Chemiluminescent immunoassays enhanced using plasmonic nanostructures

Abstract

Described herein is a chemiluminescent nanoconstruct. The chemiluminescent nanoconstruct structure may include a plasmonic nanostructure comprising having at least one localized surface plasmon resonance wavelength (λLSPR), at least one spacer coating, at least one chemiluminescent agent having a maximum emission wavelength (λEM), and at least one biorecognition element. The chemiluminescent nanoconstruct structure has a signal that is at greater than the signal of the at least one chemiluminescent agent alone.

Description

CHEMILUMINESCENT IMMUNOASSAYS ENHANCED USING PLASMONICNANOSTRUCTURESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is related to and claims priority under 35 U.S.C. § 119(e) from U.S. Patent Application No. 63 / 535, 153 filed August 29, 2023, titled “CHEMILUMINESCENT IMMUNOASSAYS ENHANCED USING PLAMSONIC NANOSTRUCTURES," which is hereby incorporated by reference in its entirety.FIELD OF INVENTIONS

[0002] The present disclosure is directed to chemiluminescent nanoconstructs for enhanced chemiluminescent immunoassays.BACKGROUND

[0003] The field of the disclosure relates generally to amplification of the signal-to-noise ratio of Chemiluminescent immunoassays (CLIA) by using plasmonic nanostructures.

[0004] 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 interrogate the component molecules accurately and quantitatively. 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.

[0005] Chemiluminescence (CL) is a useful analytical tool for the detection and quantification of a wide variety of biological materials. The usefulness of chemiluminescence is due to its simplicity and the absence of unwanted background luminescence. In chemiluminescence-based detection, no excitation source and no optical filters are required as compared to other optical techniques such as fluorescence and phosphorescence spectroscopy. Chemiluminescent-based assay, e g. chemiluminescent immunoassay (CLIA) is an immunoassay technique where the label, i.e. the true “indicator” of the analytic reaction, is a luminescent molecule. In general, luminescence is the emission of visible or near-visible (X = 300-800 nm) radiation which isgenerated when an electron transitions from an excited state to ground state. The resultant potential energy in the atom gets released in the form of light. Chemiluminescence emission is generated by photochemical reactions and is directly related to the concentration of the reactants. The chemical reactions involve the oxidation of a chemiluminescent agent by a strong oxidizing agent in the presence of a catalyst (chemical or biological). Chemiluminescent methods can be direct- using luminophore markers or indirect- using enzyme markers. In the direct methods, the luminophore markers used are acridinium and ruthenium esters with an oxidizing agent added to start the chemiluminescent reaction, while the enzymatic markers used in indirect methods are alkaline phosphatase (AP) with adamantyl 1, 2-dioxetane phenylphosphate (AMPPD) substrate and horseradish peroxidase (HRP) with luminol or acridan and their derivatives as substrates.

[0006] Immunoassays based on conventional chemiluminescence (CLIAs) are limited by weak signals and low intensity for a given observation period. The invention disclosed herein is a method of increasing luminescence signals by utilizing plasmonic-CL enhancers. The enhancers play a mediator role in the CL reaction by boosting the electronic activation, which leads to significantly increased chemiluminescence and markedly elevated analytical sensitivity of CLIAs.

[0007] Metal -enhanced chemiluminescence (MEC) was first introduced in 2006. In MEC, the excited state energy of chemiluminescent species in close-proximity (<100 nm) to metal nanoparticles is partially transferred (coupling) to surface plasmons and is subsequently emitted with the identical spectral characteristics of the chemiluminescent species in addition to the uncoupled unperturbed free-space emission. This results in a significant increase in emission intensity, which is attributed to a plasmon-based luminescent enhancement. Potentially, MEC can be used to significantly improve the sensitivity and the rapidity of the chemiluminescent technology. However, most of the MEC strategies rely on metal films serving as a substrate of the chemiluminescent assays. Using metal films not only increases the cost of the assay but also lacks the flexibility of easy-integration into existing assay formats. Moreover, this strategy has only resulted in fairly limited enhancement. Metal-nanoparticles or plasmonic nanoparticles have been used in CL assays, but have not utilized MEC. Rather, these plasmonic nanoparticles have simply acted as a catalyst for the CL reaction, and these have shown only modest improvement compared to the plasmonic-enhanced CL invention disclosed herein.

[0008] Disclosed herein are compositions of novel plasmonic nanoparticle constructs which can be used as a label for plasmonic-enhanced chemiluminescent immunoassays (PE-CLIAs), the methods of making the plasmonic nanoparticle constructs, and the methods of using them.

[0009] Importantly, the methods disclosed herein are generally applicable to a wide range of chemiluminescent immunoassay formats (direct and indirect) and assay surfaces (plate-based, membrane-based, bead-based, microfluidic-based, and even immunochromatographic assays such as lateral flow assays which are commonly used in diagnostic assays).BRIEF SUMMARY

[0010] The summary section 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.

[0011] The present disclosure is directed to a chemiluminescent nanoconstruct. The chemiluminescent nanoconstruct includes a plasmonic nanostructure having at least one localized surface plasmon resonance wavelength (XLSPR), at least one chemiluminescent agent having a maximum emission wavelength (XEM), and at least one biorecognition element. The chemiluminescent nanoconstruct may have a signal that is at least 20 times greater than a signal of the at least one chemiluminescent agent alone in the presence of an appropriate oxidizing agent such as alkaline hydrogen peroxide to trigger chemiluminescence of the chemiluminescent agent. The difference between the at least one LSPR and the XEM may be less than 50 nm.

[0012] In some embodiments, the plasmonic nanostructure is conjugated with the at least one chemiluminescent agent and the at least one biorecognition element.

[0013] In some embodiments, the plasmonic nanostructure is conjugated with at least one biorecognition element and the at least one biorecognition element is conjugated with the at least one chemiluminescent agent.

[0014] The chemiluminescent nanoconstruct may further include at least one spacer coating.

[0015] In some aspects, the plasmonic nanostructure is coated with the at least one spacer coating and the at least one spacer coating is conjugated with the at least one chemiluminescent agent. In yet other aspects, the at least one biorecognition element is conjugated to the at least one spacer coating. In yet other aspects, the at least one biorecognition element is conjugated tothe at least one spacer coating, and the chemiluminescent agent is conjugated to the at least on biorecognition element.

[0016] In some aspects, the plasmonic nanostructure is a gold nanorod coated with silver (AuNR@Ag).

[0017] In some aspects, the spacer coating comprises one or more of silane, amine, alkyl, or hydroxyl functional groups. In some embodiments, the spacer coating is one or more silane. In other embodiments, the silane comprises (3 -aminopropyl) trimethoxysilane (APTMS), trimethoxy(propyl)silane (TMPS), and / or a combination thereof.

[0018] In some aspects, wherein the spacer coating is on the plasmonic nanostructure and has a thickness of from about 0. 1 nm to about 20 nm.

[0019] In some aspects, the chemiluminescent agents comprise acridinium esters, ruthenium esters, or combinations thereof.

[0020] In some aspects, the biorecognition element includes one or more antibody, functional fragments of an antibody, oligonucleotides, aptamers, or combinations thereof. In some embodiments, the one or more antibody is functionalized with biotin. In other embodiments, the one or more antibody comprises streptavidin, neutravidin, or avidin.

[0021] The present disclosure is further directed to a chemiluminescent enhancer nanoconstruct. The chemiluminescent enhancer nanoconstruct includes a plasmonic nanostructure having at least one localized surface plasmon resonance wavelength (XLSPR), at least one enzyme capable of catalyzing the reaction of a substrate to emit light with a maximum emission wavelength (XEM), and at least one biorecognition element. The chemiluminescent enhancer nanoconstruct may produce a signal in the presence of substrate that is at least 20 times greater than a signal of the at least one enzyme and substrate alone. The difference between the at least one XLSPR and the XEM may be less than 50 nm.

[0022] In some embodiments, the plasmonic nanostructure is conjugated with the at least one enzyme capable of catalyzing the reaction of a substrate to emit light with a maximum emission wavelength (ZEM) and the at least one biorecognition element.

[0023] In some embodiments, the plasmonic nanostructure is conjugated with at least one biorecognition element and the at least one enzyme capable of catalyzing the reaction of a substrate to emit light with a maximum emission wavelength ( EM).

[0024] The chemiluminescent enhancer nanoconstruct may further include at least one spacer coating.

[0025] In some aspects, the plasmonic nanostructure is coated with the at least one spacer coating and the at least one enzyme capable of catalyzing the reaction of a substrate to emit light with a maximum emission wavelength ( EM). In yet other aspects, the at least one biorecognition element is conjugated to the at least one spacer coating. In yet other aspects, the biorecognition element is conjugated to the at least one spacer coating and the at least one enzyme capable of catalyzing the reaction of a substrate to emit light with a maximum emission wavelength (XEM).

[0026] In some aspects, the plasmonic nanostructure is a gold nanorod coated with silver (AuNR@Ag).

[0027] In some aspects, the spacer coating comprises one or more of silane, amine, alkyl, or hydroxyl functional groups. In some embodiments, the spacer coating is one or more silane. In other embodiments, the silane comprises (3 -aminopropyl) trimethoxysilane (APTMS), trimethoxy(propyl)silane (TMPS), and / or a combination thereof.

[0028] In some aspects, wherein the spacer coating is on the plasmonic nanostructure and has a thickness of from about 0.1 nm to about 20 nm.

[0029] In some aspects, the at least one enzyme capable of catalyzing the reaction of a substrate to emit light with a maximum emission wavelength (XEM) comprises alkaline phosphatase (AP) and the substrate is adamantyl 1, 2-dioxetane phenylphosphate (AMPPD). In yet other aspects, the at least one enzyme capable of catalyzing the reaction of a substrate to emit light with a maximum emission wavelength (XEM) comprises horseradish peroxidase (HRP) with luminol or acridan and their derivatives as substrates.

[0030] In some aspects, the biorecognition element includes one or more antibody, functional fragments of an antibody, oligonucleotides, aptamers, or combinations thereof. In some embodiments, the one or more antibody is functionalized with biotin. In other embodiments, the one or more antibody comprises streptavidin, neutravidin, or avidin.

[0031] The present disclosure is further directed to a method for enhancing a chemiluminescent immunoassay. The method includes adding a chemiluminescent nanoconstruct to an immunoassay. The chemiluminescent nanoconstruct includes a plasmonic nanostructure having at least one localized surface plasmon resonance wavelength (1LSPR), at least onechemiluminescent agent having a maximum emission wavelength ( EM), and at least one biorecognition element. The chemiluminescent nanoconstruct optionally includes at least one spacer coating. In some embodiments, the plasmonic nanostructure is coated with the at least one spacer coating. In some embodiments, a difference between the at least one XLSPR and the EM is less than 50 nm. In some embodiments, the spacer coating includes one or more of silane, amine, alkyl, hydroxyl functional groups. In some embodiments, the spacer coating includes one or more silane. In some embodiments, the silane includes (3 -aminopropyl) trimethoxysilane (APTMS), trimethoxy(propyl)silane (TMPS), and / or a combination thereof. In some embodiments, the spacer coating is on the nanostructure and has a thickness of from about 0.1 nm to about 20 nm. In some aspects, the at least one spacer coating is conjugated with the at least one chemiluminescent agent. In some aspects, the at least one biorecognition element is conjugated to the at least one spacer coating. In some aspects, the chemiluminescent agents include acridinium esters, ruthenium esters, or combinations thereof. In some aspects, an appropriate oxidizing agent is added to trigger chemiluminescence of the chemiluminescent agent.

[0032] In some aspects, the biorecognition element includes one or more antibody, functional fragments of an antibody, oligonucleotides, aptamers, or combinations thereof. In some embodiments, the one or more antibody is functionalized with biotin. In some embodiments, the one or more antibody includes streptavidin, neutravidin, or avidin.

[0033] In some embodiments, the plasmonic nanostructure is a gold nanorod coated with silver (AuNR@Ag).

[0034] In some aspects, the plasmonic nanostructures are functionalized with acridinium or ruthenium (or their derivatives).

[0035] In some aspects, the chemiluminescent nanoconstruct has a signal that is at least 20 times greater than a signal of the at least one chemiluminescent agent alone in the presence of an appropriate oxidizing agent to trigger chemiluminescence of the chemiluminescent agent.

[0036] In some aspects, limit of detection of the chemiluminescent immunoassay is enhanced with the chemiluminescent nanoconstruct by a factor of at least 10 relative to the an immunoassay without the chemiluminescent nanoconstruct.

[0037] The present disclosure is further directed to a method for enhancing a chemiluminescent immunoassay. The method includes adding a chemiluminescent enhancer nanoconstruct to an immunoassay containing a chemiluminescent substrate. The chemiluminescent enhancernanoconstruct includes a plasmonic nanostructure having at least one localized surface plasmon resonance wavelength (XLSPR), at least one enzyme capable of catalyzing the reaction of a substrate to emit light with a maximum emission wavelength (1EM), and at least one biorecognition element. The chemiluminescent enhancer nanoconstruct optionally includes at least one spacer coating. In some embodiments, the plasmonic nanostructure is coated with the at least one spacer coating. In some embodiments, a difference between the at least one XLSPR and the XEM is less than 50 nm. In some embodiments, the spacer coating includes one or more of silane, amine, alkyl, hydroxyl functional groups. In some embodiments, the spacer coating includes one or more silane. In some embodiments, the silane includes (3 -aminopropyl) trimethoxysilane (APTMS), trimethoxy(propyl)silane (TMPS), and / or a combination thereof. In some embodiments, the spacer coating is on the nanostructure and has a thickness of from about 0.1 nm to about 20 nm. In some aspects, the at least one spacer coating is conjugated with the at least one enzyme capable of catalyzing the reaction of a substrate to emit light with a maximum emission wavelength (XEM). In some aspects, the at least one biorecognition element is conjugated to the at least one spacer coating. In some aspects, the enzyme capable of catalyzing the reaction of a substrate to emit light with a maximum emission wavelength (XEM) comprises horseradish peroxidase or alkaline phosphatase. In some aspects the chemiluminescent substrate comprises luminol, acridan or AMPPD (or their derivatives).

[0038] In some aspects, the biorecognition element includes one or more antibody, functional fragments of an antibody, oligonucleotides, aptamers, or combinations thereof. In some embodiments, the one or more antibody is functionalized with biotin. In some embodiments, the one or more antibody includes streptavidin, neutravidin, or avidin.

[0039] In some embodiments, the plasmonic nanostructure is a gold nanorod coated with silver (AuNR@Ag).

[0040] In some aspects, the chemiluminescent enhancer nanoconstruct may produce a signal in the presence of substrate that is at least 20 times greater than a signal of the at least one enzyme and substrate alone.

[0041] In some aspects, limit of detection of the chemiluminescent immunoassay is enhanced with the chemiluminescent enhancer nanoconstruct by a factor of at least 10 relative to the an immunoassay without the chemiluminescent enhancer nanoconstruct.BRIEF DESCRIPTION OF THE FIGURES

[0042] Figure 1 shows a schematic depiction of metal-enhanced chemiluminescence (MEC). In MEC, the excited state energy of chemiluminescent species in close-proximity to metal nanoparticles is partially transferred (coupling) to surface plasmons and is subsequently emitted with the identical spectral characteristics of the chemiluminescent species in addition to the uncoupled unperturbed free-space emission. This results in a significant increase in emission intensity, which is attributed to a plasmon-based luminescent enhancement. It is pertinent to note that surface plasmons are the free electrons in metals that collectively oscillate at frequencies similar to light in the UV to visible to near-IR spectrum.

[0043] Figure 2 shows a schematic example of the preparation of the plasmonic amplifier according to some embodiments of the present disclosure. AuNRs are first synthesized followed by silver coating to obtain NR@Ag. A silane-based spacer layer is then deposited around the NR@Ag to finish the base construct of the plasmonic amplifier, it is then ready to be functionalized.

[0044] Figures 3A-3C show a few spectra of plasmonic amplifiers according to some embodiments of the present disclosure. PA430 has maximum LSPR peak overlapping with the emission spectrum of Luminol; PA450 has maximum LSPR peak overlapping with the emission spectrum of Acridan, AMPPD, Acridinium ester and their derivatives; PA650 has maximum LSPR peak overlapping with the emission spectrum of Ruthenium and its derivatives.

[0045] Figures 4A-4B show exemplary embodiments of a plasmonic nanostructure functionalized to different targeting agents for use in enhancing enzyme-based chemiluminescent immunoassays: A) functionalized with antibody conjugated with HRP (AP); B) functionalized with streptavidin conjugated with HRP (AP).

[0046] Figures 5A-5B show an illustration of the application of plasmonic amplifiers functionalized with antibodies conjugated with HRP (AP) in chemiluminescent immunoassay according to some embodiments of the present disclosure. The functionalized plasmonic amplifier can directly bind to a target analyte captured on a surface. Substrate is then added for chemiluminescence generation.

[0047] Figures 6A-6B show an illustration of the application of plasmonic amplifiers functionalized with streptavidin conjugated with HRP (AP) in chemiluminescent immunoassay according to some embodiments of the present disclosure. The functionalized plasmonic amplifier can bind to a biotinylated detection antibody which binds to a target analyte captured on a surface. Substrate is then added for chemiluminescence generation.

[0048] Figures 7A-7B show an illustration of the application of plasmonic amplifiers functionalized with a secondary antibody conjugated with HRP (AP) in chemiluminescent immunoblot (western blot) according to some embodiments of the present disclosure. The functionalized plasmonic amplifier can bind to a primary antibody which detects a target analyte captured on a surface. Substrate is then added for chemiluminescence generation.

[0049] Figures 8A-8B show an illustration of the application of plasmonic amplifiers functionalized with streptavidin conjugated with HRP (AP) in chemiluminescent immunoblot (western blot) according to some embodiments of the present disclosure. The functionalized plasmonic amplifier can bind to a biotinylated secondary antibody which binds to a primary antibody, bound to a target analyte captured on a surface. Substrate is then added for chemiluminescence generation.

[0050] Figures 9A-9B show exemplary embodiments of a plasmonic nanostructure functionalized to different targeting agents for use in enhancing acridinium ester (AE and its derivatives)-based chemiluminescent immunoassays: A) functionalized with antibody labeled with acridinium ester (AE and its derivatives); B) functionalized with streptavidin labeled with acridinium ester (AE and its derivatives) according to some embodiments of the present disclosure.

[0051] Figures 10A-10B show an illustration of the application of plasmonic amplifiers functionalized with antibodies labeled with acridinium ester (AE and its derivatives) in chemiluminescent immunoassay according to some embodiments of the present disclosure. The functionalized plasmonic amplifier can directly bind to a target analyte captured on a surface. Triggering solution (alkaline hydrogen peroxide) is then added for chemiluminescence generation.

[0052] Figure 11 shows an illustration of the application of plasmonic amplifiers functionalized with streptavidin labeled with acridinium ester (AE and its derivatives) in chemiluminescent immunoassay according to some embodiments of the present disclosure. The functionalized plasmonic amplifier can bind to a biotinylated detection antibody which binds to a target analyte. Triggering solution (alkaline hydrogen peroxide) is then added for chemiluminescence generation.

[0053] Figures 12A-12C show exemplary embodiments of a plasmonic nanostructure functionalized to different targeting agents for use in enhancing ruthenium tris-bipyridine (and its derivatives)-based electrochemiluminescent immunoassays: A) functionalized with antibodylabeled with ruthenium tris-bipyridine (and its derivatives); B) functionalized with streptavidin labeled with ruthenium tris-bipyridine (and its derivatives) according to some embodiments of the present disclosure.

[0054] Figures 13A-13B show an illustration of the application of plasmonic amplifiers functionalized with antibodies labeled with ruthenium tris-bipyridine (and its derivatives) in electrochemiluminescent immunoassay according to some embodiments of the present disclosure. The functionalized plasmonic amplifier can directly bind to a target analyte captured on a surface (electrode). Electrochemical reaction is then introduced for chemiluminescence generation.

[0055] Figures 14A-14B show an illustration of the application of plasmonic amplifiers functionalized with streptavidin labeled with ruthenium tris-bipyridine (and its derivatives) in electrochemiluminescent immunoassay according to some embodiments of the present disclosure. The functionalized plasmonic amplifier can bind to a biotinylated detection antibody which binds to a target analyte that is captured on a surface (electrode). Electrochemical reaction is then introduced for chemiluminescence generation.

[0056] Figure 15 shows exemplary embodiments of a plasmonic nanostructure functionalized with tags and biorecognition elements according to some embodiments of the present disclosure. In this case, biotin is used as a tag and antibody is used as a biorecognition element.

[0057] Figure 16 shows an illustration of the application of plasmonic amplifiers functionalized with biotinylated antibodies in (electro) chemiluminescent immunoassay according to some embodiments of the present disclosure.

[0058] Figure 17 is data showing the enhancement that can be achieved in a chemiluminescent immunoassay by employing a plasmonic amplifier functionalized with a biotinylated antibody according to some embodiments of the present disclosure.

[0059] Figure 18 is data showing the enhancement that can be achieved in a chemiluminescent immunoassay by employing a plasmonic amplifier functionalized with streptavidin-HRP according to some embodiments of the present disclosure.

[0060] Figure 19 shows a method of plasmonic-enhanced chemiluminescent Lateral flow assay with plasmonic nanostructures functionalized with antibody-HRP (AP) according to some embodiments of the present disclosure.

[0061] Figure 20 shows a method of plasmonic-enhanced chemiluminescent Lateral flow assay with plasmonic nanostructures functionalized with biotinylated detection antibody according to some embodiments of the present disclosure.

[0062] Figure 21 shows a method of plasmonic-enhanced chemiluminescent Lateral flow assay with plasmonic nanostructures functionalized with antibody-HRP (AP) and streptavidin-HRP (AP) according to some embodiments of the present disclosure.

[0063] Figure 22 shows a method of plasmonic-enhanced chemiluminescent Lateral flow assay for serology test with plasmonic nanostructures functionalized with antibody-HRP (AP) according to some embodiments of the present disclosure.

[0064] Figure 23 shows a method of plasmonic-enhanced chemiluminescent Lateral flow assay for serology test with plasmonic nanostructures functionalized with biotinylated detection antibody according to some embodiments of the present disclosure.

[0065] Figure 24 shows a method of plasmonic-enhanced chemiluminescent Lateral flow assay for serology test with plasmonic nanostructures functionalized with antibody-HRP (AP) and streptavidin-HRP (AP) according to some embodiments of the present disclosure.DETAILED DESCRIPTION

[0066] The disclosure may be understood by reference to the following detailed description, taken in conjunction with the drawings as described below. It is noted that, for purposes of illustrative clarity, certain elements in various drawings may not be drawn to scale. Several variations of the device are presented herein. It should be understood that various components, parts, and features of the different variations may be combined together and / or interchanged with one another, all of which are within the scope of the present application, even though not all variations and particular variations are shown in the drawings. It should also be understood that the mixing and matching of features, elements, and / or functions between various variations is expressly contemplated herein so that one of ordinary skill in the art would appreciate from this disclosure that the features, elements, and / or functions of one variation may be incorporated into another variation as appropriate, unless described otherwise.

[0067] Several definitions that apply throughout this disclosure will now be presented. As used herein, “about” refers to numeric values, including whole numbers, fractions, percent ages, etc., whether or not explicitly indicated. The term “about” generally refers to a range of numericalvalues, for instance, ± 0.5-1%, ± 1-5% or ± 5-10% of the recited value, that one would consider equivalent to the recited value, for example, having the same function or result.

[0068] The term “comprising” means “including, but not necessarily limited to”; it specifically indicates open-ended inclusion or membership in a so-described combination, group, series and the like. The terms “comprising” and “including” as used herein are inclusive and / or open-ended and do not exclude additional, unrecited elements or method processes. The term “consisting essentially of’ is more limiting than “comprising” but not as restrictive as “consisting of.” Specifically, the term “consisting essentially of’ limits membership to the specified materials or steps and those that do not materially affect the essential characteristics of the claimed invention.

[0069] As used herein, the term “chemiluminescent nanoconstruct” means a composite structure having at least a plasmonic nanostructure at its core with at least one localized surface plasmon resonance wavelength (XLSPR) greater than 350 nm, and at least one chemiluminescent agent conjugated to the surface of the plasmonic nanostructure, or, optionally, conjugated to at least one spacer layer which covers the surface of the plasmonic nanostructure, the at least one biorecognition element, the at least one chemiluminescent agent, or a combination thereof, wherein the chemiluminescent agent has at least one maximum emission wavelength ( EM) wherein the difference between ?cLSPR and EM is less than 50 nm. In some variations, the “chemiluminescent nanoconstruct” may further include a scaffold layer and / or a biorecognition element (e.g., MHC, pMHC, Streptavidin, etc ). The spacer layer may be between 0.1 and 20 nm thick.

[0070] As used herein, the term “chemiluminescent enhancer nanoconstruct” means a composite structure having at least a plasmonic nanostructure at its core with at least one localized surface plasmon resonance wavelength ( LSPR) greater than 350 nm, and at least one enzyme capable of catalyzing the reaction of a substrate to emit light with a maximum emission wavelength (1EM) conjugated to the surface of the plasmonic nanostructure, or, optionally, conjugated to at least one spacer layer which covers the surface of the plasmonic nanostructure, the at least one biorecognition element, or a combination thereof, wherein the chemiluminescent substrate has at least one maximum emission wavelength (XEM) wherein the difference between XLSPR and XEM is less than 50 nm. In some variations, the “chemiluminescent nanoconstruct” may further include a scaffold layer and / or a biorecognition element (e.g., MHC, pMHC, Streptavidin, etc.). The spacer layer may be between 0.1 and 20 nm thick.

[0071] As used herein, the term “substrate” or “chemiluminescent substrate” means a molecule or compound that is capable of emitting light from a chemical reaction in a reaction which is catalyzed by an enzyme.

[0072] As used herein, the term “plasmonic amplifier” or “PA” can refer to either a chemiluminescent enhancer nanoconstruct or a chemiluminescent nanoconstruct.

[0073] As used herein, the term “chemiluminescent agent” or “chemiluminescent label” means a molecule or compound that is capable of emitting light from a chemical reaction in oxidizing conditions. For example, the molecule or compound may undergo multiple oxidation reactions to form a product in the excited state, while returning to ground state emit light in a shorter wavelength.

[0074] The present disclosure is directed to a chemiluminescent nanoconstruct. The chemiluminescent nanoconstruct may include a plasmonic nanostructure having at least one localized surface plasmon resonance wavelength ( LSPR), at least one chemiluminescent agent having a maximum emission wavelength (XEM), and at least one biorecognition element. The difference between the at least one XLSPR and the XEM may be less than 50 nm.

[0075] The plasmonic nanostructure may be conjugated with the at least one chemiluminescent agent and the at least one biorecognition element may be conjugated with the at least one chemiluminescent agent.

[0076] In some embodiments, the plasmonic nanostructure may be conjugated with at least one biorecognition element and the at least one biorecognition element may be conjugated with the at least one chemiluminescent agent.

[0077] The nanoconstruct optionally may include at least one spacer coating. In some aspects, the plasmonic nanostructure may be coated with the at least one spacer coating. In other aspects, the at least one spacer coating may be conjugated with the at least one chemiluminescent agent. In yet other aspects, the at least one biorecognition element may be conjugated to the at least one spacer coating.

[0078] The chemiluminescent nanoconstruct may have a signal that is at least 10 times greater than a signal of the at least one chemiluminescent agent alone in the presence of an appropriate oxidizing agent that triggers chemiluminescence of the chemiluminescent agent. For example, the signal may be at least 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 110 times, 115 times, 120 times, 125 times, 130 times, 135 times, 140 times,145 times, 150 times, 155 times, 160 times, 165 times, 170 times, 175 times, 180 times, 185 times, 190 times, 195 times, 200 times, 210 times, 215 times, 220 times, 225 times, 230 times, 235 times, 240 times, 245 times, 250 times, 255 times, 260 times, 265 times, 270 times, 275 times, 280 times, 285 times, 290 times, 295 times, 300 times, 310 times, 315 times, 320 times, 325 times, 330 times, 335 times, 340 times, 345 times, 350 times, 355 times, 360 times, 365 times, 370 times, 375 times, 380 times, 385 times, 390 times, 395 times, 400 times, 410 times, 415 times, 420 times, 425 times, 430 times, 435 times, 440 times, 445 times, 450 times, 455 times, 460 times, 465 times, 470 times, 475 times, 480 times, 485 times, 490 times, 495 times, or 500 times greater than a signal of the at least one chemiluminescent agent alone.

[0079] The spacer coating may be at least from about 0.1 nm to about 100 nm. For example, the spacer coating may be from about 0.5 nm to about 90 nm, about 1 nm to about 80 nm, about 5 nm to about 70 nm, about 10 nm to about 60 nm, about 15 nm to about 50 nm, about 20 nm to about 45 nm, about 25 nm to about 40 nm, or from about 30 nm to about 35 nm. In some embodiments, the spacer coating may be from 0.1 nm to 20 nm.

[0080] The present disclosure is directed to using plasmonic nanostructures to create ultrasensitive chemiluminescent immunoassays. It is well known that chemiluminescence can be enhanced due to near field coupling between the chemiluminescent species and metal / plasmonic structures (Figure 1). To date, most approaches to utilize this effect to create metal / plasmonic- enhanced chemiluminescent immunoassays 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 that these approaches typically only result in marginal improvements in assay performance. This is likely due to the facts that: A) the enhancement is strongly distancedependent, and the physical geometry of the components comprising immunoassays (namely, antibodies) lead to a spacing of the chemiluminescent event that is too far from the plasmonic surface to be optimally enhanced; B) the plasmon absorption / extinction band and the chemiluminescence emission band must possess a resonant overlap which is cumbersome to achieve on a metal / plasmonic surface. 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). For plasmonicnanostructures, the distance from the metal surface for optimal chemiluminescent enhancement should be smaller than the electromagnetic decay length (EM delay length). As a rule of thumb, the EM decay length is roughly 0.2 in units of the nanostructure size. However, most of the efforts in the past trying to use plasmonic nanoparticles for enhanced-chemiluminescent assays reply on mainly gold nanospheres (<20 nm in diameter) and silver nanoparticles (<20 nm in diameter) which have roughly 4 nm EM decay length. In order to prevent plasmonic quenching of chemiluminescence, spacer (polymer layer, PEG, nucleic acids) is typically coated around the gold / silver nanoparticles before the functionalization of enzymes. This keeps the chemiluminescent event occurring on the peripheral or outside of the EM field of the metal nanoparticles, leading to minimum chemiluminescence enhancement. Thus, it would be beneficial to use plasmonic structures, such as AuNR@Ag, which have bigger dimension (long EM decay length) and stronger plasmonic field and optical tunability to match with the emission band of CL substrate.

[0081] As a non-limiting example of using AuNR@Ag-based plasmonic amplifiers for chemiluminescence, AuNRs are coated with silver first and then a spacer layer is deposited (Figure 2). The spacer layer should have versatile functional groups for protein immobilization, either covalent attachment or nonspecific attachment. An example spacer layer consists of an initiation layer using (3 -Mercaptopropyl) trimethoxysilane (MPTMS) which binds to the silver surface to provide silane groups for further sol -gel deposition for a growth layer, a mixture of (3- Aminopropyl) trimethoxysilane (APTMS) and trimethoxy(propyl)silane (TMPS). This siloxane copolymer has amine, methyl and hydroxyl functional groups that provide electrostatic, hydrophobic and hydrogen bonding interactions for further protein functionalization.

[0082] To enhance chemiluminescence, the plasmonic structure should have a localized surface plasmon resonance (LSPR) band matching the emission band of the chemiluminescent agent or substrate. The most common chemiluminescent substrates are luminol, which has emission maximum around 430 nm; acridan, which has emission maximum around 440 nm; AMPPD, which has emission maximum around 470 nm. The most common chemiluminescent agents are acridinium ester and its derivatives have emission maximum around 440 nm; ruthenium and its derivatives, which have emission maximum around 620 nm. Figure 3 shows a few spectra examples of the plasmonic amplifiers which have LSPR band around 430 nm, 450 nm, and 650 nm, which can be used to enhance the chemiluminescence from the above-mentioned chemiluminescent agents.

[0083] Enzyme-based chemiluminescence is widely used in immunoassays due to its excellent sensitivity, versatility, and availability. The two very common enzyme probes used are horseradish peroxidase (HRP) and alkaline phosphatase (AP). As shown in non-limiting examples of Figure 4, plasmonic amplifiers (PA) can be functionalized with HRP (AP) along with biorecognition elements for the integration into chemiluminescence immunoassays (CLIA). Figure 4A shows an example of functionalizing the PA with: A) Antibody conjugated with HRP (AP); B) Streptavidin conjugated with HRP (AP). As illustrated in Figure 5, it is possible to bind a plasmonic amplifier functionalized with antibody-HRP (AP) to a target analyte captured on a surface (direct or by a capture antibody). As illustrated in Figure 6, it is possible to bind a streptavidin-HRP (AP) functionalized plasmonic amplifier to an immuno-complex of an analyte- biotinylated detection antibody. For the above scenario, once the PA is added in the immunoassay, substrate solution can be later added for chemiluminescent signal generation. It is important to note that the selection of PA structures, enzyme and the substrate should match: for example, A) HRP should match with either luminol or acridan based substrate; B) AP should match with AMPPD based substrate; C) PA with LSPR max around 430 nm should match with HRP / Luminol; D) PA with LSPR max around 450 nm should match with either HRP / acridan or AP / AMPPD. Ideally, the difference between the LSPR band (LLSPR) of PA and the chemiluminescent emission (XEM) is less than about 50 nm. As another example, it is possible to simply functionalize the plasmonic nanostructure with streptavidin-HRP (AP) and add it to an immunoassay containing a biotinylated detection antibody as shown in Figure 6.

[0084] Immunoblotting techniques use antibodies (or specific ligands in related techniques) to identify target proteins among several unrelated protein species. They involve identification of protein targets via antigen-antibody (or protein-ligand) specific interactions. Proteins are typically separated by electrophoresis and transferred onto membranes (usually nitrocellulose). The membrane is overlaid with a primary antibody for a specific target and then with a secondary antibody labeled, usually with enzymes for colorimetric or chemiluminescent based detection. If PA is functionalized with secondary antibody-HRP (AP), it can be used in immunoblot (e.g. western blotting) application (Figure 7). The secondary antibody-HRP (AP) on the PA can bind to the primary antibody in the immunoblot for CL signal generation. PA functionalized with streptavidin-HRP (AP) can also be used in immunoblots if the secondary antibody is biotinylated (Figure 8).

[0085] Acridinium ester (AE) chemiluminescence technology has been used on multiple diagnostics platforms, mostly automated immunoassay systems. The advantages of AE are: A) high quantum yield for improved sensitivity and low-end precision; B) Hydrophilicity for improved efficacy of wash step for low nonspecific binding; C) Hydrolytic stability which leads to long reagent shelf life and extended onboard stability; D) Versatility due to the labeling versatility of AE to a variety of ligands or proteins; E) Small size compared to enzymes; F) Rapid kinetics, for which the light emission of AE complete in 1 to 5 seconds, enabling high throughput. AE and its derivatives can be easily labeled to antibodies or ligands, e.g., streptavidin. Once AE is introduced to the immunoassay, exposure of the AE label to an alkaline hydrogen peroxide solution (triggering solution) triggers a flash of light, which can be used for CL detection. PA can be functionalized with antibody-AE or streptavidin-AE for the easy integration to existing AE -based chemiluminescent immunoassay platforms (Figure 9). For antibody-AE functionalized PA, it can directly bind to target analyte and generate CL signal after the addition of triggering solution (Figure 10). For PA functionalized with streptavidin-AE, it can bind to biotinylated detection antibody and generate CL signal with the subsequent addition of the triggering solution (Figure 11).

[0086] Electrochemiluminescence (ECL) technology relies on labels to generate CL signals when stimulated by electricity in the appropriate chemical environment. When using a conductive substrate (e.g. carbon nanotube coated plate), this reaction is incorporated into immunoassays. For example, if using ruthenium tris-bipyridine (bpy) as label, the system involves reaction of Ru(bpy)s3+and Ru(bpy)s+to produce an excited state of Ru(bpy)s2+, a stable species which decays to the ground state by emitting a 620 nm orange emission. Ru(bpy)s3+and Ru(bpy)s+can be electrogenerated from Ru(bpy)a2+by reduction at -1.3 V and oxidation at +1.3 V. ECL is known for ultrahigh sensitivity and specificity. Ruthenium based labels can be conjugated to antibodies and ligands, e.g., streptavidin for the integration into immunoassays. PA can be functionalized with antibody-ruthenium label or streptavidin-ruthenium label for the easy integration into existing ECL immunoassay platforms (Figure 12). For antibody-ruthenium label functionalized PA, it can directly bind to target analyte and generate CL signal under electrochemical reactions (Figure 13). For PA functionalized with streptavidin-ruthenium labels, it can bind to biotinylated detection antibodies and generate CL signals under electrochemical reactions (Figure 14).

[0087] PA functionalized with a tag and a biorecognition element(s) can be used in a versatile way to enhance chemiluminescent immunoassays. Typically, a biorecognition element is used for the binding to target analyte. A tag is used to localize enzymes (HRP or AP) or chemical labels (acridinium ester or ruthenium and their derivatives) to the immunoassay complex. Tags are molecules that can be specifically targeted by the labeled species that recognize the tag: enzyme-labeled (HRP or AP) species; acridinium and its derivatives labeled-species; ruthenium and its derivatives labeled-species. These tags are attached to the biorecognition element, the plasmonic nanostructure, or a coating on the surface of the plasmonic nanostructure. Nonlimiting examples include biotin tags recognized by streptavidin, oligonucleotides recognized by complementary oligonucleotides, anti-IgG antibodies recognizing IgG, or epitope tags recognized by antibodies. As an example, biotin can be used as a tag and antibody can be used as a biorecognition element (Figure 15). The functionalized plasmonic amplifier can directly bind to a target analyte captured on a surface (electrode). Figure 16A shows a scenario when streptavidin conjugated with HRP (AP) is added to the assay and it can bind to the plasmonic amplifier functionalized with biotinylated antibody. Substrate solution is added for chemiluminescent signal generation. Figure 16B shows a scenario when streptavidin labeled with acridinium ester (and its derivatives) added to the assay and it can bind to the plasmonic amplifier functionalized with biotinylated antibody. Triggering solution (alkaline hydrogen peroxide) is added for chemiluminescent signal generation. Figure 16C shows a scenario when streptavidin labeled with ruthenium tris-bipyridine (and its derivatives) is added to the assay and it can bind to the plasmonic amplifier functionalized with biotinylated antibody. An electrochemical reaction then leads to chemiluminescence.

[0088] Data illustrating the example application of a biotinylated detection antibody functionalized plasmonic amplifier for enhanced chemiluminescent immunoassay is shown in Figure 17A. In this experiment, capture antibody (anti-human IL6, L395, Hytest Ltd) was coated on a microtiter plate and allowed to incubate for Ihr before being washed off. To determine the sensitivity and limit of detection (LOD, defined as mean+3o of the blank), serial dilutions of human IL6 of known concentration (500 pg / ml to 490 fg / ml, 2X serial diluted, in 3% BSA in IX PBS) were employed as standards. After standards incubation (Overnight in 4 °C), biotinylated detection antibodies (anti-human IL6, LI 52, Hytest Ltd) functionalized plasmonic nanostructure (plasmonic amplifier, PA) were added to the microtiter wells. To compare, a biotinylated detection antibody solution was added to the microtiter wells. Subsequently, streptavidin-horseradish peroxidase (Strep-HRP) was added to the immune-complex. In the last step, one component chemiluminescent substrate, UniGlow™ (Rockland Immunochemicals, Inc) was then added in each well to generate the CL signal, and the plate was incubated for 5 min. The CL signal was imaged using Azure Sapphire Biomolecular imager (3s exposure time). The LOD of conventional CLIA using Strep-HRP was calculated to be 23.43 pg / ml and the LOD of PA- enhanced CLIA was calculated to be 0.36 pg / ml, achieving a 65-fold improvement (Figure 17B).

[0089] Data illustrating the example application of a streptavidin-horseradish peroxidase (strep- HRP) functionalized plasmonic amplifier for enhanced chemiluminescent immunoassay is shown in Figure 18A. In this experiment, capture antibody (anti -human IL6, L395, Hytest Ltd) was coated on a microtiter plate and allowed to incubate for Ihr before being washed off. To determine the sensitivity and limit of detection (LOD, defined as mean+3o of the blank), serial dilutions of human IL6 of known concentration (1000 pg / ml to 64 fg / ml, 5X serial diluted, in 3% BS A in 1 X PBS) were employed as standards. After standards incubation (3hr in RT, 650 rpm), a biotinylated detection antibody (anti -human IL6, LI 52, Hytest Ltd) solution was added to the microtiter wells. After detection, incubation and washing, PA functionalized with strep-HRP was added to the well. To compare, strep-HRP solution was added to the microtiter wells. In the last step, one component chemiluminescent substrate, UniGlow™ (Rockland Immunochemicals, Inc) was then added in each well to generate the CL signal, and the plate was incubated for 5 min. The CL signal was imaged using Azure Sapphire Biomolecular imager (3s exposure time). The LOD of conventional CLIA using Strep-HRP was calculated to be 0.841 pg / ml and the LOD of PA-enhanced CLIA was calculated to be 0.003 pg / ml, achieving a 280-fold improvement (Figure 18B).PA-enhanced chemiluminescent lateral flow assay;

[0090] Described below are multiple versions of a lateral flow assay utilizing plasmonic amplifier enhanced chemiluminescence.PA-antibody-HRP(AP):

[0091] As shown in Figure 19, plasmonic amplifiers functionalized with antibody-HRP (AP) (PA / antibody-HRP(AP)) are deposited on one pad (conjugate pad). This pad is assembled with a sample pad, nitrocellulose membrane and absorption pad to form a lateral flow strip. Sample can be directly dropped on the sample pad and the target antigens in the sample will bind with PA / antibody-HRP(AP) to form an immune-complex on the test line where the biorecognitionelement can capture the construct. Subsequently, a substrate solution is added to the strip to generate a CL signal. This signal can then be read by a portable / benchtop reader.PA-biotinylated antibody:

[0092] As shown in Figure 20, plasmonic amplifiers functionalized with biotinylated detection antibodies are deposited on one pad (conjugate pad 1). Complimentary enzyme conjugated streptavidin (strep-HRP (AP)) is deposited on another pad (conjugate pad 2). These two pads are assembled with a sample pad, nitrocellulose membrane and absorption pad to form a lateral flow strip. Sample can be directly dropped on the sample pad and the target antigens in the sample will bind with plasmonic amplifier followed by the binding of strep-HRP (AP) to form an immuno-complex on the test line where the biorecognition element can capture the construct. Subsequently, a substrate solution is added to the strip to generate a CL signal. This signal can then be read by a portable / benchtop reader.PA-biotinylated antibody-HRP(AP) + PA-strep-HRP (AP):

[0093] As shown in Figure 21, plasmonic amplifiers functionalized with biotinylated antibodies conjugated with HRP (AP) are deposited on one pad (conjugate pad 1). Plasmonic amplifiers functionalized with streptavidin-HRP (AP) are deposited on another pad (conjugate pad 2). These two pads are assembled with a sample pad, nitrocellulose membrane and absorption pad to form a lateral flow strip. Sample can be directly drop on the sample pad and the target antigens in the sample will bind with plasmonic amplifier / biotin-HRP (AP) followed by the binding of PA / strep- HRP (AP) to form a super immuno-complex on the test line where the biorecognition element can capture the construct. Subsequently, a substrate solution is added to the strip to generate a CL signal. This signal can then be read by a portable / benchtop reader.Serology versions:

[0094] In the serology versions of the plasmonic amplifier-enhanced chemiluminescent lateral flow assays, as shown in Figures 22-24, the antibody functionalized on plasmonic amplifiers, biorecognition elements on the test line are binding to IgGs in the sample.

[0095] In one aspect, disclosed herein is a method for enhancing enzyme-based chemiluminescence immunoassays using plasmonic nanostructures, antibodies conjugated with enzymes and chemiluminescent substrate. The plasmonic nanostructures will have at least one localized surface plasmon resonance wavelength ( LSPR) greater than 350 nm and thechemiluminescent substrate agent will have at least one maximum emission wavelength (XEM). In a typical direct enzyme-based chemiluminescent immunoassay, a target molecule is captured on a substrate surface by a capture antibody. Then a detection antibody conjugated with enzyme (HRP or AP) to bind the target molecule. Chemiluminescent substrate solution is then added to generate chemiluminescent signals. In the method of plasmonic enhanced CLIA described herein, a plasmonic nanostructure functionalized with multiple detection antibody-enzyme conjguates (HRP or AP) is added to the assay and binds to the target molecule. Chemiluminescent substrate solution is then added to generate chemiluminescence. For efficient chemiluminescence enhancement, the difference between the XLSPR of the plasmonic nanostructure and the EM of the chemiluminescent substrate agent should be less than 50 nm. This leads to a significant increase in chemiluminescence by amplifying the chemiluminescence catalyzed by the enzyme conjugated to the plasmonic structures.

[0096] In one aspect, disclosed herein is a method for enhancing enzyme-based chemiluminescence immunoassays using plasmonic nanostructures, streptavidin conjugated enzymes and chemiluminescent substrates. The plasmonic nanostructures will have at least one localized surface plasmon resonance wavelength (XLSPR) greater than 350 nm and the chemiluminescent substrate agent will have at least one maximum emission wavelength (XEM). In a typical streptavidin enzyme-based chemiluminescent immunoassay, a target molecule is captured on a substrate surface between a capture antibody and a biotinylated detection antibody. Then a streptavidin-conjugated enzyme (HRP or AP) is added to this complex and it binds specifically to the biotinylated detection antibody. Chemiluminescent substrate solution is then added to generate chemiluminescence. In the method of plasmonic enhanced CLIA described herein, a plasmonic nanostructure functionalized with multiple streptavidin labeled-enzyme (HRP or AP) is added to the assay instead of streptavidin-enzyme (HRP or AP). Chemiluminescent substrate solution is then added to generate chemiluminescence. For efficient chemiluminescence enhancement, the difference between the LLSPR of the plasmonic nanostructure and the XEM of the chemiluminescent substrate agent should be less than 50 nm. This leads to a significant increase in chemiluminescence by amplifying the chemiluminescence catalyzed by the enzyme conjugated to the plasmonic structures.

[0097] In one aspect, disclosed herein is a method for enhancing enzyme-free chemiluminescence immunoassays using plasmonic nanostructures, antibodies conjugated with chemiluminescent labels. The plasmonic nanostructures will have at least one localized surfaceplasmon resonance wavelength ( LSPR) greater than 350 nm and the chemiluminescent label will have at least one maximum emission wavelength (XEM). In a typical enzyme-free chemiluminescent immunoassay, a target molecule is captured on a substrate surface by a capture antibody. Then a detection antibody conjugated with multiple chemiluminescent labels (Acridinium ester or Ruthenium and their derivatives) to bind the target molecule. Triggering solution or electrochemical reaction is then introduced to generate chemiluminescent signals. In the method of plasmonic enhanced CLIA described herein, a plasmonic nanostructure functionalized with multiple detection antibody-chemiluminescent label (Acridinium ester or Ruthenium and their derivatives) is added to the assay after the target molecule is captured by a capture antibody. Triggering solution or electrochemical reaction is then introduced to generate chemiluminescent signals. For efficient chemiluminescence enhancement, the difference between the XLSPR of the plasmonic nanostructure and the EM of the chemiluminescent substrate agent should be less than 50 nm. This leads to a significant increase in chemiluminescence generated from the chemiluminescent label (Acridinium ester or Ruthenium and their derivatives) conjugated to the plasmonic structures.

[0098] In one aspect, disclosed herein is a method for enhancing enzyme-free chemiluminescence immunoassays using plasmonic nanostructures, streptavidin conjugated with chemiluminescent labels. The plasmonic nanostructures will have at least one localized surface plasmon resonance wavelength ( LSPR) greater than 350 nm and the chemiluminescent label will have at least one maximum emission wavelength (XEM). In a typical enzyme-free chemiluminescent immunoassay, a target molecule is captured on a substrate surface between a capture antibody and a biotinylated detection antibody. Then a streptavidin conjugated with multiple chemiluminescent labels (Acridinium ester or Ruthenium and their derivatives) is added to this complex and it binds specifically to the biotinylated detection antibody. Triggering solution or electrochemical reaction is then introduced to generate chemiluminescent signals. In the method of plasmonic enhanced CLIA described herein, a plasmonic nanostructure functionalized with multiple streptavidin-chemiluminescent label (Acridinium ester or Ruthenium and their derivatives) is added to the assay after the target molecule is captured between a capture antibody and a biotinylated detection antibody. Triggering solution or electrochemical reaction is then introduced to generate chemiluminescence. For efficient chemiluminescence enhancement, the difference between the LSPR of the plasmonic nanostructure and the XEM of the chemiluminescent substrate agent should be less than 50 nm.This leads to a significant increase chemiluminescence generated from the chemiluminescent label (Acridinium ester or Ruthenium and their derivatives) conjugated to the plasmonic structures.

[0099] In one aspect disclosed herein, are plasmonic nanostructures functionalized with streptavidin-HRP (AP) that would allow these nanostructures to be easily integrated into existing chemiluminescent immunoassay workflows (indirect). In one example, streptavidin-HRP(AP) could adsorb non-specifically to the plasmonic nanostructure. In another example, streptavidin- HRP(AP) could bind to biotin-functionalized plasmonic nanostructures. These streptavidin- HRP(AP) -functionalized plasmonic nanostructures could be added to a chemiluminescent immunoassay which utilizes a biotinylated detection antibody.

[0100] In one aspect disclosed herein, are plasmonic nanostructures functionalized with streptavidin-acridinium ester (or acridinium derivatives) that would allow these nanostructures to be easily integrated into existing chemiluminescent immunoassay workflows (direct). One example of how this could be achieved is by covalently labeling streptavidin with, for example, acridinium or its derivative containing an N-Hydroxysuccinimide (NHS)-ester reactive group. In one example, streptavidin-acridinium could adsorb non-specifically to the plasmonic nanostructure. In another example, streptavidin-acridinium could bind to biotin-functionalized plasmonic nanostructures. These streptavidin-acridinium-functionalized plasmonic nanostructures could be added to a chemiluminescent immunoassay which utilizes a biotinylated detection antibody.

[0101] In one aspect disclosed herein, is a method of creating ultrasensitive chemiluminescent western blotting (Immunoblot). The general concept is to form the plasmonically-enhanced chemiluminescent structure during the performance of the western blotting. This is achieved by adding to the western blotting: a plasmonic nanostructure conjugated to secondary antibodies labeled with enzyme (HRP or AP) which specifically targets a primary antibody which binds to the target molecule of interest in the sample being interrogated and wherein subsequently the corresponding substrates are added to generate plasmonic-enhanced chemiluminescent signal.

[0102] In one aspect disclosed herein, is a method of creating ultrasensitive chemiluminescent western blotting (Immunoblot). The general concept is to form the plasmonically-enhanced chemiluminescent structure during the performance of the western blotting. This is achieved by adding to the western blotting: a plasmonic nanostructure conjugated to streptavidin labeled with enzyme (HRP or AP) which specifically targets a biotin-labeled secondary antibody which boundto a primary antibody, detecting the species of interest in the sample being interrogated and wherein subsequently the corresponding substrates are added to generate plasmonic-enhanced chemiluminescent signal.

[0103] In one aspect disclosed herein, a plasmonic amplification strategy for direct chemiluminescent assays. It should be recognized by one skilled in the art that the key components of the plasmonic amplification strategy are: 1) a plasmonic nanoparticle; 2) a biorecognition element; 3) chemiluminescent label. The plasmonic nanostructures can comprise any material which has surface plasmons that can resonate at suitable wavelengths of light, such as gold (Au), silver (Ag), copper (Cu), or combinations thereof. Suitable examples of plasmonic nanostructures include, but are not limited to, nanorods, nanocubes, nanospheres, bimetallic nanostructures (e.g. Au@Ag core-shell nanocube), nanostructures with sharp tips (e.g. nanostars), hollow nanostructures such as nanocages and nanorattles, nanobipyramids, nanoplates, self-assembled nanostructures, and nanoraspberries. In some aspects, the nanostructure is selected from the group consisting of gold core silver shell nanocuboids, nanotubes, gold nanorods, silver nanocubes, silver nanospheres, bimetallic nanostructures, gold nanorod core, silver shell (AuNR@Ag) nanocuboids, nanostructures with sharp tips, nanostars, hollow nanostructures, nanocages, nanorattles, nanobipyramids, nanoplates, self-assembled nanostructures, nanoraspberries, and combinations thereof. The biorecognition element can be any molecule that allows the plasmonic nanoparticle to specifically target a biomolecule. Nonlimiting examples include antibodies or functional fragments thereof, oligonucleotides, and aptamers. Chemiluminescent labels can be acridinium and its derivatives or ruthenium and its derivatives. Chemiluminescent labels can be conjugated to biorecognition elements mentioned above or separately conjugated to the plasmonic structure.

[0104] In one aspect disclosed herein, a plasmonic amplification strategy for indirect chemiluminescent assays. It should be recognized by one skilled in the art that the key components of the plasmonic amplification strategy are: 1) a plasmonic nanoparticle; 2) a biorecognition element; and 3) a tag which can be used to localize enzymes (HRP or AP) to the plasmonic nanoparticle. The plasmonic nanostructures can comprise any material which has surface plasmons that can resonate at suitable wavelengths of light, such as gold (Au), silver (Ag), copper (Cu), or combinations thereof. Suitable examples of plasmonic nanostructures include, but are not limited to, nanorods, nanocubes, nanospheres, bimetallic nanostructures (e.g. Au@Ag core-shell nanocube), nanostructures with sharp tips (e.g. nanostars), hollownanostructures such as nanocages and nanorattles, nanobipyramids, nanoplates, self-assembled nanostructures, and nanoraspberries. In some aspects, the nanostructure is selected from the group consisting of gold core silver shell nanocuboids, nanotubes, gold nanorods, silver nanocubes, silver nanospheres, bimetallic nanostructures, gold nanorod core, silver shell (AuNR@Ag) nanocuboids, nanostructures with sharp tips, nanostars, hollow nanostructures, nanocages, nanorattles, nanobipyramids, nanoplates, self-assembled nanostructures, nanoraspberries, and combinations thereof. The biorecognition element can be any molecule that allows the plasmonic nanoparticle to specifically target a biomolecule. Nonlimiting examples include antibodies or functional fragments thereof, oligonucleotides, and aptamers. Tags are molecules that can be specifically targeted by enzyme-labeled (HRP or AP) species that recognizes the tag. These tags are attached to the biorecognition element, the plasmonic nanostructure, or a coating on the surface of the plasmonic nanostructure. Nonlimiting examples include biotin tags recognized by streptavidin, oligonucleotides recognized by complementary oligonucleotides, anti-IgG antibodies recognizing IgG, or epitope tags recognized by antibodies.

[0105] In one aspect disclosed herein, a plasmonic amplification strategy for enzyme-free chemiluminescent assays. It should be recognized by one skilled in the art that the key components of the plasmonic amplification strategy are: 1) a plasmonic nanoparticle; 2) a biorecognition element; and 3) a tag which can be used to localize chemiluminescent labels (acridinium and its derivatives or ruthenium and its derivatives) to the plasmonic nanoparticle. The plasmonic nanostructures can comprise any material which has surface plasmons that can resonate at suitable wavelengths of light, such as gold (Au), silver (Ag), copper (Cu), or combinations thereof. Suitable examples of plasmonic nanostructures include, but are not limited to, nanorods, nanocubes, nanospheres, bimetallic nanostructures (e.g. Au@Ag core-shell nanocube), nanostructures with sharp tips (e.g. nanostars), hollow nanostructures such as nanocages and nanorattles, nanobipyramids, nanoplates, self-assembled nanostructures, and nanoraspberries. In some aspects, the nanostructure is selected from the group consisting of gold core silver shell nanocuboids, nanotubes, gold nanorods, silver nanocubes, silver nanospheres, bimetallic nanostructures, gold nanorod core, silver shell (AuNR@Ag) nanocuboids, nanostructures with sharp tips, nanostars, hollow nanostructures, nanocages, nanorattles, nanobipyramids, nanoplates, self-assembled nanostructures, nanoraspberries, and combinations thereof. The biorecognition element can be any molecule that allows the plasmonic nanoparticle to specifically target a biomolecule. Nonlimiting examples include antibodies or functionalfragments thereof, oligonucleotides, and aptamers. Tags are molecules that can be specifically targeted by acridinium and its derivatives or ruthenium and its derivatives labeled species that recognize the tag. These tags are attached to the biorecognition element, the plasmonic nanostructure, or a coating on the surface of the plasmonic nanostructure. Nonlimiting examples include biotin tags recognized by streptavidin, oligonucleotides recognized by complementary oligonucleotides, anti-IgG antibodies recognizing IgG, or epitope tags recognized by antibodies.

[0106] In one aspect disclosed herein, is a method of creating ultrasensitive chemiluminescent lateral flow immunoassays. The general concept is to form the plasmonically-enhanced chemiluminescent structure during the performance of the lateral flow immunoassay. This is achieved by adding to the lateral flow immunoassay: a plasmonic nanostructure conjugated to at least one and preferably more than one biorecognition element labeled with enzyme (HRP or AP) which specifically targets a species of interest in the sample being interrogated and wherein subsequently the corresponding substrates are added to generate chemiluminescent signal.

[0107] In one aspect disclosed herein, is a method of creating ultrasensitive chemiluminescent lateral flow immunoassays. The general concept is to form the plasmonically-enhanced chemiluminescent structure during the performance of the lateral flow immunoassay. This is achieved by adding to the lateral flow immunoassay: a plasmonic nanostructure conjugated to at least one and preferably more than one biorecognition element which specifically targets a species of interest in the sample being interrogated and wherein either the plasmonic nanostructure or the biorecognition element(s) are labeled with a tag; and an enzyme-labeled (HRP or AP) species specifically recognizing the tag wherein the corresponding chemiluminescent substrates (luminol, acridan or AMMPD) of the enzyme-labeled (HRP or AP) has an luminescent emission band overlapping with the plasmonic nanostructure’s maximal LSPR wavelength. After the addition of substrate, chemiluminescent signals will be generated.

[0108] Numerous examples are provided herein to enhance the understanding of the present disclosure. A specific set of statements are provided as follows.

[0109] Statement 1 : A chemiluminescent nanoconstruct comprising a plasmonic nanostructure having at least one localized surface plasmon resonance wavelength (XLSPR), at least one chemiluminescent agent having a maximum emission wavelength ( EM), and at least one biorecognition element, wherein the chemiluminescent nanoconstruct has a signal that is at least 20 times greater than a signal of the at least one chemiluminescent agent alone in the presenceof an appropriate oxidizing agent to trigger chemiluminescence of the chemiluminescent agent, and wherein a difference between the at least one LSPR and the EM is less than 50 nm.

[0110] Statement 2: The chemiluminescent nanoconstruct of statement 1, wherein the plasmonic nanostructure is conjugated with the at least one chemiluminescent agent and the at least one biorecognition element.

[0111] Statement 3: The chemiluminescent nanoconstruct of statement 1, wherein the plasmonic nanostructure is conjugated with at least one biorecognition element and the at least one biorecognition element is conjugated with the at least one chemiluminescent agent.

[0112] Statement 4: The chemiluminescent nanoconstruct of statement 1, further comprising at least one spacer coating; wherein the plasmonic nanostructure is coated with the at least one spacer coating.

[0113] Statement 5: The chemiluminescent nanoconstruct of statement 4, wherein the least one biorecognition element, the at least one chemiluminescent agent, or a combination thereof are conjugated to the spacer.

[0114] Statement 6: The chemiluminescent nanoconstruct of statement 1, wherein the plasmonic nanostructure is a gold nanorod coated with silver (AuNR@Ag).

[0115] Statement 7: The chemiluminescent nanoconstruct of statement 4, wherein the spacer coating comprises one or more of silane, amine, alkyl, hydroxyl functional groups.

[0116] Statement 8: The chemiluminescent nanoconstruct of statement 7, wherein the spacer coating is one or more silane.

[0117] Statement 9: The chemiluminescent nanoconstruct of statement 8, wherein the silane comprises (3 -Aminopropyl) trimethoxysilane (APTMS), trimethoxy(propyl)silane (TMPS), and / or a combination thereof.

[0118] Statement 10: The chemiluminescent nanoconstruct of statement 1, wherein the spacer coating is on the nanostructure and has a thickness of from about 0. 1 nm to about 20 nm.

[0119] Statement 11 : The chemiluminescent nanoconstruct of statement 1, wherein the chemiluminescent agents comprises acridinium esters, ruthenium esters, or combinations thereof.

[0120] Statement 12: The chemiluminescent nanoconstruct of statement 1, wherein the biorecognition element comprises one or more antibody, functional fragments of an antibody, oligonucleotides, aptamers, or streptavidin, neutravidin, avidin, or combinations thereof.

[0121] Statement 13: The chemiluminescent nanoconstruct of statement 11, wherein the one or more antibody is functionalized with biotin.

[0122] Statement 14: A method for enhancing a chemiluminescent immunoassay comprising adding a chemiluminescent nanoconstruct to an immunoassay, wherein the chemiluminescent nanoconstruct comprises a plasmonic nanostructure having at least one localized surface plasmon resonance wavelength (XLSPR), at least one chemiluminescent agent having a maximum emission wavelength (XEM) in the presence of an appropriate oxidizing agent to trigger chemiluminescence of the chemiluminescent agent, and at least one biorecognition element; and wherein a difference between the at least one XLSPR and the XEM is less than 50 nm.

[0123] Statement 15: The method of statement 14, wherein limit of detection of the chemiluminescent immunoassay is enhanced with the chemiluminescent nanoconstruct by a factor of at least 10 relative to the an immunoassay without the chemiluminescent nanoconstruct.

[0124] Statement 16: The method of statement 14, wherein the plasmonic nanostructure is coated with the at least one spacer coating.

[0125] Statement 17: The method of statement 16, wherein the spacer coating comprises one or more of silane, amine, alkyl, or hydroxyl functional groups.

[0126] Statement 18: The method of statement 17, wherein the spacer coating is one or more silane.

[0127] Statement 19: The method of statement 18, wherein the silane comprises (3- aminopropyl) trimethoxysilane (APTMS), trimethoxy(propyl)silane (TMPS), and / or a combination thereof.

[0128] Statement 20: The method of statement 16, wherein the spacer coating is on the nanostructure and has a thickness of from about 0. 1 nm to about 20 nm.

[0129] Statement 21 : The method of statement 16, wherein the at least one spacer coating is conjugated with the at least one chemiluminescent agent.

[0130] Statement 22: The method of statement 16, wherein the at least one biorecognition element is conjugated to the at least one spacer coating.

[0131] Statement 23 : The method of statement 20, wherein the chemiluminescent agents comprise acridinium esters, ruthenium esters, or combinations thereof.

[0132] Statement 24: The method of statement 14, wherein the biorecognition element comprises one or more antibody, functional fragments of an antibody, oligonucleotides, aptamers, streptavidin, avidin, neutravidin, or combinations thereof.

[0133] Statement 25: The method of statement 24, wherein the one or more antibody is functionalized with biotin.

[0134] Statement 26: The method of statement 14, wherein the chemiluminescent nanoconstruct has a signal that is at least 20 times greater than a signal of the at least one chemiluminescent agent alone in the presence of an appropriate oxidizing agent to trigger chemiluminescence of the chemiluminescent agent.

[0135] Statement 27: The method of statement 14, wherein the plasmonic nanostructure is a gold nanorod coated with silver (AuNR@Ag).

[0136] Statement 28: A chemiluminescent enhancer nanoconstruct comprising a plasmonic nanostructure having at least one localized surface plasmon resonance wavelength (kLSPR), at least one chemiluminescent agent having a maximum emission wavelength (XEM), and at least one biorecognition element, wherein the plasmonic nanostructure is coated with the at least one spacer coating; wherein the at least one spacer coating is conjugated with the at least one chemiluminescent agent; wherein the at least one biorecognition element is conjugated to the at least one spacer coating, wherein the chemiluminescent nanoconstruct has a signal that is at least 20 times greater than a signal of the at least one chemiluminescent agent alone; and wherein a difference between the at least one LSPR and the XEM is less than 50 nm.

[0137] Statement 29: The chemiluminescent enhancer nanoconstruct of statement 28, wherein the plasmonic nanostructure is conjugated with the at least one enzyme capable of catalyzing the reaction of a chemiluminescent substrate to emit light with a maximum emission wavelength (XEM) and the at least one biorecognition element.

[0138] Statement 30: The chemiluminescent enhancer nanoconstruct of statement 28, wherein the plasmonic nanostructure is conjugated with at least one biorecognition element and the at least one biorecognition element is conjugated with the at least one enzyme capable of catalyzing the reaction of a chemiluminescent substrate to emit light with a maximum emission wavelength (XEM).

[0139] Statement 31 : The chemiluminescent enhancer nanoconstruct of statement 28, further comprising at least one spacer coating; wherein the plasmonic nanostructure is coated with the at least one spacer coating.

[0140] Statement 32: The chemiluminescent enhancer nanoconstruct of statement 31, wherein the least one biorecognition element, the at least one enzyme capable of catalyzing the reaction of a chemiluminescent substrate to emit light with a maximum emission wavelength (XEM), or a combination thereof are conjugated to the spacer.

[0141] Statement 33: The chemiluminescent enhancer nanoconstruct of statement 28, wherein the plasmonic nanostructure is a gold nanorod coated with silver (AuNR@Ag).

[0142] Statement 34: The chemiluminescent enhancer nanoconstruct of statement 31, wherein the spacer coating comprises one or more of silane, amine, alkyl, hydroxyl functional groups.

[0143] Statement 35: The chemiluminescent enhancer nanoconstruct of statement 34, wherein the spacer coating is one or more silane.

[0144] Statement 36: The chemiluminescent enhancer nanoconstruct of statement 35, wherein the silane comprises (3 -aminopropyl) trimethoxy silane (APTMS), trimethoxy(propyl)silane (TMPS), and / or a combination thereof.

[0145] Statement 37: The chemiluminescent enhancer nanoconstruct of statement 31, wherein the spacer coating is on the nanostructure and has a thickness of from about 0. 1 nm to about 20 nm.

[0146] Statement 38: The chemiluminescent enhancer nanoconstruct of statement 28, wherein the biorecognition element comprises one or more antibody, functional fragments of an antibody, oligonucleotides, aptamers, streptavidin, avidin, neutravidin or combinations thereof.

[0147] Statement 39: The chemiluminescent enhancer nanoconstruct of statement 38, wherein the one or more antibody is functionalized with biotin.

[0148] Statement 40: The chemiluminescent enhancer nanoconstruct of statement 28, wherein the at least one enzyme capable of catalyzing the reaction of a chemiluminescent substrate to emit light with a maximum emission wavelength ( EM) comprises alkaline phosphatase or horseradish peroxidase.

[0149] Statement 41 : The chemiluminescent enhancer nanoconstruct of statement 40, wherein the substrate comprises adamantyl 1, 2-dioxetane phenylphosphate (AMPPD), luminol, acridan, or their derivatives.

[0150] Statement 42: A method for enhancing a chemiluminescent immunoassay comprising adding a chemiluminescent enhancer nanoconstruct to an immunoassay, wherein the chemiluminescent enhancer nanoconstruct comprises a plasmonic nanostructure having at leastone localized surface plasmon resonance wavelength ( LSPR), at least one enzyme capable of catalyzing the reaction of a chemiluminescent substrate to emit light with a maximum emission wavelength (1EM), and at least one biorecognition element; and wherein a difference between the at least one LSPR and the EM is less than 50 nm.

[0151] Statement 43: The method of statement 42, wherein limit of detection of the chemiluminescent immunoassay is enhanced with the chemiluminescent enhancer nanoconstruct by a factor of at least 10 relative to the an immunoassay without the chemiluminescent enhancer nanoconstruct in the presence of an appropriate chemiluminescent substrate.

[0152] Statement 44: The method of statement 42, wherein the plasmonic nanostructure is coated with the at least one spacer coating.

[0153] Statement 45: The method of statement 44, wherein the spacer coating comprises one or more of silane, amine, alkyl, or hydroxyl functional groups.

[0154] Statement 46: The method of statement 45, wherein the spacer coating is one or more silane.

[0155] Statement 47: The method of statement 46, wherein the silane comprises (3- Aminopropyl) trimethoxysilane (APTMS), trimethoxy(propyl)silane (TMPS), and / or a combination thereof.

[0156] Statement 48: The method of statement 44, wherein the spacer coating is on the nanostructure and has a thickness of from about 0. 1 nm to about 20 nm.

[0157] Statement 49: The method of statement 48, wherein the at least one spacer coating is conjugated with the at least one enzyme capable of catalyzing the reaction of a chemiluminescent substrate to emit light with a maximum emission wavelength (XEM).

[0158] Statement 50: The method of statement 44, wherein the at least one biorecognition element is conjugated to the at least one spacer coating.

[0159] Statement 51 : The method of statement 42, wherein the biorecognition element comprises one or more antibody, functional fragments of an antibody, oligonucleotides, aptamers, streptavidin, neutravidin, or avidin or combinations thereof.

[0160] Statement 52: The method of statement 51, wherein the one or more antibody is functionalized with biotin.

[0161] Statement 53: The method of statement 42, wherein the chemiluminescent enhancer nanoconstruct produces a signal in the presence of an appropriate chemiluminescent substrate that is at least 20 times greater than a signal of the at least one enzyme capable of catalyzing the reaction of a chemiluminescent substrate at least one substrate alone.

[0162] Statement 54: The method of statement 42, wherein the plasmonic nanostructure is a gold nanorod coated with silver (AuNR@Ag).

[0163] Statement 55: The method of statement 42, wherein the at least one enzyme capable of catalyzing the reaction of a chemiluminescent substrate to emit light with a maximum emission wavelength ( EM) comprises alkaline phosphatase or horseradish peroxidase.

[0164] Statement 56: The method of statement 55, wherein the chemiluminescent substrate comprises adamantyl 1, 2-dioxetane phenylphosphate (AMPPD), luminol, acridan, or their derivatives.

Claims

CLAIMSWhat is claimed is:

1. A chemiluminescent nanoconstruct comprising: a plasmonic nanostructure having at least one localized surface plasmon resonance wavelength (LLSPR); at least one chemiluminescent agent having a maximum emission wavelength (LEM); and at least one biorecognition element; wherein the chemiluminescent nanoconstruct has a signal that is at least 20 times greater than a signal of the at least one chemiluminescent agent alone in presence of an appropriate oxidizing reagent to trigger chemiluminescence of the chemiluminescent agent; and wherein a difference between the at least one XLSPR and the LEM is less than 50 nm.

2. The chemiluminescent nanoconstruct of claim 1, wherein the plasmonic nanostructure is conjugated with the at least one chemiluminescent agent and the at least one biorecognition element is conjugated with the at least one chemiluminescent agent.

3. The chemiluminescent nanoconstruct of claim 1, wherein the plasmonic nanostructure is conjugated with at least one biorecognition element and the at least one biorecognition element is conjugated with the at least one chemiluminescent agent.

4. The chemiluminescent nanoconstruct of claim 1, further comprising at least one spacer coating; wherein the plasmonic nanostructure is coated with the at least one spacer coating.

5. The chemiluminescent nanoconstruct of claim 4, wherein the least one biorecognition element, the at least one chemiluminescent agent, or a combination thereof are conjugated to the spacer.

6. The chemiluminescent nanoconstruct of claim 1, wherein the plasmonic nanostructure is a gold nanorod coated with silver (AuNR@Ag).

7. The chemiluminescent nanoconstruct of claim 4, wherein the spacer coating comprises one or more of silane, amine, alkyl, hydroxyl functional groups.

8. The chemiluminescent nanoconstruct of claim 7, wherein the spacer coating is one or more silane.

9. The chemiluminescent nanoconstruct of claim 8, wherein the silane comprises (3- aminopropyl) trimethoxysilane (APTMS), trimethoxy(propyl)silane (TMPS), and / or a combination thereof.

10. The chemiluminescent nanoconstruct of claim 1, wherein the spacer coating is on the nanostructure and has a thickness of from about 0.1 nm to about 20 nm.

11. The chemiluminescent nanoconstruct of claim 1, wherein the chemiluminescent agents comprises acridinium esters, ruthenium esters, or combinations thereof.

12. The chemiluminescent nanoconstruct of claim 1, wherein the biorecognition element comprises one or more antibody, functional fragments of an antibody, oligonucleotides, aptamers, streptavidin, avidin, neutravidin, or combinations thereof.

13. The chemiluminescent nanoconstruct of claim 12, wherein the one or more antibody is functionalized with biotin.

14. A method for enhancing a chemiluminescent immunoassay comprising adding a chemiluminescent nanoconstruct to an immunoassay, wherein the chemiluminescent nanoconstruct comprises a plasmonic nanostructure having at least one localized surface plasmon resonance wavelength (XLSPR), at least one chemiluminescent agent having a maximum emission wavelength (XEM), and at least one biorecognition element; and wherein a difference between the at least one XLSPR and the XEM is less than 50 nm.

15. The method of claim 14, wherein limit of detection of the chemiluminescent immunoassay is enhanced with the chemiluminescent nanoconstruct by a factor of at least 10 relative to the an immunoassay without the chemiluminescent nanoconstruct in the presence of an appropriate oxidizing agent to trigger chemiluminescence of the chemiluminescent agent.

16. The method of claim 14, wherein the plasmonic nanostructure is coated with the at least one spacer coating.

17. The method of claim 16, wherein the spacer coating comprises one or more of silane, amine, alkyl, or hydroxyl functional groups.

18. The method of claim 17, wherein the spacer coating is one or more silane.

19. The method of claim 18, wherein the silane comprises (3 -Aminopropyl) trimethoxysilane (APTMS), trimethoxy(propyl)silane (TMPS), and / or a combination thereof.

20. The method of claim 16, wherein the spacer coating is on the nanostructure and has a thickness of from about 0.1 nm to about 20 nm.

21. The method of claim 16, wherein the at least one spacer coating is conjugated with the at least one chemiluminescent agent.

22. The method of claim 16, wherein the at least one biorecognition element is conjugated to the at least one spacer coating.

23. The method of claim 14, wherein the chemiluminescent agents comprises acridinium esters, ruthenium esters, or combinations thereof.

24. The method of claim 14, wherein the biorecognition element comprises one or more antibody, functional fragments of an antibody, oligonucleotides, aptamers, streptavidin, neutravidin, avidin, or combinations thereof.

25. The method of claim 24, wherein the one or more antibody is functionalized with biotin.

26. The method of claim 14, wherein the chemiluminescent nanoconstruct has a signal that is at least 20 times greater than a signal of the at least one chemiluminescent agent alone in the presence of an appropriate oxidizing agent to trigger chemiluminescence of the chemiluminescent agent.

27. The method of claim 14, wherein the plasmonic nanostructure is a gold nanorod coated with silver (AuNR@Ag).

28. A chemiluminescent enhancer nanoconstruct comprising: a plasmonic nanostructure having at least one localized surface plasmon resonance wavelength (XLSPR);at least one enzyme capable of catalyzing the reaction of a chemiluminescent substrate to emit light with a maximum emission wavelength (XEM); and at least one biorecognition element; wherein the chemiluminescent enhancer nanoconstruct produces a signal in the presence of an appropriate chemiluminescent substrate that is at least 20 times greater than a signal of the at least one enzyme capable of catalyzing the reaction of a chemiluminescent substrate at least one substrate alone; and wherein a difference between the at least one XLSPR and the ZEM is less than 50 nm.

29. The chemiluminescent enhancer nanoconstruct of claim 28, wherein the plasmonic nanostructure is conjugated with the at least one enzyme capable of catalyzing the reaction of a chemiluminescent substrate to emit light with a maximum emission wavelength (XEM) and the at least one biorecognition element.

30. The chemiluminescent enhancer nanoconstruct of claim 28, wherein the plasmonic nanostructure is conjugated with at least one biorecognition element and the at least one biorecognition element is conjugated with the at least one enzyme capable of catalyzing the reaction of a chemiluminescent substrate to emit light with a maximum emission wavelength (AEM).

31. The chemiluminescent enhancer nanoconstruct of claim 28, further comprising at least one spacer coating; wherein the plasmonic nanostructure is coated with the at least one spacer coating.

32. The chemiluminescent enhancer nanoconstruct of claim 31, wherein the least one biorecognition element, the at least one enzyme capable of catalyzing the reaction of a chemiluminescent substrate to emit light with a maximum emission wavelength (XEM), or a combination thereof are conjugated to the spacer.

33. The chemiluminescent enhancer nanoconstruct of claim 28, wherein the plasmonic nanostructure is a gold nanorod coated with silver (AuNR@Ag).

34. The chemiluminescent enhancer nanoconstruct of claim 31, wherein the spacer coating comprises one or more of silane, amine, alkyl, hydroxyl functional groups.

35. The chemiluminescent enhancer nanoconstruct of claim 34, wherein the spacer coating is one or more silane.

36. The chemiluminescent enhancer nanoconstruct of claim 35, wherein the silane comprises (3 -aminopropyl) trimethoxysilane (APTMS), trimethoxy(propyl)silane (TMPS), and / or a combination thereof.

37. The chemiluminescent enhancer nanoconstruct of claim 31, wherein the spacer coating is on the nanostructure and has a thickness of from about 0.1 nm to about 20 nm.

38. The chemiluminescent enhancer nanoconstruct of claim 28, wherein the biorecognition element comprises one or more antibody, functional fragments of an antibody, oligonucleotides, aptamers, streptavidin, avidin, neutravidin or combinations thereof.

39. The chemiluminescent enhancer nanoconstruct of claim 38, wherein the one or more antibody is functionalized with biotin.

40. The chemiluminescent enhancer nanoconstruct of claim 28, wherein the at least one enzyme capable of catalyzing the reaction of a chemiluminescent substrate to emit light with a maximum emission wavelength (XEM) comprises alkaline phosphatase or horseradish peroxidase.

41. The chemiluminescent enhancer nanoconstruct of claim 40, wherein the substrate comprises adamantyl 1, 2-dioxetane phenylphosphate (AMPPD), luminol, acridan, or their derivatives.

42. A method for enhancing a chemiluminescent immunoassay comprising adding a chemiluminescent enhancer nanoconstruct to an immunoassay, wherein the chemiluminescent enhancer nanoconstruct comprises a plasmonic nanostructure having at least one localized surface plasmon resonance wavelength (ALSPR), at least one enzyme capable of catalyzing the reaction of a chemiluminescent substrate to emit light with a maximum emission wavelength ( EM), and at least one biorecognition element; and wherein a difference between the at least one 1LSPR and the XEM is less than 50 nm.

43. The method of claim 42, wherein limit of detection of the chemiluminescent immunoassay is enhanced with the chemiluminescent enhancer nanoconstruct by a factor of at least 10 relative to the an immunoassay without the chemiluminescent enhancer nanoconstruct in the presence of an appropriate chemiluminescent substrate.

44. The method of claim 42, wherein the plasmonic nanostructure is coated with the at least one spacer coating.

45. The method of claim 44, wherein the spacer coating comprises one or more of silane, amine, alkyl, or hydroxyl functional groups.

46. The method of claim 45, wherein the spacer coating is one or more silane.

47. The method of claim 46, wherein the silane comprises (3 -Aminopropyl) trimethoxysilane (APTMS), trimethoxy(propyl)silane (TMPS), and / or a combination thereof.

48. The method of claim 44, wherein the spacer coating is on the nanostructure and has a thickness of from about 0. 1 nm to about 20 nm.

49. The method of claim 48, wherein the at least one spacer coating is conjugated with the at least one enzyme capable of catalyzing the reaction of a chemiluminescent substrate to emit light with a maximum emission wavelength (XEM).

50. The method of claim 44, wherein the at least one biorecognition element is conjugated to the at least one spacer coating.

51. The method of claim 42, wherein the biorecognition element comprises one or more antibody, functional fragments of an antibody, oligonucleotides, aptamers, streptavidin, neutravidin, or avidin or combinations thereof.

52. The method of claim 51, wherein the one or more antibody is functionalized with biotin.

53. The method of claim 42, wherein the chemiluminescent enhancer nanoconstruct produces a signal in the presence of an appropriate chemiluminescent substrate that is at least 20 times greater than a signal of the at least one enzyme capable of catalyzing the reaction of a chemiluminescent substrate at least one substrate alone.

54. The method of claim 42, wherein the plasmonic nanostructure is a gold nanorod coated with silver (AuNR@Ag).

55. The method of claim 42, wherein the at least one enzyme capable of catalyzing the reaction of a chemiluminescent substrate to emit light with a maximum emission wavelength (AEM) comprises alkaline phosphatase or horseradish peroxidase.

56. The method of claim 55, wherein the chemiluminescent substrate comprises adamantyl 1, 2- dioxetane phenylphosphate (AMPPD), luminol, acridan, or their derivatives.

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