WO2024137691A2

WO2024137691A2 - Fluorescent immunoassays enhanced using plasmonic nanostructures

Info

Publication number: WO2024137691A2
Application number: PCT/US2023/084931
Authority: WO
Inventors: Qisheng JIANG
Original assignee: Auragent Bioscience, Llc
Priority date: 2022-12-19
Filing date: 2023-12-19
Publication date: 2024-06-27

Abstract

The present disclosure provides a method for performing an ultrasensitive fluorescent immunoassay. The ultrasensitive fluorescent includes plasmonic nanostructures.

Description

Atty Docket No.108036-780316 FLUORESCENT IMMUNOASSAYS ENHANCED USING PLASMONIC NANOSTRUCTURES CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of and incorporates by reference the content of U.S. Provisional App. No. 63/433,678 and U.S. Provisional App. No. 63/464,897, filed on December 19, 2022 and May 08, 2023 respectively. FIELD OF THE INVENTION [0002] The field of the disclosure generally relates to an ultrasensitive fluorescent immunoassay (or ultrasensitive fluoroimmunoassay). The ultrasensitive fluorescent immunoassay includes plasmonic nanostructures to provide enhanced fluorescent signal-to- noise ratio compared to an equivalent unenhanced fluoroimmunoassay. The plasmonic nanostructures may be functionalized. BACKGROUND OF THE INVENTION [0003] Detection and quantification of various biomolecules in biological fluids and tissues is of fundamental importance in biomedical research and clinical diagnostics because it is impossible to fully characterize complex, non-linear, biochemical systems without being able to accurately and quantitatively interrogate the component molecules. The relevant concentrations of molecules related to diseases like cancer, heart disease, and neurodegeneration can range in concentration over many orders of magnitude from fg/mL levels to mg/mL, and one might want to interrogate multiple markers simultaneously within the same sample, which is especially challenging. [0004] Fluoroimmunoassays are a type of immunoassay wherein a target analyte is bound to one or more antibodies specific to the target, and a fluorescent reporter molecule is bound to one of the antibodies yielding a fluorescent signal proportional to the amount of target bound. The most common embodiment of this assay is called a sandwich immunoassay. In this assay, a system contains a capture antibody specific to a target analyte and the capture antibody is bound to a substrate; a sample containing the target analyte is added to the system and the analyte binds specifically to the capture antibody; a detection antibody specific to the target analyte is then added to the system creating a capture antibody-analyte-detection antibody sandwich. In this format, the detection antibody itself can act as a fluorescent reporter molecule if it is labeled with a fluorophore. Additionally, another fluorescently-labeled molecule can be added which specifically binds to the detection antibody, and this molecule is the fluorescent ^92677326.1 - 1 - Atty Docket No.108036-780316 reporter molecule. As used herein, the term fluorescent reporter molecule means the fluorescent molecules used in traditional fluoroimmunoassays such as fluorescently labeled detection antibodies or fluorescently labeled biotin binding molecules bound to a biotinylated detection antibody. When unbound fluorescent reporter molecules are removed from the system, the system is then irradiated with an appropriate wavelength of light to excite the fluorescent molecules, and the resultant fluorescent signal is collected. The amount of target analyte in the sample is proportional to the fluorescent signal collected. [0005] Fluoroimmunoassays are useful for detection and quantification of biomarkers, but generally lack sensitivity required to detect very low-abundance molecules. Fluoroimmunoassays, like all fluorescence-based techniques, are ultimately limited in their detection sensitivity by the amount of light that can be collected during the interrogation period from the fluorescent reporter molecules. Generally, weak fluorescence signal from individual reporter molecules and the associated poor signal-to-noise ratio limits the ultimate sensitivity of current fluorescence-based assays. [0006] It is possible to improve detection sensitivity in several ways: 1) excite the fluorescent reporter molecule and collect the resultant fluorescent signal for a longer period of time; 2) improve the optical system used for detection to more efficiently collect the fluorescent signal; or 3) increase the amount of light emitted per target analyte bound either by increasing the number of fluorophores localized to the target analyte, increasing the brightness of the fluorophores, or both. The major downside of approach 1) above is that it significantly increases the time required to read out the results of the immunoassay. Moreover, if the signal of the reporter fluorophore is not significantly brighter than the non-specific background light, this approach will not improve assay sensitivity. Finally, fluorophores can only emit so much light before they are destroyed through photobleaching, which places an upper limit on the amount of light one can detect for a given fluorophore. The major downsides of approach 2) above are that a more sensitive optical system will be much more expensive and/or have a smaller field-of-view, the latter of which will also significantly increase the time required to read out the results of the immunoassay. The technology disclosed herein is approach 3) and utilizes both an increase in the number of fluorophores localized to the detection-antibody bound to the target analyte (detection antibody/analyte complex) and plasmonic enhancement of these fluorophores. This strategy significantly increases the amount of light emitted for each detection-antibody/target analyte complex, resulting in a fluoroimmunoassay with significantly higher sensitivity owing to the increased signal-to-noise ratio from each detection- ^92677326.1 - 2 - Atty Docket No.108036-780316 antibody/target analyte complex compared to a traditional immunoassay using fluorescent reporter molecules. [0007] Importantly, the methods disclosed herein are generally applicable to a wide range of immunoassay formats utilizing at least one antibody and where the signal readout is fluorescence. SUMMARY [0008] The Summary is provided to introduce a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter. [0009] The present disclosure is directed to a method for performing an ultrasensitive fluorescent immunoassay. The method includes providing an immunoassay comprising an analyte and at least one detection antibody specifically bound to the analyte, adding to the immunoassay a plasmonic nanostructure having a localized surface plasmon resonance wavelength (λLSPR) wherein the plasmonic nanostructure specifically binds to the detection antibody, adding at least one fluorescent molecule having a maximum excitation wavelength (λEX) wherein the difference between λLSPR and λEX is less than 50 nm and the at least one fluorescent molecule specifically binds to the plasmonic nanostructure, and exposing the fluorescent immunoassay to a wavelength of light suitable to excite the at least one fluorescent molecule and measuring the resultant fluorescence signal. [0010] In some aspects, the fluorescent signal intensity from the fluorescent immunoassay is more than 100-fold greater than a fluorescent immunoassay where the at least one detection antibody is conjugated directly to the at least one fluorescent molecule. [0011] In some aspects, the at least one detection antibody comprises a tag. In some aspects, at least one detection antibody includes at least one fluorescent molecule. [0012] In some embodiments, the plasmonic nanostructure is functionalized with at least one molecule that can specifically bind the tag. In some embodiments, the tag is biotin. [0013] In some embodiments, the at least one detection antibody comprises at least one fluorescent molecule. [0014] In some embodiments, the plasmonic nanostructure is functionalized with at least one biotin-binding molecule. In some aspects, the at least one biotin-binding molecule includes streptavidin, neutravidin, or avidin. ^92677326.1 - 3 - Atty Docket No.108036-780316 [0015] In some aspects, the at least one biotin-binding molecule comprises at least about 2, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 biotin-binding molecules. [0016] In some embodiments, the method further includes providing a capture antibody specific to the analyte. [0017] In some embodiments, the plasmonic nanostructure includes a gold nanorod coated with silver (AuNR@Ag). [0018] The present disclosure is also directed to a diagnostic test system. In some aspects, the diagnostic test system includes a sample region for adding a sample containing at least one type of analyte, a test region comprising at least one capture antibody that specifically binds the analyte, a region containing a detection antibody comprising a tag, a conjugate region comprising a plasmonic nanostructure functionalized to specifically bind the tag, and at least one fluorescent reagent region containing a fluorescent molecule that specifically binds the plasmonic nanostructure. [0019] In some embodiments, the at least one detection antibody includes a tag. In some embodiments, the at least one detection antibody includes at least one fluorescent molecule. In some embodiments, the plasmonic nanostructure is functionalized with at least one molecule that can specifically bind the tag. In some embodiments, the tag is biotin. [0020] In some embodiments, the plasmonic nanostructure is functionalized with at least one biotin-binding molecule, wherein the at least one biotin-binding molecule comprises streptavidin, neutravidin, or avidin. [0021] In some embodiments, the at least one biotin-binding molecule comprises at least about 2, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 biotin-binding molecules. [0022] In some embodiments, the plasmonic nanostructure includes a gold nanorod coated with silver (AuNR@Ag). [0023] These as well as other embodiments, aspects, advantages, and alternatives will become apparent to those of ordinary skill in the art by reading the following detailed description, with reference where appropriate to the accompanying drawings. Further, it should be understood that this summary and other descriptions and figures provided herein are intended to illustrate embodiments by way of example only and, as such, that numerous variations are possible. For instance, structural elements and process steps can be rearranged, combined, distributed, eliminated, or otherwise changed, while remaining within the scope of the embodiments as claimed. ^92677326.1 - 4 - Atty Docket No.108036-780316 BRIEF DESCRIPTION OF THE FIGURES [0024] FIG.1A is a pictorial representation of plasmonic nanostructures functionalized with BSA-biotin to bind a biotin binding molecule according to the present disclosure. A non- limiting example of a biotin binding molecule includes but is not limited to streptavidin. [0025] FIG.1B is a pictorial representation of plasmonic nanostructures functionalized with biotinylated antibody to bind both a target analyte or tag and streptavidin according to the present disclosure. [0026] FIG.1C is a pictorial representation of plasmonic nanostructures functionalized with streptavidin to bind to biotin according to the present disclosure. [0027] FIG. 2 is a pictorial representation of a method of enhancing a traditional fluoroimmunoassay to an ultrasensitive fluoroimmunoaasay according to the present disclosure. [0028] FIG. 3 is a pictorial representation of a method of performing an ultrasensitive fluoroimmunoassay according to the present disclosure. [0029] FIG.4A is a pictorial representation of a method of performing an ultrasensitive fluoroimmunoassay according to the present disclosure. A target analyte may be localized to a substrate surface to which a capture antibody is attached. In the next step, plasmonic nanostructures may be added which may directly conjugated to biotinylated detection antibody. This plasmonic nanostructure may bind specifically to the target analyte. In a final step, fluorescent streptavidin may be added which may specifically bind to the plasmonic nanostructure functionalized with the biotinylated detection antibody. When unbound fluorescent species may be removed, the assay may be irradiated with an appropriate wavelength of light suitable to excite the fluorescent streptavidin and the emitted fluorescent signal may be collected. The fluorescent signal may be significantly higher in the fluoroimmunoassay containing the plasmonic nanostructure as compared to the fluoroimmunoassay wherein the detection antibody is fluorescently labeled and there is no plasmonic nanostructure. [0030] FIG.4B is a pictorial representation of a method of performing an ultrasensitive fluoroimmunoassay according to the present disclosure. A target analyte may be attached to the substrate without using a detection antibody. [0031] FIG.5A is a pictorial representation of a method used to demonstrate plasmonic enhancement of a fluorescent assay according to the present disclosure. [0032] FIG.5B shows the corresponding fluorescent image of method steps illustrated in FIG.5A as a function of BSA-biotin concentration. The contrast is the same for all images. ^92677326.1 - 5 - Atty Docket No.108036-780316 [0033] FIG. 5C is a graphical representation (top) and tabulated values (bottom) of quantitative data from images in FIG. 5B showing the plasmonic enhancement may greatly enhance the signal to noise ratio of the fluorescent assay relative to an unenhanced assay. [0034] FIG.6A is a pictorial representation (top) and graphical representation (bottom) of data from a fluoroimmunoassay. A fluoroimmunoassay that does not utilize plasmonic enhancement measuring the analyte IL-6 at various concentrations is performed using a biotinylated detection antibody, and fluorescently-labeled streptavidin. [0035] FIG.6B is a pictorial representation (top) and graphical representation (bottom) from a plasmonic-enhanced fluoroimmunoassay according to the present disclosure. A fluoroimmunoassay utilizing plasmonic enhancement wherein the biotinylated detection antibody may be conjugated directly to the plasmonic nanostructure and then bound with fluorescent streptavidin is also shown illustrating a significant improvement in the LOD and the LOQ. [0036] FIG.7 is a pictorial representation of a full-strip version of plasmonic-enhanced fluorescent lateral flow immunoassay according to the present disclosure. The sample may be directly added to the test strip, then may interact with plasmonic nanostructure conjugated with biotinylated detection antibody which may bind target analyte in the sample and fluorescent streptavidin yielding a plasmonic-enhanced fluorescent structure. This complex may flow onto a region containing a capture antibody specific to the target analyte where the complex may be specifically bound. [0037] FIG. 8 is a pictorial representation of a premix/drop version of plasmonic- enhanced fluorescent lateral flow immunoassay according to the disclosure. The sample may be premixed with the plasmonic nanostructure conjugated with biotinylated detection antibody which specifically binds the target analyte. This mixture may be added to a test strip wherein it flows into a region containing fluorescent streptavidin which may bind specifically to the biotinylated detection antibodies on the plasmonic nanostructure. This complex may further flow onto a region containing a capture antibody specific to the target analyte where the complex may be specifically bound. [0038] FIG.9 is a pictorial representation of a sequential addition version of plasmonic- enhanced fluorescent lateral flow immunoassay according to the present disclosure. The sample may be added to the test strip and flows into a region containing a plasmonic nanostructure conjugated with biotinylated detection antibodies which specifically may bind the target analyte in the sample forming a complex. This complex may flow to a region containing a capture antibody specific to the target analyte where the complex may be bound. ^92677326.1 - 6 - Atty Docket No.108036-780316 Fluorescent streptavidin may then be added to the test strip where the streptavidin will localize to the plasmonic nanostructure yielding an ultrabright fluorescent complex. [0039] FIG. 10 is a pictorial representation of the full-strip version of the plasmonic- enhanced fluorescent lateral flow assay in according to the present disclosure. The assay may be used to measure the presence of an antibody in the sample which specifically may recognize a target analyte. This type of immunoassay is called a serology assay. This is a similar format to that described in FIG.7 except the target analyte replaces the capture antibody. [0040] FIG.11 is data illustrating the analytical performance of a plasmonic-enhanced fluorescent lateral flow immunoassay- for human IL6 according to the present disclosure. The performance is of a biotinylated detection antibody functionalized nanostructure and fluorescently-labeled streptavidin of FIG.7. DETAILED DESCRIPTION [0041] Example methods, devices, and systems are described herein. It should be understood that the words “example” and “exemplary” are used herein to mean “serving as an example, instance, or illustration.” Any embodiment or feature described herein as being an “example” or “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments or features. Other embodiments can be utilized, and other changes can be made, without departing from the scope of the subject matter presented herein. [0042] Articles “a” and “an” are used herein to refer to one or to more than one (i.e. at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element. [0043] “About” is used to provide flexibility to a numerical range endpoint by providing that a given value may be “slightly above” or “slightly below” the endpoint without affecting the desired result. [0044] The use herein of the terms "including," "comprising," or "having," and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”). [0045] As used herein, the transitional phrase "consisting essentially of" (and grammatical variants) is to be interpreted as encompassing the recited materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed ^92677326.1 - 7 - Atty Docket No.108036-780316 invention. Thus, the term "consisting essentially of" as used herein should not be interpreted as equivalent to "comprising." [0046] Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination. [0047] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure. [0048] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. [0049] Thus, the example embodiments described herein are not meant to be limiting. Aspects of the present disclosure, as generally described herein, and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are contemplated herein. [0050] Further, unless context suggests otherwise, the features illustrated in each of the figures may be used in combination with one another. Thus, the figures should be generally viewed as component aspects of one or more overall embodiments, with the understanding that not all illustrated features are necessary for each embodiment. [0051] The present disclosure is directed to using plasmonic nanostructures in ultrasensitive fluoroimmunoassays. Fluorescence may be enhanced due to field effects of plasmonic structures. To date, most approaches to utilize this effect to make ultrasensitive fluoroimmunoassays have centered around creating plasmonic substrates on which the assay is carried out. One major downside of this approach is that it requires fabrication of these substrates, which can be expensive and are not easily integrated into existing immunoassay workflows. Owing to this, the substrate-based approach is also limited in its flexibility, typically requiring special readers and/or assay plates to be used. Another major downside is ^92677326.1 - 8 - Atty Docket No.108036-780316 that these approaches typically only result in marginal improvements in assay performance. This is likely due to the fact that the enhancement is strongly distance-dependent, and the physical geometry of the components comprising immunoassays (namely, antibodies) lead to a spacing of the fluorescent reporter molecule that is too far from the plasmonic surface to be optimally enhanced. Moreover, plasmonic surfaces (such as silver islands or gold-coated glass) do not typically have nearly as strong a plasmonic field as nanostructures [such as gold nanorods (AuNR) or silver-coated gold nanorods (AuNR@Ag) like those described in the patent application WO2021155181A1]. For plasmonic nanostructures, the distance from the metal surface for optimal enhancement is about <20 nm, and generally, between about 1 to about 10 nm. In addition, plasmonic nanostructures such as AuNR@Ag can be tuned to have a λLSPR from about 400 nm to > about 800 nm, which significantly increases the spectral variety of fluorescent molecules that can be enhanced. [0052] In one aspect, non-fluorescent plasmonic nanostructures may be added into immunoassay systems to achieve significant enhancements in the immunoassay’s sensitivity, as defined by the LOQ and LOD of the immunoassay, as compared to an equivalent fluorescent immunoassay which does not involve plasmonic enhancement. As compared plasmonic-fluor disclosed in the art, the plasmonic nanostructures utilized in the present invention are not fluorescent, nor do they require a siloxane spacer layer to maintain the fluorescent particles at an optimal distance from the plasmonic nanostructure. Instead, the biomolecules attached to the plasmonic amplifier can themselves serve as the spacer layer and the mechanism for localizing the fluorescent species within the appropriate distance to provide plasmonic enhancement. This has a significant advantage that it is much simpler to make the plasmonic amplifier as compared to the previously described plasmonic-fluor. [0053] Some non-limiting examples of immunoassays in which plasmonic-enhanced fluorescence could be used include: immunosorbent assays wherein the capture antibody is attached to a planar substrate, the wall of a cylindrical capillary, or a porous membrane; bead- based immunoassays wherein the capture antibody is attached to a bead; blot-based assays such as Western blot where the target protein analyte is bound to a porous membrane; or a capillary electrophoresis assay where the target protein analyte is bound to the wall of a capillary. [0054] Some non-limiting examples of other assays in which plasmonic-enhanced fluorescence of the disclosed ultrasensitive fluorescent immunoassay may be used include assays for the detection of nucleic acids. Examples of such assays include nucleic acid arrays in which a capture oligonucleotide is attached to a planar substrate, the wall of a cylindrical capillary, or a porous membrane; bead-based assays in which the capture oligonucleotide is ^92677326.1 - 9 - Atty Docket No.108036-780316 attached to a bead; blot-based assays such as Northern blot where the target oligonucelotide is bound to a porous membrane; or a capillary electrophoresis assay where the target oligonucleotide is bound to the wall of a capillary. Plasmonic nanostructures [0055] As shown in FIG. 1, a variety of functionalizations of the plasmonic nanostructure may be realized to allow easy integration into immunoassays. In some embodiments, the plasmonic nanostructures are functionalized with biotin-streptavidin interaction which is commonly used in both immunoassays and nucleic acid detection. The plasmonic nanostructures may easily be functionalized with biotin by adding biotinylated bovine serum albumin (BSA) which may non-specifically adsorbs to the plasmonic nanostructure. Not only does this allow facile functionalization but also forms a layer that can enable efficient plasmonic enhancement of fluorescently-labeled streptavidin while also binding biotinylated detection antibody of the immunoassay. In various examples, the layer may be about 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, or about 10 nm thick. In at least one example, the layer may be about 3 nm thick. [0056] The plasmonic nanostructure may be functionalized with two or more biotin molecules. For example, the plasmonic nanostructure may be functionalized with at least about 2 biotin molecules, at least about 5 biotin molecules, at least about 10 biotin molecules, at least about 20 biotin molecules, or at least about 25 biotin molecules. In some embodiments, the plasmonic nanostructure may be functionalized with about at least 30 biotin molecules, about at least 40 biotin molecules, or about at least 50 biotin molecules. [0057] As shown in FIG. 1B, the plasmonic nanostructure may be directly functionalized with a biotinylated antibody which, like BSA, also non-specifically adsorbs to the plasmonic nanostructure allowing facile functionalization. In some examples, the plasmonic nanostructures may also be functionalized with a blocker, such as BSA, that prevents non-specific adsorption within an immunoassay leading to higher non-specific background. As shown in FIG. 1, the plasmonic nanostructure functionalized with biotinylated detection antibody may also functionalized with BSA to completely cover the surface of the plasmonic nanostructure. The plasmonic nanostructure may be functionalized with two or more biotinylated antibodies. For example, the plasmonic nanostructure may be functionalized with about at least 2 biotinylated antibodies, about at least 5 biotinylated antibodies, about at least 10 biotinylated antibodies, about at least 20 biotinylated antibodies, or about at least 25 biotinylated antibodies. In some embodiments, the plasmonic nanostructure may be ^92677326.1 - 10 - Atty Docket No.108036-780316 functionalized with about at least 30 biotinylated antibodies, about at least 40 biotinylated antibodies, or about at least 50 biotinylated antibodies. [0058] As shown in FIG. 1C, the plasmonic nanostructure may be functionalized with streptavidin. The plasmonic nanostructure may be functionalized directly with streptavidin non-specifically adsorbed to the surface. In some embodiments, a plasmonic nanostructure functionalized with BSA-biotin may be further functionalized with streptavidin. The streptavidin-conjugated plasmonic nanostructure may bind both the biotinylated detection antibody of the immunoassay and fluorescently labeled biotin. The streptavidin conjugated plasmonic nanostructure may be functionalized with two or more streptavidin molecules. For example, the plasmonic nanostructure may be functionalized with about at least 2 streptavidin molecules, at least 5 streptavidin molecules, at least 10 streptavidin molecules, at least 20 streptavidin molecules, or at least 25 streptavidin molecules. In some embodiments, the plasmonic nanostructure may be functionalized with about at least 30 streptavidin molecules, about at least 40 streptavidin molecules, or about at least 50 streptavidin molecules. [0059] In some embodiments the plasmonic nanostructures may be functionalized with oligonucleotides or peptide nucleic acids that would utilize molecules functionalized with complementary oligonucleotides or peptide nucleic acids for targeting specific species. The oligonucleotides may label the antibodies, plasmonic nanostructures, fluorescent species, or combinations thereof. [0060] In some embodiments the plasmonic nanostructures may be functionalized with antibodies or functional fragments thereof. An example of antibody or functional fragment includes but is not limited to functional fragments which can specifically bind the detection antibody. The detection antibodies include donkey-anti-mouse IgG wherein the detection antibody is a mouse IgG. In some embodiments, additional fluorescently-labeled detection antibodies may localize to the plasmonic nanostructure surface. [0061] In some embodiments, the plasmonic nanostructure may be functionalized with an antibody or functional fragments thereof that specifically bind to an epitope tag and the detection antibody may be labeled with the epitope tag. Additional fluorescent species containing the epitope tag may be added to the system and may localize to the plasmonic nanostructure. Nonlimiting examples of epitope tags are digoxigenin, fluorescent dyes (such as FITC), or peptide tags (such as Human influenza hemagglutinin (HA), c-myc, FLAG, or V5). [0062] The plasmonic nanostructure may also be functionalized with additional molecules to be used for an ultrasensitive fluorescent immunoassay wherein the plasmonic ^92677326.1 - 11 - Atty Docket No.108036-780316 nanostructure is functionalized to bind both a functional component as the fluorescent assay and multiple fluorescent species. The key principle is that the plasmonic nanostructure and functional components of the assay are conjugated to molecules allowing the formation of the complex containing the analyte-antibody-plasmonic nanostructure-and fluorescent species localized to the surface of the plasmonic nanostructure. [0063] The plasmonic nanostructure may have at least more than about 2, and, preferably more than about 20 binding molecules that allow association of multiple fluorescent species to within a distance of about 1 nm to about 10 nm of the plasmonic nanostructure surface. [0064] In addition, it should be recognized by those skilled in the art, that one may use combinations of functionalizations described above to achieve the same effect. For example, the plasmonic nanostructure may be functionalized with both antibodies and biotin molecules wherein the antibody may be able to specifically bind to either a detection antibody or the target analyte directly, and fluorescent streptavidin may be added to the assay and may localize to the plasmonic nanostructure. [0065] Multiple strategies and chemistries may be utilized to functionalize the plasmonic nanostructure to achieve the desired effect. Examples of functionalizations illustrated in the figures herein are easy to implement, utilize components that are already commonly used in immunoassays, and provide a distance separating the metal surface of the plasmonic nanostructure from the fluorescent species by about 1 to about 10 nm, which is optimal for plasmonic enhancement. In some examples, the distance separating the metal surface of the plasmonic nanostructure from the fluorescent species is about 1 nm to about 3 nm, about 3 nm to about 5 nm, about 5 nm to about 7 nm, or about 7 nm to about 10 nm. [0066] In some embodiments, the plasmonic nanostructures may be functionalized with proteins, polymers, siloxanes, or a combination thereof. In the case of silver or gold plasmonic nanostructures, thiol-containing molecules may covalently attach to the surface. In some embodiments, an AuNR@Ag may be functionalized with a mercaptosilane. The mercaptosilane functionalized AuNR@Ag may be further functionalized with a silane containing biotin or a silane containing a primary amine which may be covalently modified by a biotin species containing an NHS-ester. This method may be used to make a siloxane layer with a tightly controlled thickness of about 1 to about 10 nm with biotin on the surface. Ultra-sensitive fluorescent immunoassay [0067] The present disclosure is related to an ultrasensitive fluorescent immunoassay. The assay includes providing an immunoassay comprising an analyte and at least one detection ^92677326.1 - 12 - Atty Docket No.108036-780316 antibody specifically bound to the analyte, adding to the immunoassay a plasmonic nanostructure having a localized surface plasmon resonance wavelength (λLSPR) wherein the plasmonic nanostructure specifically binds to the detection antibody, adding at least one fluorescent molecule having a maximum excitation wavelength (λEX) wherein the difference between λLSPR and λEX is less than 50 nm and the at least one fluorescent molecule specifically binds to the plasmonic nanostructure, and exposing the fluorescent immunoassay to a wavelength of light suitable to excite the at least one fluorescent molecule and measuring the resultant fluorescence signal. The assay may be a plasmon enhanced immunoassay. [0068] As illustrated in Figure 2, in some embodiments the ultrasensitive fluorescent immunoassay may utilize a biotinylated plasmonic nanostructure to bind an immunoassay complex containing a biotinylated detection antibody bound with streptavidin. The streptavidin may or may not be fluorescent. As illustrated, each plasmonic nanostructure may contain two or more biotin molecules. For example, each plasmonic nanostructure may contain about at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 biotin molecules. Because streptavidin has 4 binding sites available, it may act as a bridge between the biotinylated detection antibody and the biotinylated plasmonic nanostructure. If the streptavidin is fluorescently labeled with a fluorophore having an excitation maximum (λEX), its fluorescence will be enhanced when a biotinylated plasmonic nanostructure with an λLSPR is bound, assuming the difference between a λEX and a λLSPR is less than about 50 nm. In an ideal scenario, the difference between a λEX and a λLSPR is less than about 20 nm with the λLSPR being between the dominant wavelength of an excitation source and λEX of the fluorescent molecule. An example of an excitation source may be a laser. To achieve even further signal enhancement, additional fluorescent streptavidin may be added which would then bind the biotinylated plasmonic nanostructure and all of the fluorescent streptavidins would be enhanced. [0069] Referring to FIGS 1A-1C, plasmonic nanostructures may be capable of binding many fluorescent species. For example, plasmonic nanostructures may bind to at least about 2, at least about 5, at least about 10, at least about 20, or at least about 25 per fluorescent species. In some embodiments, unbound fluorescent species may be removed at this step, the system may be irradiated with an appropriate wavelength of light to excite the fluorescent species, and fluorescent signal may be collected to complete the assay. In some embodiments, the plasmonic amplification may be continued by adding biotinylated nanostructures followed by ^92677326.1 - 13 - Atty Docket No.108036-780316 more fluorescent streptavidin. The wash and read steps may be subsequently performed to complete the fluoroimmunoassay. In some embodiments, this process may be repeated multiple times to achieve even greater fluorescent signal per capture antibody-analyte- detection antibody complex, which may result in significantly improved assay sensitivity relative to an equivalent fluoroimmunoassay that does not utilize plasmonic enhancement. [0070] As shown in FIG. 2, a target analyte may be localized to a substrate surface to which a capture antibody may be attached. In the next step, a biotinylated detection antibody may be added which specifically binds the target analyte. In the next step, a fluorescent streptavidin may be added which specifically binds to the biotinylated detection antibody. In the next step, a biotinylated plasmonic nanostructure may be added which specifically binds the fluorescent streptavidin. Finally, fluorescent strepavidins may be added which may bind to the biotinylated plasmonic nanostructure. When unbound fluorescent species are removed, the assay may be irradiated with an appropriate wavelength of light suitable to excite the fluorescent streptavidin and the emitted fluorescent signal may be collected. The fluorescent signal may be significantly higher in the fluoroimmunoassay containing the plasmonic nanostructure as compared to the fluoroimmunoassay that does not contain the plasmonic nanostructure. [0071] In another embodiment of the ultrasensitive fluorescent immunoassay, it may be possible to functionalize the plasmonic nanostructure with streptavidin and add it to an immunoassay containing biotinylated detection antibody as shown in FIG.3. The streptavidin may or may not be fluorescently labeled. If the streptavidin is not fluorescent as depicted in FIG.3, the assay will not be fluorescent until fluorescently labeled biotin is added which may localize to the streptavidin-functionalized plasmonic nanostructures. Each nanostructure may bind to two or more fluorescently labeled biotin molecules. For example, each nanostructure may bind to at least about 2, at least about 5, at least about 10, at least about 20, or at least about 25 biotin molecules. [0072] A target analyte may be localized to a substrate surface to which a capture antibody may be attached. In the next step, a streptavidin-conjugated plasmonic nanostructure may be added which specifically binds to the biotinylated detection antibody. Finally, fluorescently-labeled biotin may be added which binds specifically to the streptavidin- conjugated plasmonic nanostructure. In some embodiments, the unbound fluorescent species may be removed at this step. The system may be irradiated with an appropriate wavelength of light to excite the fluorescent species. The emitted fluorescent signal may be collected to complete the assay. When unbound fluorescent species may be removed, the assay may be ^92677326.1 - 14 - Atty Docket No.108036-780316 irradiated with an appropriate wavelength of light suitable to excite the fluorescent streptavidin and the emitted fluorescent signal may be collected. The fluorescent signal may be significantly higher in the fluoroimmunoassay containing the plasmonic nanostructure as compared to the fluoroimmunoassay wherein the streptavidin bound to the detection antibody may be fluorescent or wherein the fluorescently labeled biotin may be added without a plasmonic nanostructure. [0073] In some embodiments, the plasmonic amplification strategy may be continued by adding even more biotinylated nanostructures followed by more fluorescent streptavidin and then perform the wash and read steps to complete the fluoroimmunoassay. In some embodiments, this process may be repeated multiple times to achieve even greater fluorescent signal per capture antibody-analyte-detection antibody complex, which can result in significantly improved assay sensitivity relative to an equivalent fluoroimmunoassay that does not utilize plasmonic enhancement. [0074] FIGS. 4A and 4B illustrate plasmonic-enhanced ultrasensitive fluoroimmunoassays in some embodiments. A plasmonic nanostructure may be functionalized with biotinylated detection antibodies which may bind the target analyte directly. In this example, fluorescent streptavidin may then be added which would bind to the plasmonic nanostructure through the biotinylated detection antibodies yielding an ultrabright plasmonic- amplified fluorescent complex. Each plasmonic nanostructure may bind to at least two fluorescently-labeled streptavidins. For example, each plasmonic nanostructure may bind to at least about 2, at least about 5, at least about 10, at least about 20, or at least about 25 fluorescently-labeled streptavidin molecules. In some embodiments, the unbound fluorescent species may be removed at this step. The system may be irradiated with an appropriate wavelength of light to excite the fluorescent species. The emitted fluorescent signal may be collected to complete the assay. In some embodiments, the plasmonic amplification strategy may be continued by adding more biotinylated nanostructures followed by more fluorescent streptavidin and then perform the wash and read steps to complete the fluoroimmunoassay. This process may be repeated multiple times to achieve even greater fluorescent signal per capture antibody-analyte-detection antibody complex, which can result in significantly improved assay sensitivity relative to an equivalent fluoroimmunoassay that does not utilize plasmonic enhancement. [0075] In some aspects, the present disclosure further includes an ultrasensitive fluorescent immunoassay for the detection of nucleic acids. In a non-limiting example, an oligonucleotide complementary to a target oligonucleotide may be attached to a surface. This ^92677326.1 - 15 - Atty Docket No.108036-780316 may be referred to as the capture oligo. When a sample containing the target oligonucleotide is added to the assay, the target oligonucleotide may bind specifically to the capture oligo. Another oligonucleotide that is complementary to another portion of the target oligonucleotide may be added which may also specifically bind to the target oligonucleotide. This oligonucleotide is called a detection oligo. One may recognize that this structure, capture oligo- target oligonucleotide-detection oligo is analogous to the sandwich immunoassay complex. The capture oligo or detection oligo may be a peptide nucleic acid. The detection oligo may be biotinylated. In this case, one may use a streptavidin-conjugated plasmonic nanostructure followed by fluorescently labeled biotin to make an ultrasensitive fluorescent immunoassay for the target oligonucleotide. Analogous to the example described above for the immunoassay in FIG. 4, one could also directly attach the biotinylated target oligo to the plasmonic nanostructure. Finally, in the case of an assay for RNA in particular, it may be possible to utilize an antibody that is specific to a DNA-RNA complex such as the antibody known as S9.6. In this case, one may utilize a capture DNA oligo which may be complementary to the target RNA. An antibody such as S9.6 may be added which will specifically bind the DNA-RNA complex. At this point, S9.6 may be detection antibody and the plasmonic enhanced fluoroimmunoassay concepts such as those described above may be utilized to make an ultrasensitive fluorescent immunoassay. Plasmonic-enhanced fluorescent lateral flow assay: [0076] In one aspect, the present disclosure is directed to an ultrasensitive fluorescent lateral flow assay. Examples of the lateral flow assay utilizing plasmonic enhancement are provided in FIG.7 and FIG.8. Lateral flow assays are immunochromatographic assays which are commonly utilized in diagnostics. They are typically comprised of: a sample pad to which a sample is added; a conjugate pad containing a reporter molecule that is functionalized to specifically bind a target analyte in the sample typically with a detection antibody; a nitrocellulose membrane which contains a region with a capture antibody specific to the target analyte is bound; and an absorbent pad which facilitates capillary driven flow. The concept is that passive capillary flow introduced into the system after adding a liquid sample occurs from the sample pad to the absorbent pad. During the flow, the different immunoassay components are solubilized and allowed to interact while transported along the strip. Complexes containing the target analyte bound to the reporter molecule are captured at the test line and unbound reporters are transported past the test line to the absorbent pad. Accumulation of reporter molecules at the test line leads to the generation of some signal which is detectable and which is proportional to the amount of target analyte bound. Traditional lateral flow immunoassays ^92677326.1 - 16 - Atty Docket No.108036-780316 typically use antibody conjugated gold nanoparticles as a reporter molecule. These nanoparticles strongly absorb light and show up as a colorimetric signal on the test line. Though this signal can be read visually, the disadvantage is that the method is not very sensitive. The examples provided herein utilize the lateral flow framework, but utilize plasmon-enhanced fluorescence make an ultrasensitive lateral flow fluorescent immunoassay. [0077] One or more embodiments of the ultrasensitive lateral flow assay include a full- strip ultrasensitive fluorescent lateral flow immunoassay. An example of an ultrasensitive fluorescent lateral flow immunoassay is shown in FIG. 7. Plasmonic amplifiers may be deposited on one pad (conjugate pad 1). Complimentary fluorescent dye labeled conjugates may be deposited on another pad (conjugate pad 2). These two pads may be assembled with a sample pad, nitrocellulose membrane and absorption pad to form a lateral flow strip. Sample can be directly added to the sample pad and the analytes in the sample will bind with plasmonic amplifier followed by the binding of dye labeled conjugate to form an ultrabright fluorescent construct. This ultrabright fluorescent construct will be capture by the capture antibody localized to the test line. [0078] One or more embodiments of the ultrasensitive lateral flow assay include a premix/drop. An example of an ultrasensitive fluorescent lateral flow immunoassay is shown in FIG. 8. Plasmonic amplifiers may be incubated with sample for a time sufficient to allow binding to a target analyte. This solution may then be added to the sample pad and the analyte- bound plasmonic amplifier will flow into a region containing fluorescently labeled species which bind specifically to the plasmonic amplifier forming an ultrabright fluorescent complex. This complex may then flow in the test line where it binds specifically to the attached capture antibody. [0079] One or more embodiments of the ultrasensitive lateral flow assay include a sequential addition. An example of an ultrasensitive fluorescent lateral flow immunoassay is shown in FIG.9. In the full strip embodiment of plasmonic-enhanced fluorescent lateral flow immunoassays, shown in FIG.9, plasmonic amplifiers may be deposited on one pad (conjugate pad). This conjugate pad may be assembled with a sample pad, nitrocellulose membrane and absorption pad to form a lateral flow strip. Sample solution may be added on the sample pad and the analytes bind to the plasmonic amplifier via the capture antibody on the test line of the nitrocellulose membrane. Fluorescently labeled species which specifically bind the plasmonic amplifier may be then added to the lateral flow immunoassay where they bind to the plasmonic amplifiers to form ultrabright fluorescent constructs at the test line. ^92677326.1 - 17 - Atty Docket No.108036-780316 [0080] One or more embodiments of the ultrasensitive lateral flow assay include a serology full-strip. An example of an ultrasensitive fluorescent lateral flow immunoassay is shown in FIG.10. In the serology full-strip version of plasmonic-enhanced fluorescent lateral flow immunoassays shown in Figure 10, plasmonic amplifiers may be deposited on one pad (conjugate pad 1). Fluorescently-labeled species which specifically bind the plasmonic amplifiers may be deposited on another pad (conjugate pad 2). These two pads may be assembled with a sample pad, nitrocellulose membrane and absorption pad to form a lateral flow strip. A target analyte is deposited on the test line. The sample containing the antibody of interest (e.g. IgG, IgM, IgA, IgE) may be directly added to the sample pad and the antibodies in the sample will bind with the plasmonic amplifier conjugated to anti-target antibodies followed by the binding of a fluorescently labeled species which specifically binds the plasmonic amplifier to form an ultrabright fluorescent construct on the test line. [0081] The lateral flow immunoassay format is just one example of a system which may be used to perform the plasmonic-enhanced fluorescent assay. This is an attractive platform because of its simplicity and the passive nature of the assay wherein one simply adds sample, waits a sufficient amount of time for the assay to complete, and then reads the result. Any system or device that can automatically transport fluid to enable the combination of the reagents as illustrated in Figures 2-4 or described in the text above can be utilized to implement a plasmonic-enhanced fluorescent assay. Another attractive platform that can accomplish this is a microfluidic system, either utilizing passive flow or active flow. In a non-limiting example, a sample containing an analyte could be added to a chip containing microfluidic channels. The sample could be transported to a region of the chip containing an attached capture antibody which specifically binds the target analyte. The system could then transport biotinylated detection antibodies to the capture region where the biotinylated detection antibodies would specifically bind the target analyte. Next, fluorescent streptavidin could be transported to the capture region where it would bind the biotinylated detection antibody. Next, biotinylated plasmonic amplifiers could be transported to the capture region where they would bind the fluorescent streptavidin resulting in an ultrabright fluorescent complex. In an ideal system, each step of the assay would be followed by a wash step to reduce any non-specific background. Finally, the system could perform the plasmonic amplification step multiple times (addition of biotinylated plasmonic amplifiers followed by addition of fluorescent streptavidin) to achieve the signal-to-noise necessary for detection of the analyte if it is present in very low abundance. In addition, if the device is in a system which is capable of reading the assay (exciting with an appropriate wavelength of light and collecting emitted fluorescence), the assay can be ^92677326.1 - 18 - Atty Docket No.108036-780316 interrogated at each step wherein the fluorescent streptavidin is added and then unbound fluorescent streptavidin is removed. This would yield an assay with extreme dynamic range which is easily able to detect high abundance analytes before plasmonic enhancement and low abundance analytes after plasmonic enhancement. EXAMPLES Example 1 [0082] Biotinylated BSA (BB) was added to a microtiter plate at various concentrations and allowed to incubate for 30 minutes before being washed off. The BB serves as a proxy for an immunoassay complex containing a bound biotinylated detection antibody. After adding streptavidin labeled with the fluorescent dye 800CW (800CW-strep) having a λEX of about 780 nm, washing off unbound streptavidin, and reading with a laser scanner utilizing a 784 nm laser, the assay showed a concentration dependence of the fluorescent signal. After adding a biotinylated plasmonic nanostructure (in this example AuNR@Ag with an λLSPR of about 780 nm) and reading the assay again the fluorescent signal increased slightly due to plasmonic amplification of the previously bound 800CW-streps. FIG. 5A shows a schematic illustration of an assay without plasmonic nanoparticles. FIGS. 5B and 5C are schematic illustrations of steps in the disclosed ultrasensitive [0083] The fluorescent increase was at most 3-4 fold depending on the BB concentration. However, when additional 800CW-strep was added and unbound 800CW-strep removed, the assay was read again and the fluorescence increased more than 100-fold and saturated the 16-bit detector at the three highest concentrations. The signal-to-noise ratio (as defined by the signal for a given concentration of BB over the signal of the blank) was higher for the plasmon-enhanced assay at a BB concentration of 5 ng/mL than that of the unenhanced assay at a BB concentration of 500 ng/mL. All measurements were completed with exactly the same excitation and collection conditions. FIGS. 5B and 5C show fluorescent images and quantitative data extracted from the fluorescent images respectively. [0084] FIGS. 6A and 6B shows an example application of a biotinylated detection antibody functionalized plasmonic nanostructure for an ultrasensitive fluoroimmunoassay relative to a control. Capture antibody (anti-human IL6, L395, Hytest Ltd) was printed on a microtiter plate and allowed to incubate for 15 hr before being washed off. Serial dilutions of human IL6 of known concentration (1000 pg/ml to 457 fg/ml, in 5% milk, 1X tris-buffered saline, 0.01% Triton-X 100) were employed as standards. Standards were incubated with the capture antibody coated plate for 3 hours. After incubation of the standards, the solution was ^92677326.1 - 19 - Atty Docket No.108036-780316 removed and detection solution was added (3% BSA in 1X phosphate buffered saline) wherein the detection solution contained either plasmonic nanostructures having a λLSPR of about 650 nm (plasmonic amplifiers, PA) and which were functionalized with biotinylated detection antibody (anti-human IL6, L152, Hytest Ltd) or the biotinylated detection antibody alone. After incubation for 1 hour, the detection solution was removed, and the wells were washed 3X with 200 uL of 1X phosphate buffered saline containing 0.05% Triton X-100. Next, streptavidin labeled with IR650 (Strep-dye) having a λEX of about 650 nm was added and bound specifically to the biotinylated detection antibodies. After removing the solution containing the Strep-dye, the assay was washed 3X with 200 uL of 1X phosphate buffered saline containing 0.05% Triton X-100 and then read using a laser scanner with a 650 nm excitation. The LOD of the unenhanced fluoroimmunoassay was calculated to be 5.455 pg/ml and the LOD of plasmonic-enhanced fluoroimmunoassay was calculated to be 0.055 pg/ml, achieving a 100- fold improvement. Example 2 [0085] Recombinant human IL-6 was spiked in 1X phosphate buffered saline and diluted to a series of concentrations: 1000 pg/ml, 200 pg/ml, 40 pg/ml, 8 pg/ml. These solutions and a blank solution containing buffer but no IL-6 were added to separate lateral flow strips of the design shown in FIG.7. The plasmonic amplifiers were conjugated to biotinylated anti-IL- 6, the fluorescent species was strep-800CW, and the test line contained anti-IL-6 capture antibodies. The control line comprised biotinylated BSA. The lateral flow strips were read 20 minutes after adding the sample using a laser scanner exciting the samples at 784 nm and collecting emission through a 832nm bandpass filter with a full-width at half max of 37 nm. The analytical performance of the plasmonic-enhanced fluorescent lateral flow immunoassay for IL-6 is plotted in FIG.11 and the LOD of the test is about 1.43 pg/ml. This is comparable or even better than a standard laboratory test for IL-6 called enzyme-linked immunosorbent assay (ELISA) that takes at least 4 hours and multiple steps to run. [0086] While various examples and embodiments have been disclosed, other examples and embodiments will be apparent to those skilled in the art. The various disclosed examples and embodiments are for purposes of illustration and are not intended to be limiting, with the true scope being indicated by the following claims. ^92677326.1 - 20 -

Claims

Atty Docket No.108036-780316 CLAIMS What is claimed is: 1. A method for performing an ultrasensitive fluorescent immunoassay comprising: providing an immunoassay comprising an analyte and at least one detection antibody specifically bound to the analyte; adding to the immunoassay a plasmonic nanostructure having a localized surface plasmon resonance wavelength (λLSPR) wherein the plasmonic nanostructure specifically binds to the detection antibody; adding at least one fluorescent molecule having a maximum excitation wavelength (λEX) wherein the difference between λLSPR and λEX is less than 50 nm and the at least one fluorescent molecule specifically binds to the plasmonic nanostructure; and exposing the fluorescent immunoassay to a wavelength of light suitable to excite the at least one fluorescent molecule and measuring the resultant fluorescence signal. 2. The method of claim 1, wherein the fluorescent signal intensity from the fluorescent immunoassay is more than 100-fold greater than a fluorescent immunoassay where the at least one detection antibody is conjugated directly to the at least one fluorescent molecule. 3. The method of claim 1, wherein the at least one detection antibody comprises a tag. 4. The method of claim 1, wherein the at least one detection antibody comprises at least one fluorescent molecule. 5. The method of claim 3, wherein the plasmonic nanostructure is functionalized with at least one molecule that can specifically bind the tag. 6. The method of claim 3, wherein the tag is biotin. 7. The method of claim 6, wherein the plasmonic nanostructure is functionalized with at least one biotin-binding molecule, wherein the at least one biotin-binding molecule comprises streptavidin, neutravidin, or avidin. ^92677326.1 - 21 - Atty Docket No.108036-780316 8. The method of claim 7, wherein the at least one biotin-binding molecule comprises at least about 2, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 biotin-binding molecules. 9. The method of claim 1, further comprising providing a capture antibody specific to the analyte. 10. The method of claim 1, wherein the plasmonic nanostructure comprises a gold nanorod coated with silver (AuNR@Ag). 11. A diagnostic test system comprising: a sample region for adding a sample containing at least one type of analyte; a test region comprising at least one capture antibody that specifically binds the analyte; a region containing a detection antibody comprising a tag; a conjugate region comprising a plasmonic nanostructure functionalized to specifically bind the tag; and at least one fluorescent reagent region containing a fluorescent molecule that specifically binds the plasmonic nanostructure. 12. The diagnostic test of claim 11, wherein the plasmonic nanostructure has a localized surface plasmon resonance wavelength (λLSPR), wherein the fluorescent molecule has a maximum excitation wavelength (λEX), wherein the difference between λLSPR and λEX is less than 50 nm. 13. The diagnostic test of claim 11, wherein the at least one detection antibody comprises a tag. 14. The diagnostic test of claim 13, wherein the at least one detection antibody comprises at least one fluorescent molecule. 15. The diagnostic test of claim 13, wherein the plasmonic nanostructure is functionalized with at least one molecule that can specifically bind the tag. 16. The diagnostic test of claim 13, wherein the tag is biotin. ^92677326.1 - 22 - Atty Docket No.108036-780316 17. The diagnostic test of claim 16, wherein the plasmonic nanostructure is functionalized with at least one biotin-binding molecule, wherein the at least one biotin-binding molecule comprises streptavidin, neutravidin, or avidin. 18. The diagnostic test of claim 17, wherein the at least one biotin-binding molecule comprises at least about 2, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 biotin-binding molecules. 19. The diagnostic test of claim 11, wherein the plasmonic nanostructure comprises a gold nanorod coated with silver (AuNR@Ag). ^92677326.1 - 23 -