阅读说明：本技术 等离子体特异性结合配偶体测定中的信号放大 (Signal amplification in plasma specific binding partner assays ) 是由 R·K·梅拉 V·蒋 K·P·阿伦 A·克雷尔 于 2015-08-13 设计创作，主要内容包括：本发明涉及分析物检测装置和使用此类装置检测样品中的极少量目标分析物的方法。具体地,本发明提供一种分析物检测装置,其包括缀合至分析物结合配偶体的多个复合金属纳米结构和含有其上固定有多个捕获分子的金属纳米层的表面。还描述了制备复合纳米结构的方法。(The present invention relates to analyte detection devices and methods of using such devices to detect minute quantities of target analytes in a sample. In particular, the present invention provides an analyte detection device comprising a plurality of composite metal nanostructures conjugated to an analyte binding partner and a surface comprising a metal nanolayer having a plurality of capture molecules immobilized thereon. Methods of making the composite nanostructures are also described.)

1. An analyte detection device, comprising:

a plurality of detection conjugates, wherein the conjugates comprise composite metallic nanostructures coupled to binding partners capable of specifically binding to a target analyte;

a surface comprising a metallic nanolayer; and

a plurality of capture molecules, wherein the capture molecules are immobilized on the metal nanolayer and are capable of specifically binding to the target analyte.

2. An analyte detection device, comprising:

a plurality of detection conjugates, wherein the conjugates comprise composite metal nanostructures coupled to a target analyte;

a surface comprising a metallic nanolayer; and

a plurality of capture molecules, wherein the capture molecules are immobilized on the metal nanolayer and are capable of specifically binding to the target analyte.

3. The analyte detection device of claim 1 or 2, wherein the composite metallic nanostructure comprises at least two metals selected from gold, silver, copper, platinum, palladium, cadmium, iron, nickel, and zinc.

4. The analyte detection device of claim 1 or 2, wherein each of the composite metallic nanostructures comprises a core of a first metal and a coating of a second metal.

5. The analyte detection device of claim 4, wherein each of the composite metallic nanostructures comprises a gold-plated layer and a silver core.

6. The analyte detection device of claim 1 or 2, wherein each of the composite metal nanostructures is an alloy of a first metal and a second metal.

7. The analyte detection device of claim 1 or 2, wherein the composite metal nanostructure is a spherical nanoparticle and has a diameter of about 5nm to about 200 nm.

8. The analyte detection device of claim 1 or 2, wherein the composite metal nanostructure is a spherical nanoparticle and has a diameter of about 10nm to about 100 nm.

9. The analyte detection device of claim 1 or 2, wherein the composite metallic nanostructure is a nanoplatelet having an edge length of from about 10nm to about 800nm and a thickness of from about 1nm to about 100 nm.

10. The analyte detection device of claim 1 or 2, wherein the plurality of detection conjugates are in the form of lyophilized pellets or beads.

11. The analyte detection device of claim 1 or 2, wherein the surface is a wall, a lid, and/or a bottom of a chip, well, bead, or cuvette.

12. The analyte detection device of claim 1 or 2, wherein the metallic nanolayer is a metallic thin film.

13. The analyte detection device of claim 12, wherein the metallic thin film comprises gold, silver, copper, platinum, palladium, cadmium, zinc, or a composite thereof.

14. The analyte detection device of claim 12, wherein the metallic thin film comprises gold.

15. The analyte detection device of claim 1 or 2, wherein the metallic nanolayer comprises a plurality of metallic nanostructures immobilized on the surface.

16. The analyte detection device of claim 15, wherein the plurality of metallic nanostructures comprise gold, silver, copper, platinum, palladium, cadmium, zinc, or a composite thereof.

17. The analyte detection device of claim 15, wherein the plurality of metal nanostructures are gold nanostructures.

18. The analyte detection device of claim 1 or 2, wherein the composite nanostructure has a geometry selected from the group consisting of: spherical nanoparticles, pyramidal nanoparticles, hexagonal nanoparticles, nanoshells, nanotubes, nanorods, nanodots, nanodomains, nanowires, or combinations thereof.

19. The analyte detection device of claim 1, wherein the binding partner and/or capture molecule is an antibody, antigen, polypeptide, polynucleotide, nucleoprotein, aptamer, ligand, receptor, or hapten.

20. The analyte detection device of claim 1, wherein the binding partner is an antibody that recognizes a first epitope of the target analyte and the capture molecule is a different antibody that recognizes a second epitope of the target analyte.

21. The analyte detection device of claim 2, wherein the capture molecule is an antibody, antigen, polypeptide, polynucleotide, nucleoprotein, aptamer, ligand, receptor, or hapten.

22. The analyte detection device of claim 1 or 2, wherein the target analyte is a marker or antigen associated with an infectious disease, physiological state, or pathological condition.

23. The analyte detection device of claim 1 or 2, wherein the target analyte is canine heartworm, feline leukemia virus, canine parvovirus, C-reactive protein, giardia piriformis, ehrlichia antigen or antibody, borrelia antigen or antibody, anaplasma antigen or antibody, cancer antigen, cardiac marker antigen, thyroid stimulating hormone, thyroxine, troponin, or brain natriuretic peptide.

24. A method of detecting a target analyte in a sample, comprising:

mixing the sample with a plurality of detection conjugates, wherein the conjugates comprise composite metallic nanostructures coupled to binding partners capable of specifically binding to the target analyte present in the sample to form analyte-detection conjugate complexes;

contacting the mixture with a surface comprising a metal nanolayer on which a plurality of capture molecules are immobilized and capable of specifically binding to the target analyte present in the sample;

exposing the surface to a light source at a range of wavelengths within the ultraviolet-visible-infrared spectrum; and

measuring an optical signal from the surface, wherein a change in the optical signal indicates the presence of the target analyte in the sample.

25. The method of claim 24, wherein the optical signal is a reflectance, absorption spectrum, scattering spectrum, or emission spectrum.

26. The method of claim 24, wherein the change in the optical signal comprises a spectral peak wavelength shift.

27. The method of claim 24, wherein the presence of nanogram quantities of the target analyte is detected.

28. The method of claim 24, wherein picogram quantities of the target analyte are detected for the presence.

29. The method of claim 24, wherein the presence of femtogram quantities of the target analyte is detected.

30. The method of claim 24, wherein the surfaces are walls and bottoms of test tubes incorporated in a centrifuge rotor.

31. The method of claim 24, wherein the composite metallic nanostructure comprises at least two metals selected from the group consisting of gold, silver, copper, platinum, palladium, cadmium, iron, nickel, and zinc.

32. The method of claim 24, wherein each of the composite metallic nanostructures comprises a core of a first metal and a coating of a second metal.

33. The method of claim 32, wherein each of the composite metallic nanostructures comprises a gold-plated layer and a silver core.

34. The method of claim 24, wherein each of the composite metallic nanostructures is an alloy of a first metal and a second metal.

35. The method of claim 24, wherein the metallic nanolayer is a metallic thin film.

36. The method of claim 35, wherein the metallic thin film comprises gold.

37. The method of claim 24, wherein the metallic nanolayer comprises a plurality of metallic nanostructures immobilized on the surface.

38. The method of claim 37, wherein the plurality of metal nanostructures are gold nanostructures.

39. The method of claim 24, wherein the composite nanostructure has a geometry selected from the group consisting of: spherical nanoparticles, pyramidal nanoparticles, hexagonal nanoparticles, nanotubes, nanoshells, nanorods, nano-islands, nanodots, nanowires, or combinations thereof.

40. A method of making a composite metal nanostructure, comprising:

preparing a first solution comprising a mixture of a polymer and chloroauric acid;

preparing a second solution comprising silver or copper nanostructures; and

incubating the first solution with the second solution for a period of time, wherein the resulting mixture comprises gold-plated silver nanostructures or gold-plated copper nanostructures.

41. The method of claim 40, wherein the second solution comprises silver nanostructures and has a peak absorbance of about 550 to 750 nm.

42. The method of claim 40, wherein the polymer is polyvinylpyrrolidone, polyvinyl alcohol, polyacrylate, polyethylene glycol, or polyethyleneimine.

43. An assay complex, comprising:

a detection conjugate comprising a composite metallic nanostructure coupled to a binding partner;

a target analyte; and

a metal nanolayer-coated bead having a capture molecule immobilized thereon, wherein the binding partner of the detection conjugate binds to a first epitope on the target analyte and the capture molecule binds to a second epitope on the target analyte, thereby forming a complex comprising the detection conjugate, target analyte, and the capture molecule.

44. The assay complex of claim 43, wherein the binding partner is an antibody and the capture molecule is a different antibody.

45. The assay complex of claim 43, wherein the metal nanolayer is a metal film.

46. The assay complex of claim 45, wherein the thin metal film comprises gold, silver, copper, platinum, palladium, cadmium, zinc, or a complex thereof.

47. The assay complex of claim 46, wherein the thin metal film comprises gold.

48. The assay complex of claim 43, wherein the metal nanolayer comprises a plurality of metal nanostructures immobilized on the bead.

49. The assay complex of claim 48, wherein the plurality of metallic nanostructures comprise gold, silver, copper, platinum, palladium, cadmium, zinc, or a complex thereof.

50. The assay complex of claim 49, wherein the plurality of metal nanostructures are gold nanostructures.

51. The assay complex of claim 43, wherein the composite metal nanostructure comprises at least two metals selected from gold, silver, copper, platinum, palladium, cadmium, iron, nickel, and zinc.

52. The assay complex of claim 43, wherein the composite metal nanostructure comprises a core of a first metal and a coating of a second metal.

53. The assay composite of claim 43, wherein the composite metallic nanostructure comprises a gold-plated layer and a silver core.

54. The assay composite of claim 43, wherein the composite metal nanostructure is an alloy of a first metal and a second metal.

Technical Field

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 that is capable of detecting minute amounts of a target analyte in a sample.

Background

Current immunoassays and biomolecule-binding assays typically require multiple steps and complex equipment to perform the assay. The lack of sensitivity and the complexity involved in performing such heterogeneous assays is caused by the specific need to separate labeled from unlabeled specific binding partners.

Attempts have been made to develop assays based on the Local Surface Plasmon Resonance (LSPR) properties of noble metal nanoparticles (Tokel et al, Chem Rev., Vol.114: 5728-. LSPR is the collective oscillation of electrons in nanometer-sized structures induced by incident light. Metal nanoparticles have a strong electromagnetic response to changes in their refractive index in the immediate vicinity, so that shifts in the resonant frequency of the nanoparticle can be measured as an indication of the molecules bound to the surface of the nanoparticle. Although metal nanoparticles, particularly gold nanoparticles, are used to detect binding events in diagnostic assays, such assays are generally affected by low sensitivity and cannot be used to quantitatively monitor the kinetics of consecutive binding events.

Accordingly, there is a need for improved assay methods that use homogeneous formats while providing increased sensitivity. Assays utilizing standard laboratory techniques such as spectroscopy would also be desirable.

Disclosure of Invention

The present invention is based in part on the following findings: the composite metal nanostructure can enhance the optical signal induced by the binding of molecules to the surface of the metal nanostructure. The observed amplification greatly increases the sensitivity of detecting specific biomolecule binding events, such that sub-picogram quantities of biomolecules can be detected. Accordingly, the present invention provides analyte detection devices and methods of using such devices to detect minute quantities of a target analyte in a sample.

In one embodiment, an analyte detection device includes a plurality of detection conjugates, a surface comprising a metal nanolayer, and a plurality of capture molecules, wherein the capture molecules are immobilized on the metal nanolayer and are capable of specifically binding to a target analyte. In embodiments where the analyte detection device is configured in a sandwich assay format, the detection conjugate comprises a composite metal nanostructure coupled to a binding partner capable of specifically binding to the analyte of interest. In embodiments where the analyte detection device is configured in a direct competition assay format, the detection conjugate comprises a composite metal nanostructure coupled to the analyte of interest.

The composite metal nanostructures in the detection conjugates generally comprise at least two noble metals, transition metals, alkali metals, lanthanides, or combinations thereof. In some embodiments, the composite metallic nanostructure comprises at least two metals selected from gold, silver, copper, platinum, palladium, cadmium, iron, nickel, and zinc. In certain embodiments, each composite metallic nanostructure comprises a core of a first metal and a coating of a second metal. In some embodiments, the core may be gold-plated silver or copper. In other embodiments, the core of the first metal may be dissolved after coating such that a hollow structure consisting of the second coating metal is produced.

The metallic nanolayer deposited on the surface can be a metallic film or consist of a plurality of metallic nanostructures fixed on the surface. The metal nanolayers may also be comprised of a noble metal or a transition metal. In some embodiments, the metal nanolayer includes gold, silver, copper, platinum, palladium, cadmium, zinc, or a composite thereof. In one embodiment, the metal nanolayer includes gold. In another embodiment, the metal nanolayer includes silver. In yet another embodiment, the metal nanolayer comprises a silver nanolayer covered with a gold nanolayer.

The present invention also provides methods of detecting an analyte of interest in a sample using the analyte detection devices described herein. In one embodiment, the method comprises mixing a sample with a plurality of detection conjugates, contacting the mixture with a surface comprising a metal nanolayer having a plurality of capture molecules immobilized thereon, exposing the surface to a light source at a wavelength range within the ultraviolet-visible-infrared spectrum; and measuring an optical signal from the surface, wherein a change in the optical signal indicates the presence of the target analyte in the sample. In certain embodiments, the methods of the invention are capable of detecting femtogram to nanogram amounts of target analytes in a sample.

The present invention includes an assay complex comprising: a detection conjugate comprising a composite metal nanostructure coupled to a binding partner; a target analyte; and a metal nanolayer-coated bead having a capture molecule immobilized thereon, wherein the binding partner in the detection conjugate binds to a first epitope on the target analyte and the capture molecule binds to a second epitope on the target analyte, thereby forming a complex comprising the detection conjugate, the target analyte, and the capture molecule. In some embodiments, the composite metallic nanostructures are gold-plated silver nanostructures or gold-plated copper nanostructures and the metallic nanostructures coated on the beads comprise gold.

In another aspect, the present invention provides a method for preparing composite metal nanostructures for use in the detection apparatus and methods described herein. In one embodiment, the method comprises preparing a first solution comprising a mixture of a polymer and chloroauric acid, preparing a second solution comprising silver or copper nanostructures, and incubating the first solution with the second solution for a period of time, wherein the resulting mixture comprises gold-plated silver nanostructures or gold-plated copper nanostructures. In certain embodiments, a reducing agent, such as ascorbic acid, is added to the reaction mixture to increase the number 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.

Brief Description of Drawings

FIG. 1 is a graph of shift of peak wavelength versus acquisition time for Bovine Serum Albumin (BSA) coupled gold nanolayer sensors (channel 1) and human IgG coupled gold nanolayer sensors (channels 2-4). Arrows indicate the injection sequence and concentration of unlabeled protein a, 1mM HCl or protein a labeled with Colloidal Gold (CGC).

FIG. 2 is a graph of shift of peak wavelength versus acquisition time for an anti-CRP C7 antibody-coupled gold nanolayer sensor. Arrows indicate the injection sequence of CRP (CRP load), unlabeled anti-CRP C6 antibody at a concentration of 0 to 100ng/ml, or anti-CRP C6 antibody labeled with colloidal gold (C6-CGC) at 3 μ g/ml in different channels. When the sensor surface was occupied with unlabeled anti-CRP C6 antibody, no further C6-CGC binding was observed.

FIG. 3 graph of shift of peak wavelength versus acquisition time for anti-CRP C7 antibody-coupled gold nanolayer sensors. Arrows indicate the injection sequence of CRP (CRP load), 1. mu.g/ml anti-CRP C6 antibody labeled with colloidal gold (C6-CGC), 3. mu.g/ml C6-CGC, or 1mM HCl (acid) in different channels at concentrations of 0 to 100 ng/ml.

FIG. 4A. reflectance spectra of CRP-loaded anti-CRP C7 antibody coupled gold nanolayer sensors at 10ng/ml CRP loading at different C6-CGC concentrations in FIG. 3.

Figure 4 b-graph of shift of peak wavelength versus acquisition time for gold nanolayer sensors coupled with anti-CRP C7 antibody incubated with CRP at one of three concentrations after the introduction of 3 μ g/ml anti-CRP C6 antibody (C6-CGC) labeled with colloidal gold. The table on the right depicts the peak analysis 700 seconds after the introduction of C6-CGC.

FIG. 5 graph of shift of peak wavelength versus acquisition time for anti-CRP C7 antibody-coupled gold nanolayer sensors. Arrows indicate the injection sequence of CRP (CRP load with minimized incubation time), 3 μ g/ml anti-CRP C6 antibody labeled with colloidal gold (C6-CGC), or 1mM HCl (acid) in different channels at concentrations of 0 to 100 ng/ml.

FIG. 6 is a graph of shift of peak wavelength versus acquisition time for trace amounts in FIG. 5, immediately after introduction of 3. mu.g/ml of anti-CRP C6 antibody labeled with colloidal gold (C6-CGC). The table on the right depicts the peak analysis 700 seconds after introduction of C6-CGC compared to the peak shift obtained by CRP incubation (values are shown in fig. 4B).

Figure 7-graph of shift of peak wavelength versus acquisition time for an anti-CRP C7 antibody coupled gold nanolayer sensor incubated with CRP and C6 anti-CRP antibody conjugated to gold plated silver nanostructures at one of three concentrations. The control was a gold nanolayer sensor with immobilized Bovine Serum Albumin (BSA) instead of the C7 antibody.

Detailed Description

The present invention is based in part on the following findings: significant amplification in LSPR-based assays can be achieved by binding partners labeled with complex metal nanostructures. Accordingly, the present invention provides an analyte detection device comprising: a LSPR surface (e.g., a surface comprising a metal nanolayer), a plurality of capture molecules immobilized to the metal nanolayer, and a plurality of detection conjugates comprising composite metal nanostructures coupled to biomolecules.

The analyte detection device may be configured in a sandwich assay format or a direct competition assay format. For example, in one embodiment, an analyte detection device in a sandwich assay format comprises (i) a plurality of detection conjugates, wherein the conjugates comprise composite metal nanostructures coupled to binding partners capable of specifically binding to a target analyte, (ii) a surface comprising a metal nanolayer, and (iii) a plurality of capture molecules, wherein the capture molecules are immobilized on the metal nanolayer and capable of specifically binding to the target analyte. In another embodiment, an analyte detection device in a direct competition assay format includes (i) a plurality of detection conjugates, wherein the conjugates comprise composite metal nanostructures coupled to target analytes, (ii) a surface comprising a metal nanolayer, and (iii) a plurality of capture molecules, wherein the capture molecules are immobilized on the metal nanolayer and are capable of specifically binding to the target analytes.

The analyte detection device of the present invention includes a surface comprising a metallic nanolayer. The surface may be of any suitable size and shape, such as a chip, well, cuvette, or bead. In some embodiments, the surface is a rectangular chip. In other embodiments, the surface is a disk. In certain embodiments, the surface is the bottom, lid, and/or inner wall of a test tube (e.g., a cylindrical or rectangular test tube). In other embodiments, the surface is an array of non-metallic particles. The surface may be fabricated from a variety of materials including, but not limited to, glass, quartz, silicon, silica, polystyrene, graphite, fabric (e.g., polyethylene fabric), mesh, or film (e.g., latex, polyethylene, nylon, or polyester film).

The metallic nanolayers are preferably deposited on the surface. In some embodiments, the metal nanolayer can cover the entire surface area of a particular surface. In other embodiments, the metal nanolayer may be deposited on only a portion of the surface. For example, the surface may contain a plurality of recesses or holes and the metal nanolayers are deposited within the recesses or holes. In other embodiments, the metallic nanolayer may be applied to the surface as a plurality of spaced deposits across the surface. The optical properties of the metallic nanolayers can be tuned by varying the thickness of the nanolayers and/or the properties of the nanostructures. In one embodiment, the nanolayer consists of metallic nano-islands. In another embodiment, the nanolayers consist of nanorods. Suitable thicknesses for the metal nanolayers used in the devices and methods of the present invention include from about 0.5nm to about 100nm, from about 5nm to about 30nm, or from about 3nm to about 10 nm. Exemplary surfaces having metallic nanolayer coatings that may be used in the present apparatus and methods include those described in U.S. patent publication No. 2006/0240573, which is incorporated by reference herein in its entirety.

In certain embodiments, the metal nanolayer is a thin film of metal. Methods of depositing metal thin films on a substrate surface are known to those skilled in the art and include, but are not limited to, atomic layer deposition, pulsed laser deposition, drop casting, vapor deposition, and adsorption. See, for example, Atanaasov et al, Journal of Physics: conference Series 514 (2014); walters and Parkin, Journal of Materials Chemistry, 19: 574-590, 2009; and Gupta et al, J.Appl.Phys.92, 5264-5271, 2002, each of which is incorporated herein by reference in its entirety. The metal film may comprise other components, for example the metal film may be a polymer film, a Langmuir-Blodgett film or an oxide film. In some embodiments, the metal film comprises two layers, wherein each layer comprises a different metal. For example, the metal film may include a silver layer covered with a gold layer.

In other embodiments, the metal nanolayer comprises a plurality of metal nanostructures affixed to a surface. The metal nanostructures can be immobilized to a surface by treating a surface material with a reagent to add a chemical functional group, such as cyanide, amine, thiol, carboxyl, aldehyde, or maleimide, and reacting the metal nanostructures with the treated surface. Metal nanostructures are known to bind to such chemical functional groups with higher affinity. In some embodiments, the metal nanostructures comprising the metal nanolayer are spherical nanoparticles. Such nanoparticles have a diameter of less than about 300nm, less than about 200nm, or less than about 150 nm. In some embodiments, the spherical nanoparticles have a diameter of about 5nm to about 200nm, about 10nm to about 100nm, or about 20nm to about 60 nm. In certain embodiments, the size of the metal nanostructures used to create the metal nanolayer is similar to the size of the composite nanostructures used to detect the conjugates. In such embodiments, matching the dimensions of the two sets of nanostructures may provide an optimal wavelength shift in the reflection, emission, or scattering spectra.

The metal nanolayer (metal film or plurality of metal nanostructures) can consist essentially of a noble metal or a composite thereof. In other embodiments, the metal nanolayer (metal film or plurality of metal nanostructures) can consist essentially of a transition metal or a composite thereof. In certain embodiments, the metal nanolayer comprises a metal selected from the group consisting of gold, silver, copper, platinum, palladium, ruthenium, rhodium, osmium, iridium, titanium, chromium, cadmium, zinc, iron, cobalt, nickel, and composites thereof. In a particular embodiment, the metal nanolayer (e.g., the metal thin film or the plurality of metal nanostructures) comprises gold. In another particular embodiment, the metal nanolayer (e.g., the metal film or the plurality of metal nanostructures) comprises silver. In certain embodiments, the metal nanolayer (e.g., the metal thin film or the plurality of metal nanostructures) comprises a complex of gold and silver or gold and copper. The intensity of the LSPR peak may be increased using alkali metals (e.g., lithium, sodium, potassium, rubidium, cesium, and francium) or lanthanides (e.g., lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium). Thus, in some embodiments, the metal nanolayer (metal film or plurality of metal nanostructures) can consist essentially of one or more alkali metals or lanthanides. In other embodiments, the metal nanolayer (metal film or plurality of metal nanostructures) can consist essentially of a combination of noble metal and alkali metal or lanthanide.

The analyte detection device of the present invention further comprises a plurality of capture molecules immobilized to the metallic nanolayer deposited on the surface. The capture molecule is capable of specifically binding to the target analyte. As used herein, "specifically binds" refers to binding to a target molecule with high affinity, e.g., at least 10^-6Affinity of M. In some embodiments, the capture molecules are haptens and other small molecules, drugs, hormones, biological macromolecules including, but not limited to, antibodies or fragments thereof (e.g., Fv, Fab, (Fab)₂Single chain, CDR, etc.), antigen, receptor, ligand, polynucleotide, aptamer, polypeptide, polysaccharide, lipopolysaccharide, glycopeptide, lipoprotein, or nucleoprotein. In certain embodiments, the plurality of capture molecules are antibodies. In other embodiments, the plurality of capture molecules are antigens.

Methods of immobilizing molecules to metal nanolayers or nanostructures are known to those skilled in the art. Such methods include conjugation chemistry such as those involving 1-ethyl-3- [ 3-dimethylaminopropyl ] carbodiimide hydrochloride (EDC), sulfo-NHS coupling, hydrophobic bonding or thioether chemistry. In some embodiments, the molecule may be indirectly coupled to the metal nanolayer or nanostructure via a larger carrier molecule or protein. This indirect coupling is particularly useful when the molecules are small, such as hormones, drugs and other small molecules less than 10 kD. Preferably, the carrier protein is not capable of specifically interacting with the target analyte.

The analyte detection device of the present invention can also include a plurality of detection conjugates. Depending on the assay configuration, the detection conjugate comprises a metal nanostructure coupled to a binding partner capable of specifically binding to the target analyte or capture molecule. For example, in embodiments where the device is configured in a sandwich assay format, the detection conjugate comprises a metal nanostructure coupled or conjugated to a binding partner capable of specifically binding the target analyte. In other embodiments where the device is configured in a direct competition assay format, the detection conjugate comprises a metal nanostructure coupled or conjugated to the analyte of interest.

The binding partner can be the same type of molecule as the capture molecule, including but not limited to haptens and other small molecules, drugs, hormones, biological macromolecules such as antibodies or fragments thereof (e.g., Fv, Fab, (Fab)₂Single chain, CDR, etc.), antigen, receptor, ligand, polynucleotide, aptamer, polypeptide, polysaccharide, lipopolysaccharide, glycopeptide, lipoprotein, or nucleoprotein. In some embodiments, the binding partner is the same type of molecule as the capture molecule, but preferably binds to the target analyte at a position different from the binding site of the capture molecule. For example, the binding partner and the capture molecule may both be antibodies that recognize the target analyte, but the epitope where the binding partner binds the target analyte is separate from and ideally does not overlap with the epitope where the capture molecule binds the target analyte. Thus, in certain embodiments, the binding partner is an antibody that recognizes a first epitope of the target analyte and the capture molecule is a different antibody that recognizes a second epitope of the target analyte.

The metal nanostructures in the detection conjugate may consist essentially of a noble metal or a complex thereof. In some embodiments, the metal nanostructures in the detection conjugate can consist essentially of a transition metal or complex thereof. In some embodiments, the metal nanostructures in the detection conjugate can comprise an alkali metal or lanthanide and a noble metal or transition metal. In certain embodiments, the metal nanostructure in the detection conjugate comprises a metal selected from the group consisting of 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.

In a preferred embodiment, the metal nanostructures in the detection conjugate are composite metal nanostructures. By "composite metal nanostructures" is meant nanostructures comprising at least two noble metals, transition metals, alkali metals, or lanthanides. Two or more metals may be mixed together, such as in an alloy, or two or more metals may be present in separate portions of the nanostructure. For example, one metal may form the core of the nanostructure, while a second metal forms 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 metallic nanostructure comprises at least two metals selected from gold, silver, copper, platinum, palladium, cadmium, iron, nickel, and zinc. In a particular embodiment, the composite metallic nanostructure comprises gold and silver. In another embodiment, the composite metallic nanostructure comprises gold and copper. In another embodiment, the composite metallic nanostructure comprises silver and copper.

In some embodiments, each composite metallic nanostructure is an alloy of a first metal and a second metal. In certain embodiments, each composite metallic nanostructure comprises a core of a first metal and a coating of a second metal. In one embodiment, the core is silver and the coating is gold. In another embodiment, the core is copper and the coating is gold. In another embodiment, the core is silver and the coating is copper. In some embodiments, each composite metallic nanostructure comprises a dielectric core (e.g., silicon dioxide, gold sulfide, titanium dioxide, silicon dioxide, and polystyrene), a first coating of a first metal, and a second coating of a second metal. In a particular embodiment, the core is silica, the first coating (i.e., the undercoat) is a silver-plated layer, and the second coating is a gold-plated layer (i.e., the overcoat). In another embodiment, the core is silica, the first coating (i.e., undercoat) is a copper-plated layer, and the second coating is a gold-plated layer (i.e., overcoat).

In some embodiments, after the coating process using the second metal, the core comprising the first metal is dissolved to form a hollow structure consisting of the second metal. For example, coating of a silver core with gold nanoparticles produces a gold shell around the silver core and subsequent dissolution or degradation of the silver core, resulting in the formation of a hollow nanogold shell structure.

The metal nanostructures include spherical nanoparticles as well as nanosheets and nanoshells. The nanoplatelets have a lateral dimension (e.g., edge length) that is greater than their thickness. The nano-sheets comprise nano-discs, nano-polygons, nano-hexagons, nano-cubes, nano-rings, nano-stars and nano-prisms. In some embodiments, the metal nanostructures, including composite nanostructures, have a geometry selected from the group consisting of; spherical nanoparticles, pyramidal nanoparticles, hexagonal nanoparticles, nanotubes, nanoshells, nanorods, nanodots, nanodomains, nanowires, nanodiscs, nanocubes, or combinations thereof. Other shapes are also possible, including irregular shapes. In certain embodiments, the metal nanostructures are non-uniform in size and shape-that is, the metal nanostructures are a non-uniform mixture of nanostructures of different shapes and sizes.

For spherical nanoparticles, suitable diameter ranges include about 5nm to about 200nm, about 10nm to about 100nm, and about 20nm to about 60 nm. For nanoplatelets, the edge length can be from about 10nm to about 800nm, from about 20nm to about 500nm, from about 50nm to about 200nm, from about 30nm to about 100nm, or from about 10nm to about 300 nm. The nanoplatelets can have a thickness ranging from about 1 to about 100nm, from about 5nm to about 80nm, from about 10nm to about 50nm, or from about 5nm to about 20 nm.

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

The binding partner or target analyte may be coupled or conjugated to the metal nanostructure (e.g., composite nanostructure) using methods similar to those described above for immobilizing capture molecules to metal nanolayers. Such methods include, but are not limited to, EDC conjugation chemistry, sulfo-NHS coupling, hydrophobic bonding, or thioether chemistry. The binding partner or target analyte can be coupled to the metal nanostructure via a variety of chemical functional groups including thiols, amines, dithiols, phosphoramidites, azides, or alkynes.

In some embodiments, the one or more metals used in the metal nanolayer deposited on the surface can be the same as the one or more metals that make up the metal nanostructures in the detection conjugate. For example, in one embodiment, the metal nanostructure deposited on the surface comprises a gold film or a plurality of gold nanostructures and the detection conjugate comprises gold nanostructures. In other embodiments, the metal used for the metal nanolayer deposited on the surface is different from the metal or metals used to produce the metal nanostructures in the detection conjugate. For example, in some embodiments, the metal nanostructure deposited on the surface comprises a silver film or a plurality of silver nanostructures and the detection conjugate comprises gold nanostructures. In other embodiments, the metal nanostructure deposited on the surface comprises a gold film or a plurality of gold nanostructures and the detection conjugate comprises silver nanostructures. In certain embodiments, the metal nanostructure deposited on the surface comprises a gold film or a plurality of gold nanostructures and the detection conjugate comprises a composite nanostructure. In a related embodiment, the composite nanostructure comprises gold-plated silver nanostructures. In other specific embodiments, the metal nanostructure deposited on the surface comprises a gold film or a plurality of gold nanostructures and the detection conjugate comprises a composite nanostructure comprising gold-plated copper nanostructures. In other embodiments, the metal nanostructure deposited on the surface comprises a gold film or a plurality of gold nanostructures and the detection conjugate comprises a composite nanostructure comprising a gold-plated magnetite nanostructure. In other embodiments, the metal nanostructure deposited on the surface comprises a gold film or a plurality of gold nanostructures and the detection conjugate comprises a composite nanostructure comprising gold and an alkali metal or lanthanide.

The invention also includes kits comprising the analyte detection devices of the invention as disclosed herein. In one embodiment, a kit comprises (i) a surface comprising a metal nanolayer having a plurality of capture molecules immobilized thereon and (ii) a composition comprising a plurality of detection conjugates as described herein. In certain embodiments, the composition is packaged separately from the surface so that it can be subsequently contacted with the surface during performance of the detection method. In some embodiments, a composition comprising a plurality of detection conjugates is lyophilized, e.g., in pellet or bead form. In related embodiments, the surface containing the metal nanolayer can be a chip, a disk, or a cuvette. In a particular embodiment, the surface containing the metallic nanolayer is a cuvette suitable for use with a centrifuge rotor. In such embodiments, the metal nanolayer can be deposited on the lid, bottom, and/or walls of the cuvette.

In certain embodiments, all of the components of the analyte detection system described herein are contained within a centrifuge rotor or disk. For example, the rotor or disk may contain one or more reaction chambers in which a surface of a metal nanoparticle layer containing immobilized capture molecules and a plurality of detection conjugates is located. In one embodiment, the metal nano-layer surface is a chip positioned at the bottom of the reaction chamber. In another embodiment, the metal nanolayer is deposited directly on the floor of the reaction chamber. In yet another embodiment, the surface of the metal nanolayer is a bead (e.g., a plastic bead) coated with the metal nanolayer. In all such embodiments, the capture molecule is immobilized to the surface of the metal nanoparticle layer. In related embodiments, the plurality of detection conjugates are present in a lyophilized composition, such as a lyophilized bead or pellet.

In an alternative embodiment, the capture molecule is conjugated to a metal nanostructure, which is in the form of a colloidal suspension. A plurality of detection conjugates is added to the suspension in the presence of the test sample. If the analyte of interest is present in the sample, complex formation occurs between the detection conjugate and the suspended nanostructure containing the capture molecule, causing a change in the optical signal (e.g., a shift in the peak absorbance wavelength of the suspended nanostructure).

Thus, in some embodiments, a kit comprises a rotor or disk having one or more reaction chambers, wherein each reaction chamber comprises (i) a lyophilized composition comprising a plurality of detection conjugates as described herein and (ii) a particle coated with a metal nanolayer, wherein a plurality of capture molecules are immobilized to the metal nanolayer. Such kits provide a one-step analyte detection method whereby a test sample is contacted with a rotor or disk and applying a centrifugal force to the rotor or disk delivers the test sample to a reaction chamber where the sample is mixed with a plurality of detection conjugates and metal nanolayer-coated beads containing immobilized capture molecules. In embodiments where the rotor or disk contains more than one reaction chamber, the detection conjugate and capture molecule may be selected so that different analytes may be detected in each reaction chamber. If the rotor contains multiple reaction chambers, these rotor format detection devices may be configured in a sandwich assay format, a direct competition format, or both.

Any of the types of metal nanolayers or metal nanostructures discussed herein may be used with these rotor format detection devices. In some embodiments, the metal nanolayer coated on the bead is a gold nanolayer and the metal nanostructures in the detection conjugate are gold nanostructures. In other embodiments, the metal nanolayer coated on the bead is a silver nanolayer and the metal nanostructures in the detection conjugate are gold nanostructures. In other embodiments, the metal nanolayer coated on the particle is a gold nanolayer and the metal nanostructures in the detection conjugate are silver nanostructures. In one embodiment, the metal nanolayer coated on the particle is a silver nanolayer covered with a gold nanolayer and the metal nanostructures in the detection conjugate are gold nanostructures. In certain embodiments, the metal nanolayer coated on the bead is a gold nanolayer and the metal nanostructures in the detection conjugate are composite nanostructures. For example, in one embodiment, the composite nanostructure is a gold-plated silver nanostructure. In another embodiment, the composite nanostructure is a gold-plated copper nanostructure.

The kits of the invention may also include instructions for using the device to detect an analyte in a test sample, a device or means for collecting a biological sample, and/or an extraction buffer for obtaining a sample from solid materials such as soil, food, and biological tissue.

The invention also provides methods of detecting a target analyte in a sample. In one embodiment, a method comprises: (i) mixing a test sample with a plurality of detection conjugates as described herein; (ii) contacting the mixture with a surface comprising a metal nanolayer, wherein a plurality of capture molecules as described herein are immobilized to the metal nanolayer; (iii) exposing the surface to a light source at a wavelength range within the ultraviolet-visible-infrared spectrum; and (iv) measuring an optical signal from the surface, wherein a change in the optical signal is indicative of the presence of the target analyte in the sample.

In some embodiments, the detection method is a sandwich assay. In such embodiments, the detection conjugate comprises a metallic nanostructure coupled to a binding partner capable of specifically binding to a target analyte present in the sample to form an analyte-detection conjugate complex. The plurality of capture molecules immobilized to the surface of the metal nanolayer can also specifically bind to a target analyte present in the sample. Exposing the metal nanolayer to a light source and measuring an optical signal, wherein a change in the optical signal indicates the presence of an analyte in the sample. By way of illustration, when a sample containing a target analyte is mixed with a plurality of detection conjugates, the target analyte binds to a binding partner in the detection conjugates to form an analyte-detection conjugate complex. These complexes, in turn, bind to a plurality of capture molecules that are immobilized to the surface of the metal nanostructure via the analyte, thereby bringing the metal nanostructure in the detection conjugate into close proximity to the surface of the metal nanostructure. The amount of light absorbed or scattered by the surface of the metal nanostructure is influenced by the proximity of the metal nanostructure in the composite, thus producing an enhanced shift in the peak absorption wavelength, indicating the presence of the target analyte in the sample.

In other embodiments, the detection method is a competitive assay. In such embodiments, the detection conjugate comprises a metal nanostructure coupled to a target analyte of interest. As in the sandwich assay method, a plurality of capture molecules immobilized to the surface of the metal nanoparticle layer are capable of specifically binding to the target analyte. In this type of assay, the detection conjugate will initially bind to the capture molecule. If a sample containing the target analyte is mixed with such an initial complex, unlabeled or free target analyte in the sample will compete with the detection conjugate for binding to the capture molecule. Changes in the optical signal in this type of assay will result from the displacement of the metal nanostructures in the detection conjugate from the surface of the metal nanostructure, thereby proportionally reducing the wavelength shift of the peak absorption wavelength.

The test sample can be any type of liquid sample, including a biological sample or an extract prepared from an environmental or food sample. In a particular embodiment, the test sample is a biological sample. Biological samples include, but are not limited to, whole blood, plasma, serum, saliva, urine, pleural effusion, sweat, biliary fluid, cerebrospinal fluid, stool, vaginal fluid, sperm, ocular lens fluid, mucus, synovial fluid, peritoneal fluid, amniotic fluid, biopsy tissue, saliva, and cell lysate. The biological sample can be obtained from a human or animal subject suspected of having a disease condition, such as cancer, an infectious disease (e.g., viral, bacterial, parasitic, or fungal infection), a cardiovascular disease, a metabolic disease, an autoimmune disease, or the like. Biological samples can also be obtained from healthy subjects (e.g., humans or animals) undergoing routine medical examination.

In some embodiments of the method, the test sample is mixed with a plurality of detection conjugates and the mixture is then contacted with the surface of the metal nanoparticle layer containing the immobilized capture molecule. In other embodiments, the test sample is contacted with the surface of the metal nanoparticle layer containing the immobilized capture molecule and a plurality of detection conjugates are subsequently added. In certain embodiments, the sample, plurality of detection conjugates, and the surface of the metal nanoparticle layer containing the immobilized capture molecule are contacted simultaneously. For example, contacting the sample with both reagents simultaneously may occur in a rotor format detection device as described above.

Any analyte detection device as described above may be used in the detection methods of the invention. Thus, the various metal nanoparticle surface, capture molecules, and detection conjugates described herein are suitable for use in detection methods. For example, in some embodiments of the methods, the surface comprising the metal nanolayer is a chip, well, cuvette, or bead. In certain embodiments of the method, the surface containing the metal nanolayer is the wall and bottom of a test tube that is incorporated into or suitable for use with a centrifuge rotor. In these and other embodiments, the metallic nanolayer on the surface is a metallic film, such as a gold film. In other embodiments of the method, the metal nanostructure on the surface comprises a plurality of metal nanostructures, such as gold nanostructures, immobilized on the surface.

In certain embodiments of the detection method, the detection conjugate comprises a composite metal nanostructure coupled to a binding partner or target analyte. As described herein, the composite metal nanostructures comprise at least two noble or transition metals. In some embodiments of the method, the composite metal nanostructure comprises at least two metals selected from the group consisting of gold, silver, copper, platinum, palladium, ruthenium, rhodium, osmium, iridium, titanium, chromium, cadmium, zinc, iron, cobalt, and nickel. In other embodiments of the method, the composite metallic nanostructure comprises at least two metals selected from the group consisting of gold, silver, copper, platinum, palladium, cadmium, iron, nickel, and zinc. In a particular embodiment, the composite metallic nanostructure comprises gold and silver. In another embodiment, the composite metallic nanostructure comprises gold and copper. In another embodiment, the composite metallic nanostructure comprises silver and copper. The composite metal nanostructures used in the methods of the invention may comprise many different geometries, such as spherical nanoparticles, pyramidal nanoparticles, hexagonal nanoparticles, nanotubes, nanoshells, nanorods, nanodots, nanodomains, nanowires, nanodiscs, nanocubes, or combinations thereof.

In certain embodiments, the composite metal nanostructures used in the methods of the present invention are alloys of a first metal and a second metal. In some embodiments, the composite metallic nanostructures used in the methods of the present invention comprise a core of a first metal and a coating of a second metal. In a specific embodiment, the composite metallic nanostructure comprises a silver core and a gold-plated layer. In other embodiments, the composite metallic nanostructure comprises a copper core and a gold plating layer. In another embodiment, the core is silver and the coating is copper. In some embodiments, each composite metallic nanostructure comprises a dielectric core (e.g., silicon dioxide, gold sulfide, titanium dioxide, silicon dioxide, 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 undercoat) is a silver-plated layer, and the second coating is a gold-plated layer (i.e., the overcoat). In another embodiment, the core is silica, the first coating (i.e., undercoat) is a copper-plated layer, and the second coating is a gold-plated layer (i.e., overcoat).

The detection methods of the invention can be used to determine the qualitative or quantitative amount of a target analyte. Such methods are particularly useful for determining the approximate amount of an analyte of interest in a sample, which is particularly useful for diagnosing certain medical conditions or evaluating the efficacy of drug therapies. In one embodiment, the amount of target analyte can be determined by establishing a calibration curve for the specific analyte by measuring the change in optical signal from the surface of the metal nanoparticle layer as described herein for samples having known amounts of target analyte; determining a change in an optical signal of the test sample; and comparing the optical signal change of the test sample with the value obtained for the calibration curve. In some embodiments, determining the amount of complex between the first reagent and the second reagent comprises comparing the absorbance and/or reaction rate from the test sample to the absorbance and/or reaction rate from one sample having a known amount of complex, thereby determining the amount of complex in the test sample. The quantitative value obtained from the test sample may be compared to a predetermined threshold value, wherein the predetermined threshold value indicates an abnormal or normal level of the target analyte.

The detection method of the present invention provides a highly sensitive technique for detecting a very small amount of a target analyte in a sample. As demonstrated by the working examples, amplification of plasmon resonance-based signals from the surface of gold nanolayers can be achieved using gold nanostructure conjugates so that nanogram amounts of target analytes in a sample can be detected. Thus, in one embodiment of the method, the presence of nanogram quantities of the target analyte is detected. The inventors surprisingly found that significantly greater amplification of plasmon resonance-based signals from the surface of gold nanolayers can be achieved using composite metal nanostructure conjugates. The use of gold-plated silver nanostructures conjugated to analyte-specific antibodies enables the detection of picogram quantities of the analyte of interest, with a 1000-fold increase in sensitivity compared to the quantities obtained using gold nanostructure conjugates. See example 3. Thus, in some embodiments of the methods, the presence of picogram quantities of the analyte of interest is detected. In other embodiments of the method, the presence of a femtogram amount of the target analyte is detected. Greater sensitivity can be obtained by varying the composition and/or shape of the composite metal nanostructure and/or the surface of the metal nanostructure layer.

When incident light is applied to the metal nanostructure, conduction band electrons in the metal collectively oscillate at the same frequency of the incident electromagnetic wave. Due to such resonant oscillations, the nanostructures strongly absorb and scatter light of a specific wavelength range. For metal nanostructures comprising noble or transition metals, this wavelength range is in the ultraviolet-visible-infrared spectrum, depending on the specific composition of the nanostructure. Thus, a light source that applies electromagnetic energy suitable for use in the methods of the present invention may include any source that applies a range of wavelengths in the ultraviolet-visible spectrum or the ultraviolet-visible-infrared spectrum, including arc lamps and lasers. In some embodiments, the light source may be equipped with a monochromator so that light of a specific wavelength may be applied to the surface of the metal nano-layer.

The optical properties of the metallic nanolayers and nanostructures depend on their size, shape, and composition. For example, solid gold nanoparticlesHas an absorption peak wavelength (lambda) of about 515nm to about 560nm_{Maximum of}) Depending on the particle size. Gold spherical nanoparticles with a diameter of 30nm have an absorption maximum at about 520nm and as the particle size increases, lambda_{Maximum of}Shifting to longer wavelengths. The silver and copper particles have a lambda in the ultraviolet/blue or red region (e.g., about 350nm to about 500nm)_{Maximum of}And increased particle size results in λ_{Maximum of}Shifting to longer wavelengths. The metal nano-rod has transverse lambda_{Maximum 1}And a longitudinal direction λ_{Maximum 2}. Alloys of different metals typically exhibit absorption peaks in the mid-range between the absorption peaks of the constituent metals. For example, a nanostructure comprising an 50/50 alloy of gold and silver exhibits a λ of about 470nm_{Maximum of}And a gradual increase in the amount of gold results in a shift of the absorption peak to longer wavelengths. The sensitivity of the LSPR signal to changes in the local medium refractive index can be tuned by changing the shape or geometry of the nanostructures. For example, non-spherical 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 a particular wavelength) are tailored for a particular application by changing the size, shape, or composition of the metal nanolayers deposited on the surface or used to detect the metal nanostructures in the conjugate.

The interaction between the incident light and the surface of the metal nanolayer can be monitored as reflected light or transmitted light. The amount of absorbed or scattered incident light can be measured as an absorption spectrum in the reflective mode or an absorption spectrum in the transmissive mode. In some embodiments, the optical signal measured by the metal nanolayer can be an optical reflection, absorption spectrum, scattering spectrum, and/or emission spectrum.

The plasmon coupling between the metal nanolayer and the metal nanostructures in the detection conjugate resulting from the formation of a complex between the binding partner, the target analyte and the capture molecule produces a change in the local surface plasmon resonance spectrum of the metal nanolayer. Such changes may include, for example, increased optical extinction, increased optical reflection, and/or increased scattering and/or emission signals. In some embodiments, the change in the optical signal indicative of the presence of the target analyte in the sample comprises a shift, 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 shift in spectral peak wavelength. In one embodiment, the wavelength shift of the optical spectral peak may be a red shift (e.g., to longer wavelengths) within the 200nm to 1200nm spectral window. In another embodiment, the wavelength shift of the optical spectral peak may be a blue shift (e.g., to a shorter wavelength) within the 200nm to 1200nm spectral window. The change in optical signal can be measured at a specific point in time after the set reaction period. Additionally or alternatively, the change in optical signal over the reaction period may be measured (e.g., rate determination). Both types of measurements can be used for qualitative or quantitative analysis of the target analyte.

Various methods for measuring optical signals of different wavelengths and obtaining extinction, 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 PiccoloAnd a Vetscan analyzer (Abaxis, Inc., Union City, Calif.), a fiber optic reader (e.g., LightPath)^TMS4(LamdaGen, Menlo Park, CA)), SPR instruments (e.g., Biacore instruments available from GE Healthcare), centrifugal analyzers from Olympus, Hitachi, and the like.

The invention also includes an assay complex comprising (i) a detection conjugate comprising a composite metal nanostructure coupled to a binding partner, (ii) a target analyte, and (iii) a metal nanolayer-coated bead having a capture molecule immobilized thereon, wherein the binding partner in the detection conjugate binds to a first epitope on the target analyte and the capture molecule binds to a second epitope on the target analyte, thereby forming a complex comprising the detection conjugate, the target analyte, and the capture molecule. In some embodiments, the assay complex is contained in a test tube suitable for use with a centrifuge rotor. In other embodiments, the assay complex is contained within a reaction chamber in a centrifuge rotor or disk.

The binding partners and capture molecules in the assay complex can be any type of molecule as described above, including haptens and other small molecules, drugs, hormones, biological macromolecules such as antibodies or fragments thereof (e.g., Fv, Fab, (Fab)₂Single chain, CDR, etc.), antigen, receptor, ligand, polynucleotide, aptamer, polypeptide, polysaccharide, lipopolysaccharide, glycopeptide, lipoprotein, or nucleoprotein. In one embodiment, the binding partner is an antibody and the capture molecule is a different antibody.

The metal nanolayers and composite metal nanostructures are described in detail above. In one embodiment, the metal nanolayers coated with beads (e.g., plastic or glass beads) are gold nanolayers. In another embodiment, the metal nanolayer coating the beads is a silver nanolayer. The beads are preferably less than 0.5cm, but greater than 0.1 mm. In certain embodiments, the composite metallic nanostructures are gold-plated silver nanostructures. In other embodiments, the composite metallic nanostructures are gold-plated copper nanostructures. In other embodiments, the metallic nanostructures comprise gold doped with silver ions, copper ions, or both.

Any type of target analyte can be detected using the methods, devices and assay complexes of the invention, particularly those methods, devices and assay complexes that are effective in diagnosing disease. Target analytes may include, but are not limited to, proteins, enzymes, antigens, antibodies, peptides, nucleic acids (RNA, DNA, mRNA, miRNA), hormones, glycoproteins, polysaccharides, toxins, viruses, virions, drug molecules, haptens, or chemicals. In some embodiments, the target analyte is a marker or antigen associated with infectious diseases of humans and/or animals. In other embodiments, the analyte of interest is a marker or antigen associated with a particular physiological state or pathological condition.

In certain embodiments, the analyte of interest 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, type a, type b, hepatitis c virus, HIV virus, human papilloma virus, epstein-barr virus, rabies virus, etc.), a bacterial antigen (e.g., ehrlichia, borrelia, anaplasma, anthrax, salmonella, bacillus, etc.), a fungal antigen, or a parasitic antigen (e.g., heartworm, flagellate trichuris, plasmodium falciparum, trypanosoma africanum, trypanosoma brucei, etc.). In other embodiments, the analyte of interest 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 analyte of interest is a pregnancy-associated antigen or marker (e.g., fetal antigen, human chorionic gonadotropin).

The present invention also provides a method for preparing a composite metal nanostructure. In one embodiment, the method comprises preparing a first solution comprising a mixture of a polymer and chloroauric acid, preparing a second solution comprising silver or copper nanostructures, and incubating the first solution with the second solution for a period of time, wherein the resulting mixture comprises gold-plated silver nanostructures or gold-plated copper nanostructures. The resulting mixture preferably has a peak absorbance of from about 515nm to about 670nm or from about 520nm to about 560 nm. In one embodiment, the resulting mixture has a peak absorbance at about 530 nm.

The polymer used to prepare the first solution may be any of the following: polyvinyl pyrrolidone, polyvinyl alcohol, polyacrylates, polyethylene glycol, polyethyleneimine, polyaspartic acid, polyglutamic acid, various gums, gelatin, or mixed polymers comprising any of the foregoing. In a particular embodiment, the polymer is polyvinylpyrrolidone. Different types of coated nanostructures can be obtained by varying the molecular weight of the polymer. Suitable molecular weight ranges for the polymer include from about 5,000 daltons to about 150,000 daltons, from about 10,000 daltons to about 100,000 daltons, from 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.

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

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. In some embodiments, the halide content of the solution is less than 150 mM. In other embodiments, the halide content of the solution is in the range of about 0 to about 50 mM.

Methods of preparing solutions of silver and copper nanostructures are known to those skilled in the art. For example, the second solution comprising silver or copper nanostructures may be prepared by any of the methods described in U.S. patent publication No. 2012/0101007, U.S. patent publication No. 2014/0105982, or U.S. patent publication No. 2013/0230717, each of which is incorporated herein by reference in its entirety. In one embodiment, the second solution comprising silver or copper nanostructures is prepared by mixing a silver source or a 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 nitrate, copper (II) acetate and copper (II) trifluoroacetate. Reducing agents that can react with a silver or copper source to form nanostructures can include glucose, ascorbic acid, sodium borohydride, and basic solutions of polymers such as PVP (e.g., pH greater than 7.5). 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 is known to one of ordinary skill in the art. By way of example only, high concentrations of reducing agents may produce pentagonal and biconical nanostructures, while low concentrations of reducing agents may produce elongated nanowires or tubes. Depending on the particular shape of the nanostructures, the second solution comprising silver or copper nanostructures may have a peak absorbance of from about 550nm to about 1000nm, from about 600nm to about 700nm, from about 630nm to about 680nm, from about 750nm to about 850nm, from about 900nm to about 940nm, from about 580nm to about 620nm, or from about 550nm to about 750 nm. In certain embodiments, the second solution comprising silver nanostructures has a peak absorbance of about 600nm (i.e., 595nm to 605nm, inclusive). In some embodiments, the second solution comprising copper nanostructures has a peak absorbance of about 585nm (i.e., 580nm to 590nm, inclusive). In some embodiments, the solution comprising copper nanostructures has a greater peak absorbance (i.e., a red-shift) than the peak absorbance of a solution comprising silver nanostructures of similar size and shape.

In some embodiments, the incubation period of the first solution with the second solution is at least 12 hours. In other embodiments, the incubation period of the first solution with the second solution is greater than 24 hours, preferably greater than 48 hours, more preferably at least 72 hours. The change in 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, such as in the region of 520nm to 550nm, may indicate that gold-plated nanostructures have stabilized. In certain embodiments, the stability of the resulting nanostructures to sodium chloride (e.g., 0.25-1M) is used to indicate an appropriate coating for the nanostructures.

In certain embodiments, the present invention provides methods of synthesizing nanostructures having an optical density of greater than about 50/mL. In one embodiment, the method comprises mixing a polymer as 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 polymer to chloroauric acid, and the incubation temperature and time. The concentration of the polymer and the chloroauric acid may be in the ranges described above. The temperature may be adjusted based on the size and shape of the desired nanostructure, but may range from about 4 ℃ to about 100 ℃. Similarly, the incubation period (i.e., the first period) can be adjusted based on the desired properties of the nanostructure, but can range from about 15 minutes to one day.

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

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

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

All patent and non-patent documents referred to throughout this disclosure are incorporated by reference in their entirety for all purposes.

Examples

Example 1 amplification of LSPR Signal of gold nanoparticle conjugated analytes

The analyte detection system is prepared by providing a plastic chip having a thin film of gold nanolayers deposited thereon. Human IgG protein (100 μ g/ml) was immobilized to the gold nanolayer membrane to create a sensor surface. The control sensor was constructed by fixing bovine serum albumin to a gold nanolayer membrane. Both types of sensor surfaces were positioned within an instrument equipped with light emitting and light collecting fibers that would shine on the gold nanolayer surface and collect light reflected back from the surface.

Samples containing free protein A (10. mu.g/ml) were brought into contact with both types of sensor surfaces and changes in the reflection spectrum were measured. As shown in fig. 1, the introduction of free protein a to the sensor containing immobilized human IgG did not produce significant visible changes in the reflectance spectrum of the gold nanolayer thin film, as measured by shifts in peak wavelength.

The sensor surface was regenerated by treatment with 1mM hydrochloric acid and samples containing two different concentrations (3.5 μ g/ml and 0.175 μ g/ml) of protein a conjugated to colloidal gold nanoparticles (CGC) were contacted with the sensor surface. When protein a (i.e., the analyte of interest) is conjugated to the colloidal gold nanoparticles, the change in the reflectance spectrum of the gold nanolayer surface is exacerbated. Specifically, 3.5. mu.g/ml protein A-CGC produced a greater shift in peak wavelength compared to 10. mu.g/ml unlabeled protein A. See fig. 1, sensor 2. The amplification of the plasmon resonance signal is large enough to enable the detection of nanogram concentrations of protein a-CGC. See fig. 1, sensor 3. Changes in the reflectance spectrum of the BSA sensor are indicative of non-specific binding of protein a molecules to the sensor surface and are significantly less than the changes induced by specific binding of protein a molecules to immobilized IgG molecules.

The results of this initial experiment indicate that significant amplification of the changes in localized surface plasmon resonance signals induced by binding events at the surface of the metal nanoparticle layer can be achieved by coupling the target analyte to the colloidal gold nanoparticles. For nanogram quantities of analyte detected, an almost 60-fold increase in sensitivity was observed.

Example 2 amplification of LSPR Signal in Sandwich assay

This example describes a series of experiments designed to assess whether amplification of localized surface plasmon resonance signals using gold nanoparticle conjugates can also be achieved in sandwich assay formats in which the target analyte is not directly conjugated to the gold nanoparticles. The gold nanolayer chip surface was prepared as described in example 1. A C7 antibody directed against C-reactive protein (CRP) (100 μ g/ml) was immobilized to a gold nanolayer film deposited on the chip surface to produce an anti-CRP sensor. C6 antibody recognizing a different non-overlapping CRP epitope than C7 antibody was conjugated to colloidal gold nanoparticles (C6-CGC) for use in some experiments or in unlabeled form for other experiments.

In a first series of experiments, samples containing three different concentrations of CRP (1ng/ml, 10ng/ml, or 100ng/ml) were incubated with anti-CRP sensors for 15 to 20 minutes and the change in reflectance spectra of the gold nanolayers was monitored. As shown in fig. 2, minimal peak shifts were observed upon CRP binding to the immobilized C7 anti-CRP antibody on the sensor surface. Subsequent exposure of the sensor surface to unlabeled C6 anti-CRP antibody (1 μ g/ml) did not result in significant further peak shifts. See fig. 2. Similarly, subsequent exposure of the sensor surface to 3 μ g/ml of C6-CGC did not produce any further change in the reflectance spectrum, indicating that the bound CRP molecule may be saturated with unlabeled C6 antibody. See fig. 2.

In a second series of experiments, samples containing three different concentrations of CRP (1ng/ml, 10ng/ml or 100ng/ml) were incubated with the anti-CRP sensor for 15 to 20 minutes. Two different concentrations of C6-CGC (1. mu.g/ml and 3. mu.g/ml) were then introduced and the change in the reflection spectrum was measured (FIG. 3 and FIG. 4A). The results show that conjugating the C6 anti-CRP antibody to gold nanoparticles amplifies the peak wavelength shift compared to the unlabeled C6 antibody. Increasing the concentration of C6-CGC produced a dose-dependent shift in peak wavelength. However, the difference in signal between 1ng/ml and 10ng/ml was small (FIG. 4B).

In a third series of experiments, the effect of the analyte incubation time on the development of the signal was evaluated. The anti-CRP sensor was contacted with a sample containing 0ng/ml, 10ng/ml or 100ng/ml CRP and 3 μ g/ml C6-CGC was introduced immediately without any analyte incubation time or wash. As shown in fig. 5 and 6, shorter analyte incubation times produced smaller peak wavelength shifts.

The results of these three sets of experiments indicate that amplification of LSPR signal can be achieved using gold nanoparticle conjugates in a sandwich assay format. When the detector antibody is labeled with colloidal gold particles, an enhanced signal shift is observed, allowing the detection of nanogram concentrations of analyte, compared to unlabeled antibody.

Example 3 enhanced Signal amplification Using gold-plated silver nanostructures

To examine whether varying the type of metal used to label the binding partner affects the amplification of the LSPR signal, composite metal nanostructures were prepared. Specifically, gold-plated silver nanostructures were prepared as follows. By adding 50.0mL of deionized H₂O, 500.0. mu.L trisodium citrate (75mM), 200. mu.L AgNO₃(200mM) and 500.0. mu. L H₂O₂(27%) while stirring vigorously at room temperature to prepare silver nanostructures. Then, 500. mu.L aliquots of NaBH were added₄(200mM) was rapidly injected into the aqueous solution, causing the color to change to pale yellow. Over a period of several minutes, the color continues to change from dark yellow to red to purple and eventually stabilizes in blue. The peak absorbance of the solution was 604.5nm as determined by UV/Vis spectroscopy.

By adding 5.0mL of blue solution to 50. mu.L of polyvinylpyrrolidone (PVP MW ≈ 20% in 10,000 ethanol) and 50. mu.L of HAuCl₄(20mM) to add a gold plating layer to the silver nanostructures. After 72 hours incubation time, the sample turned dark red and had a peak absorbance of 534.5 nm. The nanoparticles were washed twice by centrifugation at 20,000rpm for 20 minutes and resuspended in 2.0mL of deionized H₂And (4) in O. The solution had a deep red color, an absorption peak at 530.3nm, and a total absorbance of 15.0OD units.

Gold-plated silver nanostructures (Au @ AgNPs) conjugated to C6 anti-CRP antibody by adding 600.0. mu.L of Au @ AgNPs and 20.0. mu. L C6 anti-CRP antibody (8.0mg/mL) to 880.0. mu.L of deionized H₂O to a final antibody concentration of 17.8. mu.g/mL/OD. After an incubation period of 2 hours at 4 ℃, the samples were centrifuged for 20 min at 30,000gAnd resuspended in 1.5mL blocking solution containing BSA in PBS (10 mg/mL). Au @ AgNP conjugated to anti-CRP C6 antibody was stored at 4 ℃ until further use.

An anti-CRP gold nanolayer sensor was prepared as described in example 2 and had a peak absorption at 530 nm. Control sensors containing gold nanolayers without any immobilized antibodies were also prepared. The sensor was equilibrated with 100 μ L PBS.

100 μ L C6 anti-CRP antibody conjugated to Au @ AgNP diluted to 1.5OD in PBS was pre-mixed with 1, 10 or 500pg/mL of CRP antigen for 1 minute. The mixture is then contacted with an anti-CRP or control sensor surface and the change in reflectance spectrum of the gold nanolayer surface is measured. The results indicate that gold-plated silver nanostructures enhanced the peak wavelength shift induced by CRP-antibody complex binding to the sensor surface (fig. 7). It is possible to detect 1pg/mL of CRP antigen using gold plated silver nanostructures, with a 1000-fold increase in sensitivity compared to that obtained using gold nanoparticles. At higher concentrations of antigen, the binding sites are saturated and no further translocation occurs.

The results of this experiment demonstrate that when composite nanostructures, such as gold-plated silver nanostructures, are used to label analyte binding partners, a significantly enhanced amplification of the LSPR signal from the surface of the metal nanostructure is achieved.

Example 4 Synthesis of higher optical Density nanostructures

Gold nanoparticles were prepared by mixing 1ml of the final volume of the following reagents in the order indicated: 0.1ml of 1% PVP-10 (1% wt/wt), 0.2ml of 0.1M gold chloride, 0.1ml of 5N NaOH, 0.4ml of water and 0.2ml of 1M ascorbic acid. After each addition, the reaction mixture was mixed. Spectroscopic measurements indicated that the reaction was largely complete after 24 hours at room temperature. This protocol produced spherical gold nanoparticles exhibiting LSPR peaks of about 535nm and a corresponding optical density of about 80 per ml. Layering with additional gold or silver is performed by adding silver nitrate or gold chloride to the preformed gold nanoparticles. Excess reagent was removed by centrifugation at 30,000g for 1-2 hours.

In a separate reaction, 0.05ml of 20% PVP (wt/wt) was mixed with 0.25ml water, 0.1ml 5N NaOH, 0.1ml 1M sodium citrate, 0.5ml 0.1M gold chloride and 1ml 1M ascorbic acid. This protocol resulted in the immediate formation of colloidal gold particles with an OD of about 90/ml and an LSPR peak of-525 nm. A linear correspondence was observed between the final OD and the gold concentration between 2.5mM gold and 25mM gold in the final reaction mixture.

It is to be understood that the disclosed invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be 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.

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. Such equivalents are intended to be encompassed by the following claims.