Signal amplification in solution-based plasmonic specific binding partner assays

Abstract

To provide a method for quantitatively monitoring a dynamics of highly sensitive and continuous coupling phenomena in a diagnosis assay by a local surface plasmon resonance (LSPR) of noble metal nanoparticles.SOLUTION: The present invention relates to a method for detecting a target analysis object in a sample, the method including the step of mixing the sample with a first detection conjugate and a second detection conjugate in a solution, the first detection conjugate and the second detection conjugate including a metal nano structure connected to a coupling parter, the coupling partner being specifically coupled to a target analysis object in the sample when there is the target analysis object in the sample, and being capable of forming a composite body with the first detection conjugate, the analysis object, and the second detection conjugate, a change in the optical signal at the time of forming the composite body showing the existence of the target analysis object in the sample. There is also described a method for preparing a nano structure and a nano alloy, and a method for preparing a nano structure and a nano alloy conjugated with the coupling partner.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 201,051, filed August 4, 2015, the contents of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The present invention relates to systems and methods for detecting a target analyte in a sample. In particular, the present invention provides a localized plasmon resonance-based analyte detection system capable of detecting trace 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 sophisticated equipment to perform the assay. The lack of sensitivity and complexity involved in performing such heterogeneous assays arises from the specific need to separate labeled from unlabeled specific binding partners.

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

[0005] Therefore, there is a need for improved assays that utilize homogeneous formats while providing increased sensitivity. Assays that utilize standard laboratory techniques, such as spectroscopy, would also be desirable. [Prior art documents] [Non-patent literature]

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

[0007] This application describes the use of localized surface plasmon resonance (LSPR) technology to perform assays for specific binding partners, including but not limited to, ligands, receptors, transcription factors, binding DNA factors, antigens, and antibodies. More specifically, this application relates to methods and materials for achieving significant amplification in such assays using composite metal nanomaterial-labeled partners.

[0008] In various embodiments described herein, the application relates to the use of composite nanomaterial-labeled partners in solution to determine binding of specific binding partners in a qualitative or quantitative manner.

[0009] In a first aspect, the present application provides a method for detecting a target analyte in a sample. In one embodiment, the method includes the steps of: mixing the sample with a first detection conjugate and a second detection conjugate, each comprising a metal nanostructure linked to a binding partner, wherein the binding partner specifically binds to the target analyte, if present in the sample, thereby allowing the formation of a complex between the first detection conjugate, the analyte, and the second detection conjugate; exposing the complex to a light source having a 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 the target analyte in the sample. In a representative embodiment, the metal nanostructure in the first detection conjugate and / or the second detection conjugate is a composite metal nanostructure. In another representative embodiment, the mixing step is performed in the presence of a polymeric material selected from polyethylene glycol (PEG), polyvinylpyrrolidone, polyallylamine, polyethyleneimine, polylysine, polyacrylic acid, polyvinyl alcohol, and polyaspartic acid. In a preferred embodiment, the polymeric material is PEG. In yet another exemplary embodiment, the mixing step is carried out in the presence of a polysaccharide. In some embodiments, the polysaccharide is selected from maltodextrin, corn syrup, and polyglucose. In a preferred embodiment, the polysaccharide is maltodextrin. 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, casein, gelatin, ovalbumin, and gamma-globulin. In a preferred embodiment, the blocking agent is bovine serum albumin.

[0010] In some embodiments, the detection conjugate comprises a binding partner capable of specifically binding to the target analyte. In certain embodiments, the binding partner is a biopolymer, including but not limited to, haptens and other small molecules, drugs, hormones, antibodies or fragments thereof (e.g., Fv, Fab, (Fab)2, single chain, CDR, etc.), antigens, receptors, ligands, polynucleotides, aptamers, polypeptides, polysaccharides, lipopolysaccharides, glycopeptides, lipoproteins, or nucleoproteins. In certain representative embodiments, the binding partner is an antibody. In other representative embodiments, the binding partner is an antigen. In some embodiments, the detection conjugates (e.g., the first detection conjugate and the second detection conjugate) comprise binding partners that are the same type of molecule.

[0011] In some embodiments, the metal nanostructures in the detection conjugate can be composed of a noble metal or a complex thereof. In some embodiments, the metal nanostructures in the detection conjugate may be composed of a transition metal or a complex thereof. In some embodiments, the metal nanostructures in the detection conjugate may include an alkali metal or a lanthanide in combination with a noble or transition metal. In certain embodiments, the metal nanostructures in the detection conjugate comprise a metal selected from gold, silver, copper, platinum, palladium, ruthenium, rhodium, osmium, iridium, titanium, chromium, cadmium, zinc, iron, cobalt, nickel, and complexes thereof. In an exemplary embodiment, the metal nanostructures are gold nanostructures. In another exemplary embodiment, the metal nanostructures are silver nanostructures.

[0012] In a preferred embodiment, the metal nanostructure in the detection conjugate is a composite metal nanostructure comprising at least two noble metals, transition metals, alkali metals, or lanthanides. 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 a representative embodiment, the composite metal nanostructure comprises gold and silver.

[0013] In one exemplary embodiment, the first binding partner is conjugated to a gold or composite nanoparticle, and the second binding partner is conjugated to another composite nanomaterial containing two metals selected from the group consisting of gold, silver, copper, platinum, palladium, cadmium, and zinc. In another exemplary embodiment, the first binding partner is conjugated to a nanoparticle containing silver and gold, and the second binding partner is conjugated to a nanoparticle comprising gold and copper.

[0014] As described herein, significant signal amplification is achievable in a variety of assays. In certain embodiments, the assays are direct, indirect, sandwich, competitive, and secondary labeling assays. In certain further embodiments, these assays may use absorbance, scattering, and / or reflectance measurements to monitor specific binding events.

[0015] In certain embodiments, the methods of the present invention are capable of detecting femtogram to nanogram amounts of a target analyte in a sample.

[0016] As described above, the present application relates to the use of nanomaterial-labeled partners, e.g., antibodies conjugated to composite metal nanostructures, in solution to determine binding of specific binding partners in a qualitative or quantitative manner. In some embodiments, the solution contains one or more of a polysaccharide (e.g., maltodextrin), trehalose, a polymeric material (e.g., PEG), a blocking agent (e.g., bovine serum albumin), and / or sodium chloride. In exemplary embodiments, one or more solution components, e.g., maltodextrin, may be provided in lyophilized form, e.g., as beads or pellets. For example, one or more solution components may be provided as beads or pellets in a spectrophotometric cuvette or in one or more reaction chambers of an analytical rotor. The beads or pellets may be suspended upon addition of a liquid, e.g., water, saline solution, liquid sample, etc. In one embodiment, the solution contains maltodextrin at a final concentration of about 2% to about 20% weight / volume (wt / vol). In another embodiment, the solution contains maltodextrin at a final concentration of about 4% to about 15% weight / volume. In yet another embodiment, the solution contains maltodextrin at a final concentration of about 5% to about 10% weight / volume. In some embodiments, the sensitivity of the assay is improved when maltodextrin is added to the solution compared to when the assay is performed in a solution containing another sugar, such as sucrose or ficoll.

[0017] In another aspect, the present invention provides an analyte detection device for utilizing the methods described herein to detect a target analyte in a sample. Suitable analyte detection devices may include, but are not limited to, spectrophotometric cuvettes, analytical rotors, microwell plates, clinical analyzers (e.g., Cobas Fara), or flow chambers. Fiber optic tips or transparent gels may also be utilized to implement the detection methods disclosed herein. In exemplary embodiments, the analyte detection device is selected from spectrophotometric cuvettes and analytical rotors.

[0018] In a preferred embodiment, the components of the analyte detection device are contained in a centrifugal rotor or disk. In some embodiments, the rotor or disk may contain one or more reaction chambers in which multiple detection conjugates are located. In certain embodiments, the detection conjugates are present in the form of a lyophilized composition, such as lyophilized beads or pellets. In some embodiments, the analyte detection device includes one or more reaction chambers, each of which contains multiple detection conjugates (e.g., a first detection conjugate and a second detection conjugate), and the detection conjugates are linked to metal nanoparticles, e.g., composite metal nanostructures. In embodiments in which the rotor or disk includes two or more reaction chambers, the detection conjugates can be selected so that different analytes can be detected in each reaction chamber.

[0019] In yet another aspect, the present invention provides a kit comprising the analyte detection device of the present invention. In one embodiment, the kit comprises a plurality of detection conjugates (e.g., a first detection conjugate and a second detection conjugate), wherein the detection conjugates are linked to metal nanoparticles, e.g., composite metal nanostructures. In some embodiments, one or more of the detection conjugates may be lyophilized. In one embodiment, all of the detection conjugates are lyophilized. In a representative embodiment, the metal nanostructures in the first detection conjugate and / or the second detection conjugate are composite metal nanostructures.

[0020] In yet another aspect, the present invention provides a method for preparing composite metal nanostructures for use in the detection devices and methods described herein. In one embodiment, the method includes preparing a first solution containing a mixture of a polymer and chloroauric acid, preparing a second solution containing silver and copper nanostructures, and incubating the first solution with the second solution for a period of time, where the resulting mixture contains gold-coated silver nanostructures or gold-coated copper nanostructures. In certain embodiments, a reducing agent such as ascorbic acid is added to the reaction mixture to increase the amount of nanostructures produced. In one embodiment, the polymer in the first solution is polyvinylpyrrolidone. In another embodiment, the polymer in the first solution is polyvinyl alcohol. In another embodiment, the method includes preparing a first solution containing a mixture of a detergent such as CHAPS and chloroauric acid, and a solution containing a silver or copper salt, and incubating the first solution with a second solution containing a reducing agent such as ascorbic acid to induce the formation of composite nanostructures. The size and shape of the nanostructures can be varied by changing the ratio of metals used, the concentration of detergent, and finally the amount of ascorbic acid used. [The present invention 1001] (a) mixing a sample with a first detection conjugate and a second detection conjugate, wherein the first detection conjugate and the second detection conjugate comprise a composite metallic nanostructure linked to a binding partner, which specifically binds to a target analyte, if present in the sample, and is capable of forming a complex between the first detection conjugate, the analyte, and the second detection conjugate; (b) exposing the complex to a light source having a wavelength range within the ultraviolet-visible-infrared spectrum; and (c) measuring an optical signal from the complex, wherein a change in the optical signal indicates the presence of the target analyte in the sample. 1. A method for detecting a target analyte in a sample, comprising: [The present invention 1002] 1001. The method of claim 1001, wherein the optical signal is a reflectance, absorbance spectrum, scattering spectrum, or emission spectrum. [The present invention 1003] 1001. The method of claim 1001, wherein the change in the optical signal comprises a spectral peak wavelength shift and / or a full spectrum wavelength shift. [The present invention 1004] The method of the present invention 1003, wherein the total spectral wavelength shift is the difference spectrum. [The present invention 1005] 1001. The method of claim 1001, wherein the presence of nanogram amounts of the target analyte is detected. [The present invention 1006] 1001. The method of claim 1001, wherein the presence of picogram amounts of the target analyte is detected. [The present invention 1007] 1001. The method of claim 1001, wherein the presence of femtogram amounts of a target analyte is detected. [The present invention 1008] 1001. The method of claim 1001, wherein step (a) is carried out in a spectrophotometric cuvette, an analytical rotor, a microwell plate, a clinical analyzer, a flow chamber, on the tip of an optical fiber, or in a transparent gel. [The present invention 1009] 1001. The method of claim 1001, wherein the composite metallic nanostructure comprises at least two metals selected from gold, silver, copper, platinum, palladium, cadmium, iron, nickel, and zinc. [The present invention 1010] 1001. The method of claim 1001, wherein each of the composite metallic nanostructures comprises a core of a first metal and a coating of a second metal. [The present invention 1011] The method of claim 1009, wherein each of the composite metallic nanostructures comprises a gold coating and a silver core. [The present invention 1012] The method of claim 1009, wherein each of the composite metallic nanostructures comprises a silver coating and a gold core. [The present invention 1013] 1001. The method of claim 1001, wherein each of the composite metallic nanostructures is an alloy of a first metal and a second metal. [The present invention 1014] 1001. The method of claim 1001, wherein the composite metallic nanostructure has a geometric structure selected from a spherical nanoparticle, a pyramidal nanoparticle, a hexagonal nanoparticle, a nanotube, a nanostar, a nanoshell, a nanorod, a nanoisland, a nanodot, a nanowire, or a combination thereof. [The present invention 1015] 1001. The method of claim 1001, wherein the binding partner is a biopolymer. [The present invention 1016] The method of claim 1015, wherein the biopolymer comprises an antibody or fragment thereof, an antigen, a receptor, a ligand, a polynucleotide, an aptamer, a polypeptide, a polysaccharide, a lipopolysaccharide, a glycopeptide, a lipoprotein, or a nucleoprotein. [The present invention 1017] The method of claim 1016, wherein the biopolymer is an antibody. [The present invention 1018] The method of claim 1016, wherein the biopolymer is an antigen. [The present invention 1019] 1002. The method of claim 1001, wherein the first detection conjugate and the second detection conjugate comprise binding partners that are antibodies. [The present invention 1020] The method of claim 1019, wherein said antibodies bind to different epitopes of the target analyte. [The present invention 1021] 1001. The method of claim 1001, wherein the target analyte is selected from a protein, an enzyme, an antigen, an antibody, a peptide, a nucleic acid, a hormone, a glycoprotein, a polysaccharide, a toxin, a virus, a virus particle, a drug molecule, a hapten, and a chemical. [The present invention 1022] 1001. The method of claim 1001, wherein the target analyte is a pathogenic antigen or an antibody to a pathogenic antigen. [The present invention 1023] The method of claim 1022, wherein the pathogenic antigen is a viral antigen. [The present invention 1024] The method of claim 1023, 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. [The present invention 1025] The method of claim 1022, wherein the pathogenic antigen is a bacterial antigen. [The present invention 1026] 1025. The method of claim 1025, wherein the bacterial antigen is selected from Ehrlichia, Borrelia, Anaplasma, Salmonella, Bacillus, and Rickettsia. [The present invention 1027] 1026. The method of claim 1026, wherein the bacterial antigen is selected from Ehrlichia canis, Ehrlichia chafeensis, Ehrlichia ewingii, Borrelia burgdorferi, Anaplasma platys, Anaplasma phagocytophilum, Salmonella enterica, Bacillus anthracis, and Rickettsia rickettsii. [The present invention 1028] The method of claim 1022, wherein the pathogenic antigen is a fungal antigen or a parasitic antigen. [The present invention 1029] 1028. The method of claim 1028, wherein the fungal or parasitic antigen is selected from Dirofilaria immitis, Giardia lamblia, Plasmodium falciparum, African trypanosomiasis, and Trypanosoma brucei. [The present invention 1030] 1001. The process of claim 1001, wherein the mixing in step (a) is carried out in the presence of a polymeric material selected from polyethylene glycol, polyvinylpyrrolidone, polyallylamine, polyethyleneimine, polylysine, polyacrylic acid, polyvinyl alcohol, and polyaspartic acid. [The present invention 1031] The method of claim 1030, wherein the polymeric material is polyethylene glycol. [The present invention 1032] 1001. The method of claim 1001, wherein the mixing in step (a) is carried out in the presence of a polysaccharide. [The present invention 1033] The method of claim 1032, wherein the polysaccharide is selected from maltodextrin, corn syrup, and polyglucose. [The present invention 1034] The method of claim 1033, wherein the polysaccharide is maltodextrin. [This invention 1035] The method of claim 1034, wherein the final concentration of maltodextrin in the reaction mixture is from about 2% to about 20% weight / volume. [The present invention 1036] The method of claim 1035, wherein the final concentration of maltodextrin in the reaction mixture is about 5% to about 10% weight / volume. [This invention 1037] 1001. The method of claim 1001, wherein the mixing in step (a) is carried out in the presence of a blocking agent. [The present invention 1038] 1037. The method of claim 1037, wherein the blocking agent is selected from bovine serum albumin, casein, gelatin, ovalbumin, and gamma-globulin. [This invention 1039] The method of claim 1038, wherein the blocking agent is bovine serum albumin. [The present invention 1040] The method of claim 1039, wherein the final concentration of bovine serum albumin in the reaction mixture is about 1% to about 5% weight / volume. [The present invention 1041] a first detection conjugate comprising a metallic nanostructure linked to a binding partner capable of specifically binding to a target analyte, if present, in the sample; a second detection conjugate comprising a metallic nanostructure linked to a binding partner capable of specifically binding to the target analyte, if present in the sample; 1. An analyte detection device comprising: The metal nanostructure in the first detection conjugate and / or the second detection conjugate is a composite metal nanostructure; Analyte detection devices. [The present invention 1042] The analyte detection device of the present invention 1041, which is a spectrophotometric cuvette, an analytical rotor, a microwell plate, or a flow chamber. [This invention 1043] The analyte detection device of the present invention 1042, which is an analytical rotor. [This invention 1044] The analyte detection device of the present invention 1043, wherein the analytical rotor comprises one or more reaction chambers in which a first detection conjugate and a second detection conjugate are disposed. [This invention 1045] The analyte detection device of the present invention 1041, wherein the first detection conjugate and / or the second detection conjugate are lyophilized. [The present invention 1046] An analyte detection device of the present invention 1041 configured to receive a test sample. [This invention 1047] An analyte detection device according to claim 1041, configured to expose a complex of a first detection conjugate, the analyte, and a second detection conjugate to a light source in a wavelength range within the ultraviolet-visible-infrared spectrum. [This invention 1048] 1047. The analyte detection device of claim 1047, wherein said device is further configured to measure an optical signal from said complex, wherein a change in the optical signal indicates the presence of a target analyte in said sample. [Brief explanation of the drawings]

[0021] [Figure 1] The basic principle of the LSPR immunoassay described herein is illustrated. Metal nanoparticles themselves exhibit optical spectra that depend on the metal composition, diameter, shape, and the nature of the dispersion medium. Small changes in the surface of the nanoparticles due to initial primary and subsequent secondary binding induce gradual changes in the characteristics of light interacting with the nanoconjugate. These changes can be recorded by an appropriate spectrometer, providing qualitative and quantitative information. [Figure 2] An example of a receptor with multiple ligand binding sites is shown: an antibody labeled with a nanoparticle causes a spectral shift upon binding to an antigen. [Figure 3] LSPR coupling behavior between different nanoparticle types is shown when the receptor has multiple binding sites or when the receptors have different binding sites. [Figure 4] This figure illustrates the effect of polyethylene glycol (PEG) on LSPR signal. The LSPR signal increases substantially in the presence of PEG. This figure shows a 10-fold increase in LSPR signal upon addition of PEG to a reaction medium containing 2.5 ng of heartworm antigen and anti-heartworm polyclonal antibody. [Figure 5] This figure shows the increased wavelength shift achieved by utilizing blue gold nanostars. In this figure, the antibody conjugated to blue nanostars provided a two-fold increase in wavelength shift rate when compared to the antibody conjugated to red colloidal gold. The experiment was set up using 2.5 ng of crude heartworm extract as the antigen, which was then reacted with either a commercially available conjugate prepared using red colloidal gold or the novel blue conjugate prepared according to the present invention. Polyethylene glycol was used in both types of conjugates. [Figure 6] The blue colloidal conjugate of chicken anti-Protein A reacts with Protein A over a wide concentration range, demonstrating that the reaction rate is linear over time. [Figure 7A] This shows a substantial improvement in analyte detection when LSPR technology is used in the solution phase (Figure 7A) compared to the solid phase (Figure 7B). Reactions were performed using a Nicoya chip in the solid phase or a Nicoya cuvette assembly in the liquid phase. The same Nicoya spectrometer was used in both experiments. The CRP response in the cuvette assay (solution phase) was approximately 6-8 times higher than in the solid phase. [Figure 7B] This shows a substantial improvement in analyte detection when LSPR technology is used in the solution phase (Figure 7A) compared to the solid phase (Figure 7B). Reactions were performed using a Nicoya chip in the solid phase or a Nicoya cuvette assembly in the liquid phase. The same Nicoya spectrometer was used in both experiments. The CRP response in the cuvette assay (solution phase) was approximately 6-8 times higher than in the solid phase. [Figure 8A] 1 shows the detection of TSH in solution phase using a colloidal gold-conjugated monoclonal anti-TSH antibody. [Figure 8B] 1 shows the detection of TSH in solution phase using a colloidal gold-conjugated monoclonal anti-TSH antibody. [Figure 9A] Comparison of TSH detection without PEG in the reaction medium. Two monoclonal antibodies (C1 and C6) were used as colloidal gold conjugates. The ratio of the two conjugates was varied, with the optimal signal obtained at 30% C1 and 70% C6. [Figure 9B] Comparison of TSH detection using PEG in the reaction medium. Two monoclonal antibodies (C1 and C6) were used as colloidal gold conjugates. The ratio of the two conjugates was varied, with the optimal signal obtained at 30% C1 and 70% C6. [Figure 9C] 1 shows a comparison of the TSH LSPR peak shift of detection when PEG is included in the reaction medium, demonstrating that PEG enhances analyte detection in the TSH assay at 500 seconds. [Figure 10]Optical spectra of gold / silver alloy nanoparticles synthesized as follows: gold chloride was reacted with CTAB before the addition of silver nitrate, followed by ascorbic acid, and finally sodium hydroxide. [Figure 11] 1 shows a linear blue shift in λmax with increasing silver content in the nanoalloy particles. [Figure 12] Immunoreactivity of mouse IgG conjugates with gold and gold / silver alloy nanoparticles was demonstrated. The conjugates were synthesized by passive adsorption of mouse IgG onto gold or alloy particles. They were tested for reactivity with Protein A stripped on lateral flow nitrocellulose strips. [Figure 13] The optical spectrum of gold / silver nanostars capped with CHAPS is shown. Gold chloride is added to CHAPS prior to the addition of silver nitrate and trisodium citrate. Nanostar formation is induced by the addition of a reducing solution containing ascorbic acid, CHAPS, and trisodium citrate. A red shift in λmax occurs up to a certain silver concentration, followed by a blue shift. Thus, nanostars of different sizes can be produced by varying the gold-to-silver ratio in the reaction medium. [Figure 14] 1 shows the peak shift to red upon binding of mouse IgG to gold-only nanoparticles prepared in the absence of silver. [Figure 15] 1 shows a larger peak shift to the red upon binding of mouse IgG to gold / silver nanostars prepared in the presence of approximately 37.5% silver. [Figure 16] 1 shows the highly favorable effect of maltodextrin on the LSPR signal. [Figure 17] We show how maltodextrin and BSA reduce sedimentation in the analytical rotor and maintain a high LSPR signal. [Figure 18] 1 shows pg / ml detection of TSH under various concentrations of BSA, PEG and maltodextrin. DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description of the Invention The present invention is based in part on the discovery that significant amplification in LSPR-based assays can be achieved by composite metallic nanostructure-labeled binding partners. Accordingly, the present invention provides methods for analyte detection that utilize multiple detection conjugates comprising composite metallic nanostructures linked to biomolecules.

[0023] The present invention overcomes the challenges of current immunoassays, ligand-receptor binding assays, nucleic acid-protein binding assays, or other specific binding partner assays, which generally require multiple steps and sophisticated equipment to perform such steps. The lack of sensitivity and complexity involved in performing such heterogeneous assays arises from the specific need to separate labeled from unlabeled specific binding partners. The present invention overcomes such limitations by performing all steps involved in the assay in a homogeneous format, in which separation of reacted and unreacted assay components is unnecessary, because the binding event alters the LSPR characteristics, which are measured in real time by any spectroscopic technique used by those skilled in the art of spectroscopy. The one-pot assay of the present invention, which does not require separation, uses plasmonic coupling and related effects to provide amplification of the final LSPR modulation signal.

[0024] As will be apparent to those skilled in the art, the present invention may be applied to the detection of various antigenic analytes, such as those associated with infectious diseases in both humans and animals, for example, antigens associated with infectious diseases and antibodies raised in response thereto. Beyond the detection of antigens and antibodies, 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 factors. Furthermore, using appropriate conjugates of metal nanoparticles with specific binding partners, RNA-RNA, RNA-DNA, DNA-DNA, or protein-nucleic acid interactions can be detected.

[0025] As provided herein, the invention describes the use of metal nanoparticles in solution (as opposed to being attached to a surface via chemical or physical deposition) to determine the binding of specific binding partners in a qualitative or quantitative manner. Changes in the characteristics of light that interacts with the unbound and binding partner-containing regions attached to the metal nanoparticles can be measured, allowing both qualitative and quantitative interactions between specific binding partners to be determined by appropriate detectors.

[0026] In a first aspect, the present application provides a method for detecting a target analyte in a sample. In some embodiments, the method includes mixing a sample with a plurality of detection conjugates comprising metal nanostructures linked to binding partners. In one embodiment, the method includes: a first detection conjugate and a second detection conjugate comprising metal nanostructures linked to binding partners that, when present, specifically bind to a target analyte in the sample and are capable of forming a complex between the first detection conjugate, the analyte, and the second detection conjugate; exposing the complex to a light source having a 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 the target analyte in the sample. In a representative embodiment, the metal nanostructures in the first detection conjugate and / or the second detection conjugate are composite metal nanostructures. In another representative embodiment, the mixing step is performed in the presence of a polymeric material selected from polyethylene glycol (PEG), polyvinylpyrrolidone, polyallylamine, polyethyleneimine, polylysine, polyacrylic acid, polyvinyl alcohol, and polyaspartic acid. In a preferred embodiment, the polymeric material is PEG. In yet another exemplary embodiment, the mixing step is carried out in the presence of a polysaccharide. In some embodiments, the polysaccharide is selected from maltodextrin, corn syrup, and polyglucose. In a preferred embodiment, the polysaccharide is maltodextrin. 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, casein, gelatin, ovalbumin, and gamma-globulin. In a preferred embodiment, the blocking agent is bovine serum albumin.

[0027] In various embodiments described herein, the methods of the present invention can be configured in sandwich assay formats, direct assay formats, indirect assay formats, and competitive and secondary labeling formats.

[0028] In some embodiments, the detection method is a sandwich assay. In such embodiments, the detection conjugate comprises a metallic nanostructure linked to a binding partner capable of specifically binding to a target analyte when present in a sample. For example, in one embodiment, a sandwich assay format method includes a first detection conjugate and a second detection conjugate, each comprising a metallic nanostructure linked to a binding partner that specifically binds to a target analyte in a sample when present and is capable of forming a complex between the first detection conjugate, the analyte, and the second detection conjugate. In a representative embodiment, the metallic nanostructure in the first detection conjugate and / or the second detection conjugate is a composite metallic nanostructure. The complex is exposed to a light source, and an optical signal is measured, where a change in the optical signal indicates the presence of the analyte in the sample. For example, when a sample containing a target analyte is mixed with the first and second detection conjugates, the target analyte binds to the binding partner of the detection conjugate, and a complex is formed between the first detection conjugate, the analyte, and the second detection conjugate. Such complexation brings the metallic nanostructures in the detection conjugate into close proximity to one another, i.e., plasmon-plasmon coupling. The amount of light absorbed, scattered, or transmitted by the metallic nanostructures is affected by the proximity of the metallic nanostructures in the complex, thus resulting in an enhanced shift in the peak absorption wavelength, which indicates the presence of the target analyte in the sample.

[0029] In other embodiments, the detection method is a competitive assay. In such embodiments, the first detection conjugate comprises a metal nanostructure bound to a target analyte of interest. Similar to the sandwich assay method, the second detection conjugate is capable of specifically binding 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 initial complexes, 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 will be due to the displacement of the metal nanostructure in the first detection conjugate from the second detection conjugate, which will proportionally reduce the wavelength shift at the peak absorption wavelength.

[0030] As described above, the methods of the present invention may utilize multiple detection conjugates. The detection conjugate comprises a metal nanostructure linked to a binding partner capable of specifically binding to a target analyte or another detection conjugate, depending on the assay configuration. For example, in embodiments in which the method is configured in a sandwich assay format, the detection conjugate comprises a metal nanostructure linked or conjugated to a binding partner capable of specifically binding 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 comprises a metal nanostructure linked or conjugated to a target analyte. In a representative embodiment, the metal nanostructure in the first detection conjugate and / or the second detection conjugate is a composite metal nanostructure.

[0031] In some embodiments, the detection conjugate comprises a binding partner capable of specifically binding to the target analyte. As used herein, "specific binding" refers to binding with high affinity, e.g., at least 10 -6It refers to binding to a target molecule with an affinity of M. In some embodiments, the binding partner is a biopolymer, including but not limited to, haptens and other small molecules, drugs, hormones, antibodies or fragments thereof (e.g., Fv, Fab, (Fab)2, single chain, 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.

[0032] In some embodiments, the detection conjugates, for example, the first detection conjugate and the second detection conjugate, are the same type of molecule, but preferably contain binding partners that bind to the target analyte at different positions.For example, the first detection conjugate and the second detection conjugate can both be antibodies that recognize the target analyte, but the epitope to which the first detection conjugate binds to the target analyte is different from the epitope to which the second detection conjugate binds to the target analyte, and ideally does not overlap.Thus, in certain embodiments, the first detection conjugate contains an antibody that recognizes a first epitope of the target analyte, and the second detection conjugate contains a different antibody that recognizes a second epitope of the target analyte.In various embodiments described herein, the first detection conjugate can contain a monoclonal antibody that recognizes a first epitope of the target analyte. In further embodiments, the second detection conjugate may comprise a monoclonal antibody that recognizes a second epitope of the target analyte that 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 comprise a polyclonal antibody. For example, the second detection conjugate may comprise a monoclonal antibody, while the first detection conjugate comprises a polyclonal antibody. In some embodiments, the first detection conjugate comprises a polyclonal antibody, and the second detection conjugate comprises a polyclonal antibody.

[0033] The metal nanostructures in the detection conjugate can be composed of a noble metal or a complex thereof. In some embodiments, the metal nanostructures in the detection conjugate may be composed of a transition metal or a complex thereof. In some embodiments, the metal nanostructures in the detection conjugate may include an alkali metal or a lanthanide in combination with a noble or transition metal. In certain embodiments, the metal nanostructures in the detection conjugate comprise a metal selected from gold, silver, copper, platinum, palladium, ruthenium, rhodium, osmium, iridium, titanium, chromium, cadmium, zinc, iron, cobalt, nickel, and complexes thereof. In one embodiment, the metal nanostructures are gold nanostructures. In another embodiment, the metal nanostructures are silver nanostructures.

[0034] In a preferred embodiment, the metal nanostructure in the detection conjugate is a composite metal nanostructure. "Composite metal nanostructure" refers to a nanostructure comprising at least two noble metals, transition metals, alkali metals, or lanthanides. Two or more metals may be mixed together as an alloy, or the two or more metals may be present in separate portions of the nanostructure. For example, one metal may form the core of the nanostructure, and the second metal may form the outer 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 specific 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. The composite metal nanostructures used in the methods of the present invention can include a number of different geometries, such as spherical nanoparticles, pyramidal nanoparticles, hexagonal nanoparticles, nanotubes, nanostars, nanoshells, nanorods, nanodots, nanoislands, nanowires, nanodisks, nanocubes, or combinations thereof. In an exemplary embodiment, the composite metal nanostructure is selected from nanostars and nanorods.

[0035] In certain embodiments, the composite metal nanostructures used in the methods of the invention are alloys of a first metal and a second metal. In some embodiments, the composite metal nanostructures used in the methods of the invention comprise a core of a first metal and a coating of a second metal. In certain embodiments, the composite metal nanostructures comprise a silver core and a gold coating. In other embodiments, the composite metal nanostructures comprise a copper core and a gold coating. In another embodiment, the core is silver and the coating is copper. In some embodiments, each composite metal nanostructure comprises a dielectric core (e.g., silicon dioxide, gold sulfide, titanium dioxide, silica, and polystyrene), a first coating of a first metal, and a second coating of a second metal. In one particular embodiment of the detection method, the core is silica, the first coating (i.e., the inner coating) is a silver coating, and the second coating (i.e., the outer coating). In another embodiment, the core is silica, the first coating (i.e., the inner coating) is a copper coating, and the second coating is a gold coating (i.e., the outer coating).

[0036] In some embodiments, following the coating process with the second metal, the core containing the first metal is dissolved to create a hollow structure composed of the second metal. For example, coating a silver core with gold nanoparticles creates a gold shell around the silver core, which is then dissolved or decomposed to form a hollow nanogold shell structure.

[0037] Metal nanostructures include spherical nanoparticles, as well as nanoplates and nanoshells. Nanoplates have a lateral dimension (e.g., edge length) that is greater than their thickness. Nanoplates include nanodisks, nanopolygons, nanohexagons, nanocubes, nanorings, nanostars, and nanoprisms. In some embodiments, metal nanostructures, including composite nanostructures, have a geometry selected from spherical nanoparticles, pyramidal nanoparticles, hexagonal nanoparticles, nanotubes, nanostars, nanoshells, nanorods, nanodots, nanoislands, nanowires, nanodisks, nanocubes, or combinations thereof. Other shapes, including irregular shapes, are possible. In certain embodiments, the diameter and shape of the metal nanostructures are not uniform, i.e., the metal nanostructures are a heterogeneous mixture of nanostructures of different shapes and diameters. In an exemplary embodiment, the metal nanostructures are nanostars. In another exemplary embodiment, the metal nanostructures are nanorods. In another exemplary embodiment, the metal nanostructures are composite nanospheres.

[0038] For spherical nanoparticles, suitable diameter ranges include about 5 nm to about 200 nm, about 10 nm to about 100 nm, and about 20 nm to about 60 nm. For nanorods, suitable diameter ranges include about 5 nm to about 50 nm, about 8 nm to about 30 nm, and about 10 nm to about 25 nm. Furthermore, for nanorods, suitable length ranges include about 25 nm to about 150 nm, about 40 nm to about 120 nm, and about 50 nm to about 100 nm. In some embodiments, the aspect ratio of the nanorods, i.e., length / diameter, is 2 to 10. For nanoplates, the edge length may be about 10 nm to about 800 nm, about 20 nm to about 500 nm, about 50 nm to about 200 nm, about 30 nm to about 100 nm, or about 10 nm to about 300 nm. The thickness of the nanoplates can range from about 1 to about 100 nm, from about 5 nm to about 80 nm, from about 10 nm to about 50 nm, or from about 5 nm to about 20 nm.

[0039] In some embodiments, the nanoplates have an aspect ratio greater than 2. The aspect ratio is the ratio of edge length to thickness. Preferably, the nanoplates have an aspect ratio of about 2 to about 25, about 3 to about 20, about 5 to about 10, about 2 to about 15, or about 10 to about 30.

[0040] Methods for conjugating molecules to metal nanostructures are known to those skilled in the art. Such methods include conjugation chemistries such as those involving 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), sulfo-NHS coupling, hydrophobic bonds, or thioether chemistry. In some embodiments, binding partners or target analytes can be linked to metal nanostructures via various chemical functionalities, including thiols, amines, dithiols, acyl phosphoramidites, azides, or alkynes. In some embodiments, the molecule can be indirectly linked to the metal nanostructure via a larger carrier molecule or protein. Such indirect coupling is particularly useful when the molecule is small, such as hormones, drugs, or other small molecules less than 10 kDa. Preferably, the carrier protein is incapable of specific interaction with the target analyte. In some embodiments, Protein A or Protein G or Protein A / G may be conjugated or linked to the nanoparticles.

[0041] In some embodiments, the metal utilized in the first detection conjugate can be the same as the metal from which the metal nanostructures in the second detection conjugate are fabricated. For example, in one embodiment, the first detection conjugate comprises gold nanostructures, and the second detection conjugate comprises gold nanostructures. In other embodiments, the metal utilized in the first detection conjugate is different from the metal used to fabricate the metal nanostructures in the second detection conjugate. For example, in some embodiments, the first detection conjugate comprises silver nanostructures, and the second detection conjugate comprises gold nanostructures. In other embodiments, the first detection conjugate comprises gold nanostructures, and the second detection conjugate comprises silver nanostructures. In certain embodiments, the first detection conjugate comprises gold nanostructures, and the second detection conjugate comprises composite nanostructures. In related embodiments, the composite nanostructures comprise gold-coated silver nanostructures. In other specific embodiments, the first detection conjugate comprises gold nanostructures, and the second detection conjugate comprises composite nanostructures comprising gold-coated copper nanostructures. In yet other embodiments, the first detection conjugate comprises a gold nanostructure and the second detection conjugate comprises a composite nanostructure comprising gold-coated magnetite nanostructures, hi yet other embodiments, the first detection conjugate comprises a gold nanostructure and the second detection conjugate comprises a composite nanostructure comprising gold and an alkali metal or lanthanide.

[0042] In certain embodiments, the diameter of the metallic nanostructures used to fabricate the first detection conjugate is similar to the diameter of the metallic nanostructures used in the second detection conjugate, and in such embodiments, matching the diameters of the two sets of nanostructures can provide an optimal wavelength shift in reflectance, emission, or scattering spectra.

[0043] In some embodiments, the reaction environment can be adjusted by appropriate buffers, ionic strength, and other promoters. In a preferred embodiment, the reaction environment includes polyethylene glycol (PEG), which can enhance the intensity of the LSPR signal, as described herein. Other similar polymeric materials, including but not limited to polyvinylpyrrolidone, polyallylamine, polyethyleneimine, polylysine, polyacrylic acid, polyvinyl alcohol, and polyaspartic acid, can also be used.

[0044] The present invention also provides analyte detection devices for utilizing the methods described herein to detect target analytes in a sample. Suitable analyte detection devices may include, but are not limited to, spectrophotometric cuvettes, analytical rotors, microwell plates, or flow chambers. As will be appreciated by those skilled in the art, fiber optic tips or transparent gels may also be utilized to implement the detection methods disclosed herein.

[0045] In certain embodiments, all components of the analyte detection device described herein are contained in a centrifugal rotor or disk. For example, the rotor or disk may contain one or more reaction chambers in which multiple detection conjugates are located. In some embodiments, the detection conjugates are present in the form of a lyophilized composition, such as lyophilized beads or pellets. In some embodiments, the analyte detection device includes a rotor or disk containing one or more reaction chambers, each reaction chamber containing multiple detection conjugates (e.g., a first detection conjugate and a second detection conjugate), the detection conjugates being linked to metal nanoparticles. Such devices provide a one-step analyte detection assay, whereby a test sample contacts the rotor or disk, and application of centrifugal force to the rotor or disk delivers the test sample to the reaction chamber, where it is mixed with the first detection conjugate and the second detection conjugate. In embodiments in which the rotor or disk contains two or more reaction chambers, the detection conjugates can be selected so that different analytes can be detected in each reaction chamber. These rotor format detection devices can be configured in a sandwich assay format, a direct competition format, or both if the rotor contains multiple reaction chambers.

[0046] Any of the types of metal nanostructures described herein can be used with these rotor format detection devices. In some embodiments, the first detection conjugate comprises gold nanostructures, and the metal nanostructures in the second detection conjugate are gold nanostructures. In other embodiments, the first detection conjugate comprises silver nanostructures, and the metal nanostructures in the second detection conjugate are gold nanostructures. In still other embodiments, the first detection conjugate comprises gold nanostructures, and the second detection conjugate comprises a composite nanostructure. For example, in one embodiment, the composite nanostructure is a gold-coated silver nanostructure. In another embodiment, the composite nanostructure is a gold-coated copper nanostructure.

[0047] The present invention also includes kits comprising the analyte detection devices of the present invention disclosed herein. In one embodiment, the kit includes multiple detection conjugates (e.g., a first detection conjugate and a second detection conjugate), wherein the detection conjugates are linked to metal nanoparticles. In some embodiments, one or more of the detection conjugates may be lyophilized, e.g., in the form of pellets or beads. In one embodiment, all of the detection conjugates are lyophilized. In further embodiments, the kit may include one or more additional reagents. In some embodiments, one or more additional reagents are provided in lyophilized form. In some embodiments, the kit may include a blocking agent, a sugar, a polymeric facilitating material, sodium chloride, and / or combinations thereof. A "blocking agent" is an agent that prevents association of the detectable agent and / or analyte with proteins present in a sample. Blocking agents are typically proteins themselves, and may include, but are not limited to, bovine serum albumin, 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 include maltodextrin and trehalose. In some embodiments, the polymeric facilitating material is PEG.

[0048] Kits of the invention may include instructions for using the device to detect an analyte in a test sample, devices or tools for collecting biological samples, and / or extraction buffers for obtaining samples from solid materials such as soil, food, and biological tissue.

[0049] As described herein, a test sample can be any type of liquid sample, including biological samples or extracts prepared from environmental or food samples. In a specific 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, feces, vaginal fluid, sperm, ocular lens fluid, mucous membranes, synovial fluid, peritoneal fluid, amniotic fluid, biopsy tissue, saliva, and cell lysates. Biological samples can be obtained from human or animal subjects suspected of having a disease state such as cancer, infectious disease (e.g., viral, bacterial, parasitic, or fungal infection), cardiovascular disease, metabolic disease, or autoimmune disease. Biological samples can also be obtained from healthy subjects (e.g., humans or animals) undergoing routine health checkups.

[0050] In some embodiments of the method, the test sample is mixed with a first detection conjugate, and the mixture is then contacted with a second detection conjugate. In certain embodiments, the sample, the first detection conjugate, and the second detection conjugate are contacted simultaneously. For example, contact of the sample with both reagents can occur simultaneously in a rotor format detection device described herein.

[0051] As described above, the present application relates to the use of composite nanomaterial-labeled partners in solution to determine the binding of specific binding partners in a qualitative or quantitative manner. The inventors surprisingly found that the sensitivity of solution-based assays is significantly enhanced when a polysaccharide, e.g., maltodextrin, is added to the solution compared to the addition of other sugars, such as sucrose, trehalose, or ficoll. In centrifugal rotor formats, low-speed centrifugation is required to deliver the sample into the analysis chamber. The addition of a polysaccharide, e.g., maltodextrin, to the solution is particularly effective in preventing aggregation and precipitation of composite nanomaterial-labeled partners, e.g., antibodies conjugated to gold-silver nanostars, during and after centrifugation. This improvement in sensitivity compared to other sugars, such as sucrose, trehalose, or ficoll, was unexpected. Increased assay sensitivity is achieved through reduced aggregation and precipitation. Thus, in some embodiments, the methods of the present invention are carried out in a solution containing a polysaccharide, e.g., maltodextrin, corn syrup, or polyglucose.

[0052] In one embodiment, the solution contains a polysaccharide at a final concentration of about 2% to about 20% weight / volume. In another embodiment, the solution contains a polysaccharide at a final concentration of about 4% to about 15% weight / volume. In yet another embodiment, the solution contains a polysaccharide at a final concentration of about 5% to about 10% weight / volume. In representative embodiments, the solution contains a polysaccharide at a final concentration of about 5%, 6%, 7%, 8%, 9%, or 10%, including all values ​​therebetween. In various embodiments described herein, the sensitivity of the assay may be improved when a polysaccharide is added to the solution compared to when the assay is performed in a solution containing another sugar, such as sucrose or ficoll. In a representative embodiment, the polysaccharide is maltodextrin.

[0053] In one embodiment, the solution contains a blocking agent at a final concentration of about 0.1% to about 20% weight / volume. In another embodiment, the solution contains a blocking agent at a final concentration of about 0.5% to about 10% weight / volume. In yet another embodiment, the solution contains a blocking agent at a final concentration of about 1% to about 5% weight / volume. In representative embodiments, the solution contains a blocking agent at a final concentration of about 1%, 2%, 3%, 4%, or 5%, including all values ​​therebetween. In various embodiments described herein, the sensitivity of an assay may be improved when a blocking agent is added to the solution compared to when the assay is performed in the absence of a blocking agent. In some embodiments, the blocking agent is selected from bovine serum albumin, casein, gelatin, ovalbumin, and gamma-globulin. In a representative embodiment, the blocking agent is bovine serum albumin.

[0054] In some embodiments, the solution comprises one or more of maltodextrin, trehalose, PEG, a blocking agent (e.g., bovine serum albumin), 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 will suspend upon addition of a liquid, e.g., water, saline solution, or liquid sample. For example, one or more of the solution components may be provided in a spectrophotometric cuvette or reaction chamber of an analytical rotor as beads that will suspend in solution after addition of the liquid.

[0055] In additional embodiments, the LSPR signal can be substantially increased by mixing the first and second detection conjugates with the analyte in the presence of a polymeric facilitating material selected from polyethylene glycol, polyvinylpyrrolidone, polyallylamine, polyethyleneimine, polylysine, polyacrylic acid, polyvinyl alcohol, and polyaspartic acid. In a representative embodiment, the polymeric material is polyethylene glycol (PEG). In one embodiment, the reaction mixture contains a polymeric 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 a polymeric 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 a polymeric 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 a polymeric material, e.g., PEG, at a final concentration of about 2 mg / mL to about 8 mg / mL. In representative embodiments, the reaction mixture contains a polymeric material, eg, PEG, at a final concentration of about 2, 3, 4, 5, 6, 7, or 8 mg / mL, including all values ​​therebetween.

[0056] The detection methods of the present invention may be used to determine the qualitative or quantitative amount of a target analyte. Such methods are particularly useful for determining the approximate amount of a target analyte in a sample, which can be used, inter alia, to diagnose certain medical conditions or evaluate the effectiveness of 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 the metal nanoparticles described herein for samples with known amounts 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 the value obtained for the standard curve. In some embodiments, determining the amount of a complex between a first reagent and a second reagent includes comparing the absorbance ratio and / or reaction rate from the test sample with the absorbance ratio and / or reaction rate from a sample with 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 value indicating either an abnormal or normal level of the target analyte.

[0057] The detection methods of the present invention provide highly sensitive techniques for detecting minute amounts of target analytes in a sample. In some embodiments, amplification of the plasmon resonance-based signal can be achieved by gold nanostructure conjugates, such that nanogram amounts of the target analyte can be detected in a sample. Thus, in one embodiment of this method, the presence of nanogram amounts of the target analyte is detected. In some embodiments, the plasmon resonance-based signal from a detection conjugate comprising gold nanoparticles can be amplified using a composite metal nanostructure detection conjugate. The use of gold-coated silver nanostructures conjugated to analyte-specific antibodies can enable the detection of picogram amounts of the target analyte. Thus, in some embodiments of this 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. Greater sensitivity can be achieved by altering the composition and / or shape of the composite metal nanostructure.

[0058] When incident light is applied to a metal nanostructure, the 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 nanostructures strongly absorb and scatter light in a specific wavelength range. For metal nanostructures containing noble or transition metals, this wavelength range lies in the ultraviolet-visible-infrared spectrum, depending on the specific composition of the nanostructure. Thus, light sources for applying electromagnetic energy suitable for use in the methods of the present invention can include any light source capable of applying wavelength ranges within the ultraviolet-visible or ultraviolet-visible-infrared spectrum, including arc lamps and lasers. In some embodiments, the light source may be equipped with a monochromator to apply specific wavelengths of light.

[0059] The optical properties of metal nanostructures depend on their size, shape, and composition. For example, solid gold nanoparticles have an absorption peak wavelength (λ) between about 515 nm and about 560 nm, depending on their particle size. max) A spherical gold nanoparticle with a diameter of 30 nm has an absorption maximum at about 520 nm, but max The λ shifts to longer wavelengths as particle diameter increases. Silver and copper particles have λ in the ultraviolet / blue or red region (e.g., from about 350 nm to about 500 nm). max However, as the particle diameter increases, λ max is shifted to longer wavelengths. The metal nanorods are max1 and the longitudinal direction λ max2 Alloys of different metals typically exhibit an absorption peak in the mid-range between the absorption peaks of the metals involved. For example, nanostructures comprising a 50 / 50 alloy of gold and silver have a λ of about 480 nm. max However, as the amount of gold increases, the absorption peak shifts to longer wavelengths. The sensitivity of the LSPR signal to changes in the refractive index of the local medium can be tuned by changing the shape or geometry of the nanostructure. For example, nanospherical particles (e.g., nanoprisms, nanorods, nanoshells, etc.) have increased LSPR sensitivity compared to spheres. In some embodiments, the optical properties (e.g., absorption / scattering at specific wavelengths) are tailored for specific applications by changing the diameter, shape, or composition of the metal nanostructures utilized in the detection conjugate.

[0060] The interaction between the incident light and the metallic nanostructures can be monitored as reflected or transmitted light. The amount of incident light that is absorbed or scattered can be measured as an absorption spectrum in reflection mode or an absorption spectrum in transmission mode. In some embodiments, the optical signal measured from the metallic nanostructures can be an optical reflection, absorbance spectrum, scattering spectrum, and / or emission spectrum.

[0061] Plasmon coupling between the metallic nanostructures in the detection conjugate resulting from complex formation between the binding partner and the target analyte results in a change in the localized surface plasmon resonance spectrum of the metallic nanostructure. For example, such changes can include an increase in optical absorbance, an increase in optical reflectance, and / or an increase in scattering and / or emission signal. In some embodiments, the change in the optical signal indicative of the presence of the target analyte in the sample includes a shift, an increase or decrease in optical scattering, or a combination of these features. In certain embodiments, the change in the optical signal indicative of the presence of the target analyte in the sample is a wavelength shift in the spectral peak. In certain other embodiments, the change in the optical signal indicative of the presence of the target analyte in the sample is a wavelength shift at a position other than the peak. For example, the change in the optical signal indicative of the presence of the target analyte in the sample can be a midpoint spectral wavelength shift, a wavelength-by-wavelength spectral wavelength shift, or a full spectral wavelength shift, such as a difference spectrum. In one embodiment, the wavelength shift in the optical spectral peak can be a red shift (e.g., a shift to longer wavelengths) within the spectral window of 200 nm to 1200 nm. In another embodiment, the wavelength shift in the optical spectrum peak can be a blue shift (e.g., a shift to shorter wavelengths) within the spectral window region of 200 nm to 1200 nm. The change in the optical signal can be measured at a specific time point after a set reaction time. Additionally, or alternatively, the change in the optical signal over the reaction period (e.g., rate determination) can be measured. Both types of measurements can be used for qualitative or quantitative analysis of target analytes.

[0062] Various means for measuring optical signals at different wavelengths and obtaining absorbance, 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), centrifugal analyzers from Olympus, Hitachi, etc.

[0063] The present invention also includes an assay complex comprising (i) a first detection conjugate comprising a metal nanostructure linked to a binding partner, (ii) a target analyte, and (iii) a second detection conjugate comprising a metal nanostructure linked to a binding partner, wherein the binding partner of the first detection conjugate binds to a first epitope on the target analyte and the binding partner of 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 within a reaction chamber in a centrifuge rotor or disk.

[0064] Any type of target analyte can be detected using the methods, devices, and assay complexes of the present invention, particularly those important in disease diagnosis. Target analytes can include, but are not limited to, proteins, enzymes, antigens, antibodies, peptides, nucleic acids (RNA, DNA, mRNA, miRNA), hormones, glycoproteins, polysaccharides, toxins, viruses, virus particles, drug molecules, haptens, or chemicals. 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 particular physiological or pathological state.

[0065] In certain embodiments, the target analyte is a pathogenic antigen or an antibody to a pathogenic antigen. For example, the pathogenic antigen can be a viral antigen (e.g., feline leukemia virus, canine parvovirus, foot-and-mouth disease virus, influenza virus, hepatitis A, B, or C virus, HIV virus, human papillomavirus, Epstein-Barr virus, rabies virus, etc.), a bacterial antigen (e.g., Ehrlichia, Borrelia, Anaplasma, Salmonella, Bacillus, Rickettsia, etc.), a fungal antigen, or a parasitic antigen (e.g., Heartworm, Giardia lamblia, Plasmodium falciparum, African trypanosomiasis, Trypanosoma brucei, etc.). In certain embodiments, the bacterial antigen can be Ehrlichia canis, Ehrlichia chafeensis, Ehrlichia ewingii, Borrelia burgdorferi, Anaplasma platys, Anaplasma phagocytophilum, Salmonella enterica, Bacillus anthracis, and Rickettsia rickettsii. In other embodiments, the target analyte is a disease-associated antigen or an antibody to a disease-associated antigen.Disease-associated antigens include, but are not limited to, cancer-associated antigens or markers (e.g., PSA, AFP, CA125, CA15-3, CA19-9, CEA, NY-ESO-1, MUC1, GM3, GD2, ERBB2, etc.), cardiovascular disease-associated antigens or markers (e.g., troponin, C-reactive protein, brain natriuretic peptide, CKMB, fatty acid-binding protein, etc.), metabolism-associated antigens or markers (e.g., thyroid-stimulating hormone, thyroxine, leptin, insulin), or autoimmune disease-associated 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-associated antigen or marker (e.g., fetal antigen, human chorionic gonadotropin).

[0066] The present invention also provides a method for preparing composite metal nanostructures. In one embodiment, the method includes preparing a first solution containing a mixture of a polymer and chloroauric acid, preparing a second solution containing silver and copper nanostructures, and incubating the first solution with the second solution for a period of time, whereby the resulting mixture contains gold-coated silver nanostructures or gold-coated copper nanostructures. The resulting mixture preferably has a peak absorbance between about 515 nm and about 670 nm or between about 520 nm and about 560 nm. In one embodiment, the resulting mixture has a peak absorbance between about 530 nm and about 545 nm. In another embodiment, the method includes preparing a first solution containing a mixture of a detergent, such as CHAPS, and chloroauric acid, and a solution containing a silver or copper salt, and incubating the first solution with a second solution containing a reducing agent, such as ascorbic acid, to induce the formation of composite nanostructures. The size and shape of the nanostructures can be varied by changing the ratio of metals used, the concentration of detergent, and finally the amount of ascorbic acid used.

[0067] The polymer used in preparing the first solution can be any one of polyvinylpyrrolidone, polyvinyl alcohol, polyacrylate, polyethylene glycol, polyethyleneimine, polyaspartic acid, polyglutamic acid, various gums, gelatin, or a mixed polymer containing any of the above. In one specific embodiment, the polymer is polyvinylpyrrolidone. By varying the molecular weight of the polymer, various types of coating nanostructures can be obtained. Suitable molecular weight ranges for the polymer include about 5,000 daltons to about 150,000 daltons, about 10,000 daltons to about 100,000 daltons, and about 20,000 daltons to about 80,000 daltons. In some embodiments, the polymer has a molecular weight of less than 50,000 daltons. In other embodiments, the polymer has a molecular weight of less than 20,000 daltons. In certain embodiments, the polymer has a molecular weight of about 10,000 daltons.

[0068] The characteristics of the gold coating can be controlled by adjusting the polymer:chloroauric acid concentration ratio. For example, the polymer:chloroauric acid concentration ratio can be about 100:1 to about 1:100, about 2:1 to about 5:1, or about 1.5:1 to about 8:1. In some embodiments, the polymer:chloroauric acid concentration ratio is 1:1. Suitable polymer concentrations include, but are not limited to, about 0.1% to about 20% wt / wt in water or ethanol. Suitable chloroauric acid concentrations include, but are not limited to, about 0.001 M to about 1.0 M, about 0.010 M to about 0.500 M, and about 0.050 M to about 0.100 M.

[0069] Coating efficiency and thickness can also be affected by the pH and halide content of the coating solution (i.e., the first solution). In certain embodiments, the pH of the solution is maintained in the range of about 3 to about 14. The halide content of the solution is less than 150 mM in some embodiments. In other embodiments, the halide content of the solution is in the range of about 0 to about 50 mM.

[0070] Methods for preparing solutions of silver and copper nanostructures are known to those skilled in the art. For example, the second solution containing silver or copper nanostructures can be prepared by any of the methods described in U.S. Patent Application Publication No. 2012 / 0101007, U.S. Patent Application Publication No. 2014 / 0105982, or U.S. Patent Application Publication No. 2013 / 0230717, each of which is incorporated herein by reference in its entirety. In one embodiment, the second solution containing silver or copper nanostructures is prepared by mixing a silver or copper source with a reducing agent. Suitable silver sources include silver salts such as silver nitrate. Suitable copper sources include copper(II) sulfate, copper(II) chloride, copper(II) hydroxide and copper(II) nitrate, copper(II) acetate, and copper(II) trifluoroacetate. Reducing agents that can react with silver or copper sources to form nanostructures can include alkaline solutions (e.g., pH greater than 7.5) of glucose, ascorbic acid, sodium borohydride, and polymers such as PVP. In certain embodiments, the reducing agent is ascorbic acid. The desired shape and optical spectral peaks of the silver or copper nanostructures can be achieved by adjusting the ratio or concentration of the reactants, as known to those skilled in the art. By way of example only, high concentrations of reducing agent can result in pentagonal and bipyramidal nanostructures, while low concentrations of reducing agent can result in elongated nanowires or tubes. Depending on the specific shape of the nanostructures, the second solution containing silver or copper nanostructures may have a peak absorbance between about 540 nm and about 1000 nm, between about 600 nm and about 700 nm, between about 630 nm and about 680 nm, between about 750 nm and about 850 nm, between about 900 nm and about 940 nm, between about 580 nm and about 620 nm, or between about 550 nm and about 750 nm. In certain embodiments, the second solution containing silver nanostructures has a peak absorbance between about 600 nm (i.e., between 595 nm and 605 nm, inclusive). In some embodiments, the second solution containing copper nanostructures has a peak absorbance between about 585 nm (i.e., between 580 nm and 590 nm, inclusive).In some embodiments, the peak absorbance of a solution containing copper nanostructures is higher (ie, red-shifted) than the peak absorbance of a solution containing silver nanostructures of similar size and shape.

[0071] In some embodiments, the incubation time of the first solution with the second solution is at least 12 hours. In other embodiments, the incubation time of the first solution with the second solution is longer than 24 hours, preferably longer than 48 hours, and more preferably at least 72 hours. Changes in the peak absorbance of the reaction mixture can be monitored during the incubation period to adjust the incubation time accordingly. For example, a shift in peak absorbance to shorter wavelengths, e.g., the 520-550 nm region, can indicate stability of the gold-coated nanostructures. In certain embodiments, the stability of the resulting nanostructures to sodium chloride (e.g., 0.25-1 M) is used to indicate adequate coating of the nanostructures. CTAB-coated particles, such as nanorods, are resistant to sodium chloride.

[0072] In certain embodiments, the present invention provides a method for synthesizing nanostructures having an optical density greater than about 50 / mL. In one embodiment, the method includes mixing a polymer described herein with chloroauric acid, stirring the mixture at a set temperature for a first period of time, adding ascorbic acid to the mixture, and incubating the mixture for a second period of time. The size and shape of the nanostructures are determined by the concentration ratio of the polymer to chloroauric acid and the temperature and time of incubation. The concentrations of the polymer and chloroauric acid can be within the ranges described above. The temperature can be adjusted based on the desired size and shape of the nanostructures, but may range from about 4°C to about 100°C. Similarly, the incubation period (i.e., the first period of time) can be adjusted based on the desired properties of the nanostructures, but may range from about 15 minutes to 1 day.

[0073] In some embodiments, after the first incubation period, about 0.1 to 1 part (e.g., about 1 to 5 M) ascorbic acid is added to the mixture. The second incubation period after the addition of ascorbic acid can be about 1 to about 24 hours. Without being bound by theory, the addition of ascorbic acid results in a substantial increase in the amount of nanostructures produced.

[0074] In certain embodiments, the method further comprises adding or doping the mixture with about 1 to about 100 parts gold chloride (e.g., about 0.001M to 1M) or silver nitrate (e.g., about 0.001M to 1M) or other metal (e.g., a noble metal, transition metal, alkali metal, or lanthanide). This doping step can further increase the resonance strength of the resulting nanostructures. In some embodiments, the gold chloride, silver nitrate, or other metal is added to the mixture before ascorbic acid is added to the reaction. In other embodiments, the gold chloride, silver nitrate, or other metal is added to the mixture after the addition of ascorbic acid. The order of addition of the metal and ascorbic acid may be adjusted to tailor the resulting nanostructures to a desired shape and diameter.

[0075] In some embodiments, the present disclosure provides methods for synthesizing composite nanoparticles. In certain embodiments, silver / gold nanoparticles are synthesized in a single container by adding, sequentially and with vigorous mixing, predetermined amounts of the following reagents: (1) a surfactant (e.g., ionic [anionic, cationic, or zwitterionic] or nonionic) or capping agent, such as 3-((3-cholamidopropyl)dimethylamino)-1-propanesulfonate (CHAPS), SDS, Tween, Triton, or any sulfobetaine detergent; (2) gold chloride; (3) water; (4) silver nitrate; (5) trisodium citrate; and finally (6) ascorbic acid, which is added to initiate nanoparticle formation. In other embodiments, nanoparticles are synthesized in a single container by adding predetermined amounts of the following in the following order: (1) a surfactant or capping agent, such as CHAPS, SDS, Tween, Triton, CTAB, or any sulfobetaine detergent; (2) gold chloride; (3) silver nitrate; (4) trisodium citrate; (5) water; and (6) a reductant. In some embodiments, the reductant is composed of CHAPS, ascorbic acid, trisodium citrate, and water. In further embodiments, the reductant is composed 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, about 1 mL of a 1% (wt / wt) aqueous CHAPS solution is mixed sequentially with about 0.25 mL of 0.1 M gold chloride, about 0.5 mL of 0.02 M silver nitrate, about 0.05 mL of 1 M trisodium citrate, about 6.2 mL of water, and about 2 mL of reductant. Varying the concentrations of various active ingredients, such as metal salts, capping agents, and reductants, as well as the pH of the solution, results in different particle types (e.g., nanospheres, nanostars, or nanorods) and different compositions of nanoparticles.

[0076] In some embodiments, nanostars are formed by sequentially mixing water, cetyltrimethylammonium bromide (CTAB), gold chloride, ascorbic acid, and preformed 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 geometry of the nanoparticles and, therefore, their optical spectra.

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

[0078] In some embodiments, the diameter, and therefore the optical properties, can be altered by varying the concentrations of surfactants or capping agents, ascorbic acid, trisodium citrate, gold chloride, and / or silver nitrate. The diameter of the synthesized nanostars increases with increasing silver content up to a certain point, then decreases. These changes are reflected in the LSPR peak of the synthesized nanostars, where the peak red-shifts as the silver / gold ratio increases, but then blue-shifts at a gold:silver molar ratio of 5:2. The final concentration of the selected detergent in the reaction mixture can be varied from 0.05 to 5%, with higher detergent concentrations resulting in smaller particles predominating. Increasing the concentration of ascorbic acid produces smaller nanostars, with the final ascorbic acid concentration varying from 0.05 to 0.2M. Similarly, increasing the concentration of trisodium citrate from 10 mM to 100 mM decreases the diameter of the nanostars.

[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 by sequentially mixing water, CTAB, gold chloride (0.5 mM to 5 mM), silver nitrate (20% to 80% gold), ascorbic acid (10 mM to 200 mM) or a reductant containing ascorbic acid, trisodium citrate, and CHAPS, and NaOH (50% to 200% ascorbic acid). In further embodiments, nanoalloys are formed by mixing approximately 0.825 ml of water, approximately 0.1 ml of 20% CTAB prepared in isopropanol, approximately 0.025 ml of 0.1 M gold chloride, approximately 0.005 to 0.025 ml of 0.1 M silver nitrate, approximately 0.05 ml of 1 M ascorbic acid, and approximately 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 a higher content of synthesized nanostars. Acidic pH favors the formation of nanorods, and decreasing pH results in higher aspect ratios.

[0080] This invention is further illustrated by the following additional examples, which should not be construed as limiting. Those of skill in the art, in light of the present disclosure, will appreciate that many changes can be made to the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0081] All patent and non-patent literature referenced throughout this disclosure is hereby incorporated by reference in its entirety for all purposes. [Example]

[0082] Example 1. Direct assay utilizing amplification of LSPR signal In this example, a blank reaction is constructed by adding a solution containing a colloidal conjugate of an anti-heartworm polyclonal antibody, an appropriate dispersion medium such as phosphate-buffered saline, and a sample lacking heartworm antigen. The spectral change is recorded over a period of time. A positive control set is then constructed by adding a known amount of heartworm antigen to the reaction solution used in the blank solution. Alternatively, a blank reaction is recorded, and then a known amount of heartworm antigen is added to prepare a calibration curve relating the shift in the spectral scan to the concentration of the antigen. This calibration curve is then used to calculate the amount of heartworm antigen in the unknown sample. The shift in the spectral scan is determined by: (i) λ max (2) the difference spectrum between positive and negative samples, or (3) the derivative spectrum.

[0083] Example 2. Sandwich assay utilizing LSPR signal amplification Sandwich assays are most appropriate when the analyte exhibits at least two distinct binding sites (antigen epitopes), each of which binds to a specific binding partner. Thus, in this example, an antibody directed against one epitope of CRP is immobilized on gold and / or silver nanoparticles, and a second antibody directed against a non-overlapping epitope is labeled with colloidal gold and / or silver. This configuration allows for the measurement of CRP antigen, since the amount of CRP antigen in the sample determines the extent of the spectral change. Spectral changes are observed even when the second antibody is unlabeled, but the spectral changes are several orders of magnitude lower. The metal composition of the nanoparticles can be varied to optimize the reaction conditions.

[0084] Example 3. Running the assay in a rotor Direct competitive assay or sandwich assay can be carried out in a centrifugal rotor, such as the rotor described in U.S. Patent No. 5,061,381, U.S. Patent No. 5,122,284, U.S. Patent No. 5,186,844, U.S. Patent No. 5,304,348, U.S. Patent No. 5,457,053 and U.S. Patent No. 5,693,233.In this case, the nanoparticle conjugate of two paired monoclonal antibodies or a polyclonal antibody mixture that binds to two or more epitopes is added as lyophilized beads.Solution phase LSPR assay works with both monoclonal and polyclonal antibodies.

[0085] Example 4. Enhancement of LSPR signal with polyethylene glycol or similar polymers The data presented in Figures 4 and 9A-9C show that the LSPR signal increases substantially in the presence of polyethylene glycol. PEGs with different molecular weights may be used at optimized concentrations to obtain the desired selectivity in a given assay. PEG may be substituted with polyvinylpyrrolidone or similar polymeric materials to obtain optimized reaction conditions for a given nanoparticle and / or specific binding partner combination.

[0086] Example 5. Improving assay sensitivity by adding maltodextrin Multiple experiments were performed to determine the influence of various sugars and other agents to minimize precipitation effects and maintain the LSPR signal. As shown in Figure 16, maltodextrin surprisingly increased the signal, which was further improved by the presence of BSA. In contrast, when trehalose, sorbitol, or cyclodextrin was added, the intensity of the LSPR signal was not as strong (data not shown). In addition, the addition of maltodextrin and BSA also resolved the precipitation problem (Figure 17). Approximately 50 pg / ml of TSH was detectable using specific amounts of PEG, BSA, and maltodextrin (Figure 18).

[0087] Example 6. Synthesis of gold nanostars and gold-silver alloy particles and their uses A novel method was utilized to synthesize nanomaterials for use in plasmonic assays, such as the solution-phase plasmonic assay described herein.

[0088] CHAPS-coated nanostars or CTAB-coated nanostars were prepared using the following method. For CHAPS-coated nanostars, 1 ml of a 1% (wt / wt) aqueous solution of CHAPS (3-((3-cholamidopropyl)dimethylamino)-1-propanesulfonate) was prepared in a suitable container. 0.25 ml of 0.1 M gold chloride, 0.5 ml of 0.02 M silver nitrate, 0.05 ml of 1 M trisodium citrate, 6.2 ml of water, and finally 2 ml of the reductant (200 mg CHAPS, 4 g ascorbic acid, 117.6 mg trisodium citrate, 15.68 g water) were continuously stirred in the container and thoroughly mixed for at least 1 hour. After dilution to 1:20 in water, optical spectra were read. In some embodiments, the size, and therefore the optical properties, can be altered by varying the concentrations of CHAPS, ascorbic acid, trisodium citrate, gold chloride, and silver nitrate.

[0089] For CTAB-coated nanostars, cetyltrimethylammonium bromide (CTAB) was dissolved in isopropanol at a concentration of 20% (wt / wt). All other reagents were aqueous. Nanostar formation was induced by sequentially mixing 0.825 ml of water, 0.1 ml of 20% CTAB, 0.025 ml of 0.1 M gold chloride, 0.05 ml of 1 M ascorbic acid, and finally 0.05 ml of preformed gold nanosphere seeds. In some embodiments, the diameter, and therefore the optical properties, can be modified by changing the concentrations of the components. When new seeds are used to seed the nanorods, nanorods were fabricated from an aqueous solution of CTAB at 30 °C. A CTAB solution prepared in isopropanol can be used at room temperature, but it favors the synthesis of nanostars over nanorods.

[0090] Gold-silver nanoalloys were synthesized under alkaline reduction conditions by mixing CTAB dissolved in isopropanol with gold chloride and silver nitrate. Nanoalloy formation was induced by sequentially mixing water (giving a total reaction volume of 1 ml), 0.2 ml of 20% CTAB (in isopropanol), 0.025 ml of 0.1 M gold chloride, 0-0.05 ml of 0.02 M silver nitrate, 0.02 ml of a reductant containing ascorbic acid, CHAPS, and trisodium citrate, and finally 0.05 ml of 1 M NaOH. In some embodiments, the reductant contains CHAPS, trisodium citrate, and ascorbic acid. The acidic pH favors the production of nanostars and nanorods, depending on the age of the seeds.

[0091] The optical spectra of gold / silver alloy nanoparticles synthesized by reacting gold chloride with CTAB before the addition of silver nitrate, followed by ascorbic acid, and finally sodium hydroxide are provided in Figure 10. As shown in Figure 11, increasing the silver content in the nanoalloy particles leads to an increase in λ max A linear blue shift is obtained in , and a red shift is obtained with increasing gold content in the nanoalloy particles.

[0092] To prepare CHAPS-capped gold / silver nanostars, gold chloride was added to CHAPS prior to the addition of silver nitrate and trisodium citrate. Nanostar formation was induced by the addition of a reducing solution containing ascorbic acid, CHAPS, and trisodium citrate. The optical spectrum of the CHAPS-capped gold / silver nanostars is shown in Figure 13. λ max was red-shifted up to a certain silver concentration and then blue-shifted thereafter. Thus, by varying the gold-to-silver ratio in the reaction medium, nanostars of different diameters were produced.

[0093] Antibodies were loaded onto nanostars or nanoalloys using the following method: An appropriate volume of nanostar or nanoalloy solution was centrifuged at an appropriate g-force. The supernatant was carefully removed and replaced with an equal volume of 1% CHAPS. Diluted 1:20 in water and λ max The spectra and OD were read. In a 2ml microtube, water, 0.5M borate (pH 9.2), 1% CHAPS, the washed nanostars / nanoalloys from step 1, and the desired antibody were added in that order. The volumes of the solutions were adjusted so that the final CHAPS concentration was 0.1%, the borate was 0.05-0.1M, the particle OD was 2 per ml, and the antibody concentration was 1-10 μg / OD. After a 5-10 minute incubation, an equal volume of conjugate diluent CG (3x PBS, 1% BSA, 2% CHAPS, and 0.1% sodium azide) was added, mixed thoroughly, and centrifuged at 5000g for 10 minutes. The supernatant was removed, and the conjugate was resuspended in conjugate diluent CG to its original volume. The centrifugation step was repeated once, and the final pellet was resuspended in 1 / 5 of the original volume of conjugate diluent CG. The OD spectrum of a 1:10 dilution was read. At this point, the conjugate is ready for treatment or use in immunoassays. As known in the literature, antibodies are attached to as-synthesized nanoparticles by adding the antibody to a diluted solution of the nanoparticles.

[0094] Figure 12 shows the immunoreactivity of mouse IgG conjugates with gold and gold / silver (50% / 50% equimolar) alloy nanoparticles. Conjugates synthesized by passive adsorption of mouse IgG onto gold or alloy particles were tested for reactivity with protein A stripped on lateral flow nitrocellulose strips.

[0095] The gold / silver nanostars exhibited a larger peak shift to the red upon binding of mouse IgG compared to gold-only nanoparticles prepared in the absence of silver. The peak shift to the red upon binding of mouse IgG to gold-only nanoparticles is shown in Figure 14, and the larger peak shift to the red upon binding of mouse IgG to gold / silver nanostars prepared in the presence of approximately 37.5% silver is shown in Figure 15.

[0096] In some embodiments, centrifugation conditions, ionic strength, pH, and antibody-to-nanomaterial ratios may be optimized for each type of antibody-nanomaterial combination. Alternative methods for conjugation using covalent bonds are well known to those skilled in the art.

[0097] It is understood that the disclosed invention is not limited to the particular methodology, protocols, and materials described, which may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0098] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. 1. A kit comprising an analyte detection device and one or more instructions for using the device to detect an analyte in a test sample, the analyte detection device comprising: a first detection conjugate comprising a metallic nanostructure linked to a binding partner capable of specifically binding to a target analyte, if present, in the sample; a second detection conjugate comprising a metallic nanostructure linked to a binding partner capable of specifically binding to the target analyte, if present in the sample; Including, the metallic nanostructures of each of the first detection conjugate and the second detection conjugate are comprised of an alloy of gold and silver; kit.

2. 10. The kit of claim 1, wherein the kit further comprises one or more extraction buffers for obtaining a test sample from a solid material.

3. 10. The kit of claim 1, wherein at least one of the first detection conjugate or the second detection conjugate is lyophilized.

4. The kit of claim 1 , wherein the analyte detection device comprises a rotor containing one or more reaction chambers.

5. The kit of claim 1 , wherein the analyte detection device is configured to receive a test sample.

6. 10. The kit of claim 1, wherein the kit comprises one or more additional reagents selected from the group consisting of a blocking agent, a sugar, a polymeric facilitating material, and sodium chloride.

7. 7. The kit of claim 6, wherein the sugar comprises maltodextrin, corn syrup, polyglucose, or trehalose.

8. The kit of claim 6 , wherein the blocking agent comprises bovine serum albumin, casein, gelatin, or ovalbumin.

9. The kit of claim 6 , wherein the polymeric facilitating material comprises polyethylene glycol (PEG).

10. The kit of claim 1 , wherein the test sample is mixed with the first detection conjugate in the analyte detection device.

11. 10. The kit of claim 1, wherein the test sample is mixed with the first detection conjugate and the second detection conjugate in the analyte detection device.

12. 10. The kit of claim 1, wherein the analyte detection device is configured to expose a complex of the first detection conjugate, the analyte, and the second detection conjugate to a light source in a wavelength range within the ultraviolet-visible-infrared spectrum.

13. 13. The kit of claim 12, wherein the analyte detection device is further configured to measure an optical signal from the complex, wherein a change in the optical signal indicates the presence of a target analyte in the test sample.

14. 10. The kit of claim 1, wherein the presence of nanogram amounts of a target analyte in the test sample is detected.

15. The kit of claim 1 , wherein the binding partner of each of the first detection conjugate and the second detection conjugate is a biopolymer.

16. The kit of claim 15, wherein the biopolymer is selected from an antibody or fragment thereof, an antigen, a receptor, a ligand, a polynucleotide, an aptamer, a polypeptide, a polysaccharide, a lipopolysaccharide, a glycopeptide, a lipoprotein, or a nucleoprotein.

17. 17. The kit of claim 16, wherein the first detection conjugate and the second detection conjugate each comprise a binding partner that is an antibody.

18. 18. The kit of claim 17, wherein the antibodies of the first detection conjugate and the second detection conjugate bind to different epitopes of the target analyte.

19. 17. The kit of claim 16, wherein the target analyte is a pathogenic antigen or an antibody to a pathogenic antigen.

20. The pathogenic antigen is a bacterial antigen, and the bacterial antigen is selected from the group consisting of Ehrlichia canis, Ehrlichia chafeensis, Ehrlichia ewingii, Borrelia burgdorferi, Anaplasma platys, Anaplasma phagocytophilum, Salmonella enterica, Bacillus anthracis, and Rickettsia rickettsii.

20. The kit of claim 19, wherein the .

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