)-1-propanesulfonate (CHAPS) and sulfobetaine surfactants.

54. The reaction mixture according to claim 53, wherein CHAPS is present at a concentration of approximately 0.1% to approximately 1%.

55. The reaction mixture according to claim 54, wherein CHAPS is present at a concentration of approximately 0.5%.

56. The reaction mixture according to claim 52, wherein the average diameter of the nanostructure is approximately 70 nm.

57. The reaction mixture according to claim 52, wherein the average diameter of the nanostructure is about 90 nm.

58. The reaction mixture according to claim 52, further comprising a sample containing a target analyte.

59. The reaction mixture according to claim 58, wherein the target analyte is selected from proteins, enzymes, antigens, antibodies, peptides, nucleic acids, hormones, glycoproteins, polysaccharides, toxins, viruses, viral particles, drug molecules, haptens, and chemical substances.

60. The reaction mixture according to claim 52, wherein the binding partner is a biopolymer.

61. The reaction mixture according to claim 60, wherein the biopolymer is selected from antibodies or fragments thereof, antigens, receptors, ligands, polynucleotides, aptamers, polypeptides, polysaccharides, lipopolysaccharides, glycopeptides, lipoproteins, or nucleoproteins.

62. The reaction mixture according to claim 60, wherein the biopolymer is an antibody.

63. The reaction mixture according to claim 52, wherein the metal nanostructure is a gold metal nanostructure.

64. The reaction mixture according to claim 52, comprising a first detection conjugate and a second detection conjugate, wherein the first and second detection conjugates comprise binding partners that are first and second antibodies.

65. The reaction mixture according to claim 64, wherein the first and second antibodies bind to different and non-overlapping epitopes on the target analyte.

66. A method for preparing a conjugate comprising a binding partner and a metal nanostructure, suitable for detecting changes in optical signals based on the presence of a target analyte, wherein the metal nanostructure comprises a plurality of protrusions, the diameter of the metal nanostructure is at least about 50 nm, and the method comprises the following steps: (a) A step of mixing a solution containing the metal nanostructure with a solution containing the binding partner to form a binding partner-nanostructure conjugate; (b) Blocking the conjugate with BSA, PEG, Biolipidure® reagent, or a combination thereof; (c) the step of centrifuging the conjugate; and (d) Resuspending the conjugate in a diluent comprising a buffer selected from the group consisting of PBS, TBS, and borate; a blocking agent selected from the group consisting of BSA, PEG, Biolipidure® reagent, or a combination thereof; and CHAPS.

67. The method according to claim 66, wherein the binding partner is an antibody.

68. The method according to claim 67, wherein the antibody is an antibody containing a hydrophobic region.

69. The method according to claim 66, wherein the solution comprises a viscosity enhancer selected from trehalose, maltodextrin, sucrose, sorbitol, mannitol, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), cyclodextrin, methylcellulose, dextran, and Ficol.

70. The method according to claim 66, wherein step (c) includes centrifugation at approximately 2,000 g or more.

71. The method according to claim 66, wherein the average diameter of the nanostructure is approximately 70 nm.

72. The method according to claim 66, wherein the average diameter of the nanostructure is about 90 nm.

73. The method according to claim 66, further comprising a freeze-drying step after step (d).

74. The method according to claim 73, wherein the freeze-drying step includes distributing the conjugate in liquid nitrogen and freeze-drying using vacuum and temperature cycling.

75. The method according to claim 66, wherein the solution further comprises EDTA or EGTA.

76. The method according to claim 66, wherein the solution further comprises MgCl2 or NaSCN.

77. The method according to claim 66, wherein the solution further comprises EDTA and MgCl2.

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