Ultrabright fluorescent nanoconstructs as versatile enhancers

Abstract

To provide an approach to address the low sensitivity of fluorescent assays.SOLUTION: Disclosed herein is a fluorescent nanoconstruct that includes: a plasmonic nanostructure having at least one localized surface plasmon resonance wavelength (λLSPR); at least one spacer coating; and at least one fluorescent agent having a maximum excitation wavelength (λEX). The fluorescent intensity of the fluorescent nanoconstruct is at least 500 times greater than the fluorescent intensity of the at least one fluorescent agent alone.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 741,237, filed October 4, 2018, and U.S. Provisional Application No. 62 / 879,824, filed July 29, 2019, the entire contents of which are incorporated herein by reference.

[0002] Federal Government Support This invention was made with federal support under awards CBET1254399 awarded by the National Science Foundation and CA141521 awarded by the National Institutes of Health. The federal government has certain rights in this invention. [Background technology]

[0003] The field of this disclosure generally relates to plasmonic-fluor (PF), an ultrabright fluorescent nanoconstruct that can be used to enhance biological assays. Specifically, the present invention relates to the use of a novel combination of a plasmonic nanostructure, a spacer layer, and a fluorophore, which results in a nanoconstruct that is spectrally similar to the fluorophore but at least 500 times brighter than the individual fluorophores alone. These ultrabright fluorescent nanoconstructs can be conjugated to at least one biorecognition element and used to enhance the detection performance and improve the detection limits of various biological assays and processes.

[0004] The relevant concentrations of biomolecules or biomarkers associated with diseases such as cancer, heart disease, inflammation, and neurological disorders can range over many orders of magnitude, from the μg / ml level to sub-fg / ml. Due to the lack of sensitive bioanalytical techniques, some of these biomolecules or biomarkers may remain unidentified. Utilizing small sample volumes to perform multiple detections in precious biological fluids, such as inhaled air condensate, ocular fluid, cerebrospinal fluid, or serum from neonates or small animal models, where sample dilution to further reduce concentrations is still highly desirable. As a foundation for biomedical science and clinical research, fluorescence-based bioanalytical methods are widely used for the detection, quantification, and imaging of a wide range of biological analytes. Several methods have been explored to improve the sensitivity of fluorescent immunoassays, including increasing antibody affinity, reducing background fluorescence, facilitating mass transfer, and increasing substrate surface area. However, weak fluorescent signals and the poor signal-to-noise ratios of their associated fluorescent labels remain challenges, limiting the maximum sensitivity of current fluorescence-based assays.

[0005] Most previous plasmon-enhanced fluorescence assays rely on the design of plasmon-active substrates by either the deposition of metal islands or the adsorption of plasmonic nanostructures. These methods, of course, require the use of specialized surfaces and potentially major modifications of the readout device and bioassay protocol. Therefore, the methods are not easily applicable to many different systems or bioassays.

[0006] Although some plasmon-enhanced fluorescence assays have attempted to use solution-phase particles, these particles suffer from instability, highly nonspecific binding that causes unacceptable background signals, and, most importantly, poor fluorescence enhancement, typically less than 10-fold.

[0007] Fluorescent probes and fluorescence analysis methods have been used in biomedical research not only as imaging tools for visualizing the location and dynamics of cells and various subcellular species and molecular interactions in cells and tissues, but also as labels in fluorescence immunoassays for the detection and quantification of molecular biomarkers. Fluorescence-based techniques have fundamentally transformed biology and life sciences by elucidating genomic, transcriptomic, and proteomic signatures of disease onset, progression, and response to treatment. However, “weak signals” have been a persistent and recurring problem in a range of detection and imaging techniques that rely on fluorescence. Overcoming this fundamental challenge without the use of specialized reagents, equipment, or major modifications to well-established procedures has been the subject of extensive research in the field of biomedical optics. For example, there is an urgent need for ultrasensitive fluorescence immunoassays that can be widely adopted by most biological and clinical laboratories to detect low-abundance target species.

[0008] Although fluorescence offers several advantages over assay detection schemes such as colorimetric ELISA or chemiluminescence—multiplexing, high dynamic range, and broad platform applicability (i.e., it can be used intracellularly, on cell surfaces, in tissues, on plates, on beads, in solution, etc.)—fluorescence is fundamentally limited by its weak signal. In plate-based assays, improved sensitivity of fluorescence detection has been achieved using complex schemes such as poly-HRP, PCR-ELISA, avidin-biotin-complex (ABC) ELISA, and tyramide signal amplification (TSA). All of these are more complex, more expensive, and generally have a poorer dynamic range than the assay types they replace. To achieve very high detection sensitivity, complex techniques such as digital ELISA (Quanterix Simoa System) or electrochemiluminescence (Meso Scale Discovery) require specialized substrates, equipment, and workflows, respectively.

[0009] Generally, if an assay uses an antibody or streptavidin labeled with HRP that catalyzes a reaction that converts a substrate into either a luminescent species (as in chemiluminescence) or a species that absorbs light at a certain wavelength (as in ELISA), the performance of the assay can be improved by using antibody- or streptavidin-conjugated Plasmon Fluor. Examples of such assays are membrane-based immunoassays such as ELISA (colorimetric and chemiluminescent) and Western blots (colorimetric and chemiluminescent).

[0010] Improving the signal-to-noise ratio of assays without completely deviating from existing assay protocols would alleviate the stringent requirements for high sensitivity and large photodetectors, reduce implementation costs, eliminate inconsistencies between laboratories and platforms, and potentially advance these technologies to point-of-care, in-field, and resource-limited environments. Various techniques, including multiple fluorophore labeling, rolling-cycle amplification, and photonic crystal enhancement, have been introduced to improve the signal-to-noise ratio of fluorescence-based imaging and detection techniques. Despite improvements in sensitivity, these techniques have not been widely adopted in research and clinical settings. Most of these techniques require significant modifications to existing practices, such as additional steps that significantly extend overall operation time, dedicated and expensive readout systems, unconventional data processing and analysis, or temperature-sensitive reagents that require strictly controlled shipping and storage conditions.

[0011] Enhanced emission of fluorophores in the vicinity of plasmonic nanostructures is due in part to the enhanced electromagnetic field at the surface of the plasmonic nanostructure (local excitation region) and reduced fluorescence lifetime due to coupling between the excited fluorophore and the nanostructure's surface plasmons. While various plasmonic substrates, such as metal nanoislands, have been shown to provide moderate fluorescence enhancement, these plasmon-active surfaces require the use of prefabricated substrates, typically glass slides on which metal nanostructures are deposited, instead of standard or sometimes non-interchangeable biological analysis and imaging platforms. The need for specialized substrates limits inter-platform and inter-laboratory consistency and seamless integration with widely used biological analysis procedures, significantly limiting their widespread application in biomedical research and clinical settings. Non-traditional bioconjugation procedures and the poor stability of biomolecules (e.g., antibodies) immobilized on metal surfaces pose further challenges to their widespread application. Solution-phase plasmon-enhanced fluorescence solutions have generally been limited by poor fluorescence enhancement and unstable particles. Summary of the Invention [Problem to be solved by the invention]

[0012] Fluorescence-based multiplex microarrays are used in expression profiling, drug target binding assays, and high-throughput proteomics. Compared to single platforms such as enzyme-linked immunosorbent assays (ELISAs), this technique allows researchers and clinicians to test a large number of biomarkers in parallel, enabling patient stratification and multifactorial disease monitoring with limited sample volume. This allows for multiple individual biomarker assays while minimizing assay costs and time. Furthermore, high-throughput biomarker profiling enables personalized medicine using comprehensive molecular fingerprinting of diseases, further improving diagnostic resolution between closely related disease phenotypes. For example, combining urinary levels of multiple biomarkers has proven to significantly increase the sensitivity and specificity for diagnosing kidney disease compared to individual biomarkers. However, despite the availability of various commercial products, this multiplex method suffers from lower sensitivity and a relatively high limit of detection (LOD) compared to ELISAs, hindering its widespread application.

[0013] One approach to addressing the low sensitivity of various fluorescent assays is disclosed in U.S. Provisional Application No. 62 / 590,877, filed November 27, 2017, entitled "Plasmonic Film as a Universal Fluorescent Enhancer," which is incorporated herein by reference in its entirety. In this approach, plasmonic nanostructures are deposited on a polymer film surface, and the polymer film is then placed, with the plasmonic structures facing downward, over the top of a plate or assay that has been pre-coated with fluorophores. By placing the film in this orientation, the plasmonic nanostructures are brought into close proximity with the fluorophores, resulting in excellent fluorescence enhancement and a significant increase in sensitivity of the assay. Some assay techniques, while very useful, are not compatible with this approach (e.g., the assay is not performed on a flat, rigid surface such as a microplate or glass slide). Therefore, there is a need to develop methods that address each of these shortcomings while being compatible with more assay techniques. [Means for solving the problem]

[0014] In one embodiment, a fluorescent nanoconstruct is disclosed herein. The nanoconstruct generally comprises a plasmonic nanostructure having at least one localized surface plasmon resonance wavelength (λLSPR), at least one spacer coating, and at least one fluorescent agent having a maximum excitation wavelength (λEX). The fluorescent nanoconstruct has a fluorescence intensity at least 500 times greater than that of the at least one fluorescent agent alone.

[0015] In another aspect, a method for constructing a fluorescent nanoconstruct is disclosed herein. The method generally includes coating a plasmonic nanostructure with at least one spacer coating, optionally coating the at least one spacer coating with a functional layer, conjugating a fluorescent agent to either the at least one spacer coating or the functional layer, and optionally conjugating a biorecognition element to either the at least one spacer coating or the functional layer.

[0016] In yet another aspect, disclosed herein are methods for detecting an analyte using an assay, which generally include adding a fluorescent nanoconstruct to the assay to generate a fluorescent signal, and detecting the analyte by analyzing the fluorescent signal. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is an exemplary embodiment of the fluorescence intensity of conventional Cy3 and Plasmon-Fluor-Cy3 at various molar concentrations thereof according to the present disclosure.

[0018] [Figure 2] FIG. 2 is an exemplary embodiment of the fluorescence intensity of conventional Fluor-800CW and Plasmon-Fluor-800CW at various molar concentrations thereof according to the present disclosure.

[0019] [Figure 3] FIG. 3 is an exemplary embodiment of the fluorescence intensity of conventional FITC and Plasmon-Fluor-FITC at various molar concentrations thereof according to the present disclosure.

[0020] [Figure 4] FIG. 4 is an exemplary embodiment of a Plasmon-Fluor design according to the present disclosure.

[0021] [Figure 5]Figure 5 shows an exemplary embodiment of the normalized extinction spectra of aqueous solutions of three representative plasmonic nanostructures (from left to right: Au@Ag-490, AuNR-670, and AuNR-760) according to the present disclosure. The extinction spectra of Au@Ag-490, AuNR-670, and AuNR-760 show significant overlap with the absorption (excitation) spectra of FITC, 680LT, and 800CW, respectively, and their appropriate excitation wavelengths.

[0022] [Figure 6] 6A and 6B are exemplary embodiments of the importance of overlapping absorbance of plasmonic particles and the absorbance / excitation spectra of conjugated dyes according to the present disclosure.

[0023] [Figure 7] Figure 7A shows an exemplary embodiment of the fluorescence intensity map (left) and enhancement factor (right) of the resulting AuNRs and AuNRs with a polymer spacer layer according to the present disclosure. Figure 7B shows an exemplary embodiment of the fluorescence lifetime of a conventional fluorophore (800CW) and a fluorescent nanoconstruct according to the present disclosure (AuNR-800CW).

[0024] [Figure 8] Figure 8A is an exemplary embodiment of multiple confocal laser scanning microscopy images showing fluorescent signals corresponding to a protein biomarker (ErbB2) overexpressed on the surface of breast cancer cells by probing the biomarker using various dilutions of ErbB2 primary antibody (top: with particle enhancement; bottom: without particle enhancement) in accordance with the present disclosure. Fluorescent signals are still present even after a 100,000-fold dilution of the ErbB2 primary antibody with nanostructure enhancement. Figure 8B is an exemplary embodiment of the mean fluorescent intensity of labeled breast cancer cells with and without particle enhancement in accordance with the present disclosure.

[0025] [Figure 9]9A and 9B are exemplary embodiments of histograms of fluorescence intensity corresponding to ErbB2 receptor obtained using Fluor and fluorescent nanoconstructs according to the present disclosure.

[0026] [Figure 10] FIG. 10 is an exemplary embodiment of a Plasmon-Fluor design according to the present disclosure.

[0027] [Figure 11] FIG. 11 is an exemplary embodiment of an alternative design of a Plasmon-Fluor according to the present disclosure.

[0028] [Figure 12] FIG. 12 is an exemplary embodiment of an additional alternative design of a Plasmon-Fluor according to the present disclosure.

[0029] [Figure 13] Figure 13 shows another exemplary embodiment of a plasmonic nanostructure according to the present disclosure. Plasmonic nanostructures (gold nanorods coated with silver) are embedded in a dielectric material matrix. The dielectric material matrix is ​​coated with a functional layer (blue clouds). A targeting agent (pink "y"-shaped, e.g., an antibody) is conjugated to the functional layer.

[0030] [Figure 14] FIG. 14 is an exemplary embodiment of the extinction spectrum (excitation maximum=784 nm) of a Plasmon-Fluor conjugated to an IRDye 800CW in accordance with the present disclosure.

[0031] [Figure 15] FIG. 15 is an exemplary embodiment of the mismatch between the LSPR maximum of the Plasmon-Fluor and the excitation maximum of the IRDye 800CW, according to the present disclosure.

[0032] [Figure 16]FIG. 16 is an exemplary embodiment of the extinction spectrum (excitation maximum=550 nm) of Plasmon-Fluor (AuNR@Ag cuboid plasmonic nanostructure) conjugated to Cy3 according to the present disclosure.

[0033] [Figure 17] FIG. 17 is an exemplary embodiment of the mismatch between the LSPR maximum of Plasmon-Fluor (AuNR@Ag cuboid plasmonic nanostructures) and the excitation maximum of Cy3, according to the present disclosure.

[0034] [Figure 18] Figure 18 is an exemplary embodiment of a plasmonic nanostructure according to the present disclosure. The plasmonic nanostructure is coated in a dielectric matrix of a specific thickness (green shell). Fluorophores (red star shapes) are directly attached to the outer surface of the dielectric matrix. Biorecognition elements (pink "y" shapes, e.g., antibodies) can be directly conjugated to a spacer, which can be coated with a functional layer material (blue cloud).

[0035] [Figure 19] FIG. 19 is an exemplary embodiment of a plot showing the standard curve (dose-dependent colorimetric signal) of a human NGAL ELISA that takes 280 minutes to complete, according to the present disclosure.

[0036] [Figure 20] FIG. 20 is an exemplary embodiment of a plot showing human NGAL dose-dependent fluorescence intensity from p-FLISA performed within 20 minutes according to the present disclosure.

[0037] [Figure 21] FIG. 21 is an exemplary embodiment of NGAL concentrations in urine samples from renal patients and healthy volunteers determined using p-FLISA completed within 20 minutes according to the present disclosure.

[0038] [Figure 22]FIG. 22 is an exemplary embodiment of a plot showing the correlation between concentrations of human NGAL determined using ELISA (280 minute assay) and p-FLISA (20 minute) according to the present disclosure.

[0039] [Figure 23] FIG. 23 is an exemplary embodiment of a general sandwich immunoassay using biotinylated plasmon-fluor in accordance with the present disclosure.

[0040] [Figure 24] FIG. 24 is an exemplary embodiment of enhancement of a general sandwich immunoassay using streptavidin-conjugated plasmon-fluor in accordance with the present disclosure.

[0041] [Figure 25] FIG. 25 is an exemplary embodiment of a general sandwich immunoassay method using a plasmon-fluor conjugated secondary antibody in accordance with the present disclosure, where the antibody conjugated to the plasmon-fluor recognizes the detection antibody.

[0042] [Figure 26] FIG. 26 is an exemplary embodiment of a general sandwich immunoassay method using a plasmon-fluor conjugated primary antibody in accordance with the present disclosure, where the antibody conjugated to the plasmon-fluor recognizes the analyte.

[0043] [Figure 27] 27A is an exemplary embodiment of a TEM image of gold nanorods (AuNR) used as nanostructures in a Plasmon Fluor-800CW according to the present disclosure, and FIG. 27B is an exemplary embodiment of a finite-difference time-domain (FDTD) simulation showing the distribution of electric field strength around the AuNR according to the present disclosure.

[0044] [Figure 28]FIG. 28 is an exemplary embodiment of a schematic diagram showing the steps involved in forming polymer spacers on the surface of plasmonic nanostructure AuNRs according to the present disclosure.

[0045] [Figure 29] FIG. 29 is an exemplary embodiment of AFM images illustrating the increase in diameter of AuNR / polymer under increasing amounts of monomers (MPTMS, TMPS, and APTMS) according to the present disclosure.

[0046] [Figure 30] FIG. 30 is an exemplary embodiment of the UV-visible spectra of AuNR under various polymerization conditions according to the present disclosure.

[0047] [Figure 31] FIG. 31 is an exemplary embodiment of a plot showing the increase in diameter of AuNRs (2x greater than the thickness of the polymer layer) under each polymerization condition as measured from AFM images, according to the present disclosure.

[0048] [Figure 32] FIG. 32 is an exemplary embodiment of the zeta potential of AuNR, AuNR / MPTMS, AuNR / MPTMS / polysiloxane (AuNR / polymer), and Plasmon-Fluor-800CW (AuNR / polymer / BSA-biotin-800CW) in accordance with the present disclosure.

[0049] [Figure 33] 33A and 33B are exemplary embodiments of PF-800CW TEM and extinction spectra according to the present disclosure.

[0050] [Figure 34] FIG. 34 is an exemplary embodiment of a schematic diagram (not to scale) showing a model system based on binding events occurring in a fluorophore-labeled immunosorbent assay according to the present disclosure.

[0051] [Figure 35]FIG. 35 is an exemplary embodiment of various capacities of core AuNR plasmonic nanostructures to enhance 800 CW according to the present disclosure.

[0052] [Figure 36] FIG. 36 is an exemplary embodiment of various capacities of core AuNR plasmonic nanostructures to enhance 800 CW according to the present disclosure.

[0053] [Figure 37] FIG. 37 is an exemplary embodiment of an extinction spectrum for an AuNR@Ag cuboid according to the present disclosure.

[0054] [Figure 38] FIG. 38 is an exemplary embodiment of a plasmonic nanostructure suitable for enhancing fluorophores that can be excited at 488 nm (Au@Ag-490), 658 nm (AuNR-670), and 784 nm (AuNR-760) according to the present disclosure.

[0055] [Figure 39] 39A and 39B are exemplary embodiments of TEM of PF-532 (Cy3) according to the present disclosure.

[0056] [Figure 40] FIG. 40 is an exemplary embodiment of the extinction spectrum of PF-532 (Cy3) according to the present disclosure.

[0057] [Figure 41] FIG. 41 is an exemplary embodiment of the fluorescence enhancement factor obtained using Plasmon-Fluor-800CW with various polymer spacer thicknesses according to the present disclosure.

[0058] [Figure 42]Figure 42 is an exemplary embodiment of plasmon-enhanced fluorescence and colloidal stability of Plasmon-Fluor according to the present disclosure. Error bars represent standard deviation (n≧3 independent tests). Data statistically significant P-value=0.0013, **P<0.01 (by two-tailed unpaired t-test with Welch's correction). The plot on the left of Figure 42 shows the stability of Plasmon-Fluor suspensions stored at 4°C and reconstituted from lyophilized powder. Error bars represent standard deviation (n=6 replicates). NS: not significant. P-value>0.9999 (by one-way ANOVA with Tukey's post-hoc test). Figure 42 also shows photographs illustrating the lyophilized powder of Plasmon-Fluor before and after reconstitution.

[0059] [Figure 43] FIG. 43 is an exemplary embodiment of a schematic illustrating the concept of conventional FLISA (800CW) and Plasmon-Fluor-800CW enhanced FLISA (p-FLISA) performed in standard 96-well plates according to the present disclosure.

[0060] [Figure 44] FIG. 44 is an exemplary embodiment of fluorescence intensity maps of human IL-6 FLISA and p-FLISA at various analyte concentrations according to the present disclosure.

[0061] [Figure 45] FIG. 45 is an exemplary embodiment of a fluorescence intensity map (with enlarged scale bars) of human IL-6 FLISA and p-FLISA, and a photograph of the colorimetric signal of the "gold standard" human IL-6 ELISA, according to the present disclosure.

[0062] [Figure 46] FIG. 46 is an exemplary embodiment of individual data points, means and standard deviations from human IL-6 FLISA, p-FLISA and ELISA according to the present disclosure.

[0063] [Figure 47]FIG. 47 is an exemplary embodiment of a plot of human IL-6 dose-dependent fluorescence intensity from a conventional FLISA according to the present disclosure.

[0064] [Figure 48] FIG. 48 is an exemplary embodiment of the LOD of a conventional IL-6 FLISA according to the present disclosure.

[0065] [Figure 49] FIG. 49 is an exemplary embodiment of a human IL-6 dose-dependent fluorescence intensity plot from p-FLISA according to the present disclosure.

[0066] [Figure 50] Figure 50A is an exemplary embodiment of IL-6 dose-dependent fluorescence intensity from p-FLISA according to the present disclosure, and Figure 50B is an exemplary embodiment of non-specific binding of Plasmon-Fluor-800CW according to the present disclosure.

[0067] [Figure 51] FIG. 51 is an exemplary embodiment of an SEM image of the bottom surface of a 96-well plate after IL-6 p-FLISA, according to the present disclosure.

[0068] [Figure 52] FIG. 52 is an exemplary embodiment of a plot showing a standard curve for a human IL-6 ELISA according to the present disclosure.

[0069] [Figure 53] FIG. 53 is an exemplary embodiment of IL-6 concentrations in human serum samples (diluted 10-fold) measured using p-FLISA according to the present disclosure.

[0070] [Figure 54] FIG. 54 is an exemplary embodiment of a schematic illustrating the concept of using Plasmon-Fluor-Cy3 to enhance the sensitivity of a bead-based immunoassay (eg, a Luminex assay) according to the present disclosure.

[0071] [Figure 55] 55A and 55B are exemplary embodiments of TEM images of plasmonic-Fluor-Cy3 utilizing AuNR@Ag as plasmonic nanostructures in accordance with the present disclosure.

[0072] [Figure 56] Figure 56A is an exemplary embodiment of a fluorescence microscope image of an individual Plasmon-Fluor-Cy3 according to the present disclosure. Figure 56B is an exemplary embodiment of an SEM image of an individual Plasmon-Fluor-Cy3 shown in Figure 56A according to the present disclosure. Figure 56C is an exemplary embodiment of a magnified SEM image corresponding to the square shown in Figures 56A and 56B showing a single Plasmon-Fluor-Cy3 (single nanocuboid) according to the present disclosure.

[0073] [Figure 57] FIG. 57 is an exemplary embodiment of an SEM image of microbeads before and after being interrogated with Plasmon-Fluor-Cy3 according to the present disclosure.

[0074] [Figure 58] Figure 58A is an exemplary embodiment of a microscope brightfield and fluorescence image of a Luminex microbead before interrogation with Plasmon-Fluor-Cy3 according to the present disclosure, and Figure 58B is an exemplary embodiment of a microscope brightfield and fluorescence image of a Luminex microbead after interrogation with Plasmon-Fluor-Cy3 according to the present disclosure.

[0075] [Figure 59]Figure 59(A-D) are exemplary embodiments of images of Luminex microbeads after staining with Plasmon-Fluor-Cy3 according to the present disclosure. Figure 59A is a fluorescence image of Luminex microbeads after staining with Plasmon-Fluor-Cy3 showing the barcodes of the microbeads (excited with a 633 nm laser) of various emission intensities. Figure 59B is a fluorescence image of Luminex microbeads after staining with Plasmon-Fluor-Cy3 showing the fluorescence of bound Cy3 (excited with a 543 nm laser). Figure 59C is a brightfield image of the microbeads. Figure 59D is a composite image of brightfield and fluorescence shown in Figure 59(A-C).

[0076] [Figure 60] FIG. 60 is an exemplary embodiment of a fluorescence image of a microbead before and after interrogation with Plasmon-Fluor-Cy3 according to the present disclosure.

[0077] [Figure 61] FIG. 61 is an exemplary embodiment of a mouse IL-6 standard curve obtained before (left) and after (right) application of Plasmon-Fluor-Cy3, according to the present disclosure.

[0078] [Figure 62] FIG. 62 is an exemplary embodiment of a mouse TNF-α standard curve obtained before (left) and after (right) application of Plasmon-Fluor-Cy3, according to the present disclosure.

[0079] [Figure 63] FIG. 63 is an exemplary embodiment of individual data points, means, and standard deviations from a mouse IL-6 Luminex, a plasmon-fluor-Cy3 enhanced mouse IL-6 Luminex, a mouse TNF-α Luminex, and a plasmon-fluor-Cy3 enhanced mouse TNF-α Luminex assay according to the present disclosure.

[0080] [Figure 64]Figure 64A is an exemplary embodiment of a plot showing the LOD of an unenhanced bead-based fluorescent immunoassay (Luminex) for mouse IL-6 according to the present disclosure. Figure 64B is an exemplary embodiment of a plot showing the LOD of an unenhanced bead-based fluorescent immunoassay (Luminex) for TNF-alpha according to the present disclosure.

[0081] [Figure 65] FIG. 65 is an exemplary embodiment showing how biotinylated plasmon-fluor can be used to enhance an exemplary multiplexed microarray according to the present disclosure.

[0082] [Figure 66] FIG. 66 is an exemplary embodiment of a method of using streptavidin-conjugated plasmon-fluor to enhance a typical multiplexed microarray according to the present disclosure.

[0083] [Figure 67] Figure 67 (A-B) are exemplary embodiments of identifying the specific analyte (or control) of each pair of fluorescent spots for a kidney biomarker array according to the present disclosure. The fluorescent spots shown in Figure 67A are identified by their coordinates in Figure 67B.

[0084] [Figure 68] FIG. 68 is an exemplary embodiment of an SEM image showing uniform distribution of Plasmon-Fluor-800CW (several highlighted by yellow circles) on and within the subsurface region of a nitrocellulose membrane in accordance with the present disclosure.

[0085] [Figure 69] FIG. 69 is an exemplary embodiment of a fluorescence intensity map representing a kidney disease protein biomarker profile of a kidney disease patient obtained using a conventional fluorophore (streptavidin-800CW) with a fluorescence intensity scale bar of 0 to 13, in accordance with the present disclosure.

[0086] [Figure 70] FIG. 70 is an exemplary embodiment of a fluorescence intensity map representing the kidney disease protein biomarker profile of FIG. 45 including a fluorescence intensity scale bar of 0 to 5000 according to the present disclosure.

[0087] [Figure 71] FIG. 71 is an exemplary embodiment of a fluorescence intensity map representing the kidney disease protein biomarker profile of the kidney disease patient shown in FIGS. 69 and 70 after addition of Plasmon-Fluor-800CW, including a fluorescence intensity scale bar of 0 to 5000, in accordance with the present disclosure.

[0088] [Figure 72] Figure 72A is an exemplary embodiment of a pair of fluorescent spots of a kidney biomarker array shown in Figure 67A according to the present disclosure. Figure 72B is an exemplary embodiment of an SEM image of a nitrocellulose membrane in a negative control region (blue rectangle shown in the lower right corner of Figure 72A, corresponding to coordinates F23 and F24 shown in Figure 67A) according to the present disclosure.

[0089] [Figure 73] FIG. 73 is an exemplary embodiment of individual data points, mean and standard deviation using Plasmon-Fluor according to the present disclosure.

[0090] [Figure 74] FIG. 74 is an exemplary embodiment of individual data points, mean and standard deviation without Plasmon-Fluor according to the present disclosure.

[0091] [Figure 75] FIG. 75 is an exemplary embodiment of a photograph taken from a cell phone showing the color change of a nitrocellulose membrane with a urine sample from a kidney disease patient after addition of Plasmon-Fluor-800CW in accordance with the present disclosure.

[0092] [Figure 76] Figure 76A is an exemplary embodiment of a 40-plex cytokine microarray arrangement according to the present disclosure. Figure 76B is an exemplary embodiment of a fluorescence map of a cytokine microarray obtained using a conventional fluorophore (streptavidin-800CW) according to the present disclosure. Figure 76C is an exemplary embodiment of a fluorescence map of a cytokine microarray obtained after the addition of Plasmon-Fluor-800CW according to the present disclosure. Figure 76D is an exemplary embodiment of a plot showing the fluorescence intensity corresponding to each cytokine obtained using a conventional fluorophore (streptavidin-800CW) according to the present disclosure. Figure 76E is an exemplary embodiment of a plot showing the fluorescence intensity corresponding to each cytokine obtained after the addition of Plasmon-Fluor-800CW according to the present disclosure. Figure 76F is an exemplary embodiment of dark-field scattering of Plasmon-Fluor-800CW (AuNR) absorbed on a cytokine microarray according to the present disclosure.

[0093] [Figure 77] FIG. 77 is an exemplary embodiment of a plot showing the correlation between two readout modes (fluorescence readouts vs. colorimetric readouts) of a kidney biomarker array according to the present disclosure.

[0094] [Figure 78] FIG. 78 shows an exemplary embodiment of confocal laser scanning microscopy (CLSM) images of breast cancer cells (SK-BR-3) examined with conventional Fluor (800CW, top row) and Plasmon-Fluor-800CW (bottom row) according to the present disclosure at various concentrations of ErbB2 primary antibody.

[0095] [Figure 79]Figure 79A is an exemplary embodiment of a bright field microscope image of SK-BR-3 cells before (top) and after (bottom) labeling with Plasmon-Fluor-800CW according to the present disclosure. Figure 79B is an exemplary embodiment of an SEM image of conventional Fluor-labeled SK-BR-3 cells according to the present disclosure. Figure 79C is an exemplary embodiment of an SEM image of Plasmon-Fluor-800CW-labeled SK-BR-3 cells according to the present disclosure, with the inset showing Plasmon-Fluor distributed uniformly on the cell membrane surface.

[0096] [Figure 80] FIG. 80 is an exemplary embodiment of a plot showing the fluorescence intensity of SK-BR-3 cells stained with conventional Fluor and Plasmon-Fluor-800CW in accordance with the present disclosure.

[0097] [Figure 81] Figure 81A is an exemplary embodiment of a confocal laser scanning microscopy (CLSM) image of ErbB2-stained breast cancer cells (SK-BR-3) obtained using a conventional immunocytochemistry procedure (cells are sequentially labeled with biotinylated primary antibody and streptavidin-Fluor (800CW)) at various dilutions of the ERbB2 primary antibody, according to the present disclosure. Figure 81B is an exemplary embodiment of a confocal laser scanning microscopy (CLSM) image of ErbB2-stained breast cancer cells (SK-BR-3) after the addition of Plasmon-Fluor-800CW at various dilutions of the ERbB2 primary antibody, according to the present disclosure.

[0098] [Figure 82] FIG. 82 is an exemplary embodiment of fluorescence mapping of SK-BR-3 cells cultured in a 6-well plate according to the present disclosure.

[0099] [Figure 83]FIG. 83 is an exemplary embodiment of a schematic diagram showing flow cytometry of ErbB2 stained SK-BR-3 cells examined by conventional Fluor (680LT) followed by Plasmon-Fluor-680LT in accordance with the present disclosure.

[0100] [Figure 84] 84A and 84B are exemplary embodiments of a TEM image and extinction spectrum of 680LT according to the present disclosure.

[0101] [Figure 85] Figure 85A is an exemplary embodiment of a photograph showing the color change of SK-BR-3 cells (top: pellet; bottom: suspension) after labeling with Plasmon-Fluor-680LT according to the present disclosure. Figure 85B is an exemplary embodiment of the visible-NIR extinction spectra of Plasmon-Fluor-680LT labeled SK-BR-3 cell suspension under various dilutions of ErbB2 primary antibody according to the present disclosure.

[0102] [Figure 86] FIG. 86 is an exemplary embodiment of a pseudocolor plot of side and forward scatter of SK-BR-3 cells before (left) and after (right) labeling with Plasmon-Fluor-680LT (including an example of a gating strategy to include single cells) according to the present disclosure.

[0103] [Figure 87] FIG. 87 is an exemplary embodiment of a flow contour plot (including outliers) of fluorescence versus forward scatter (vertically offset for clarity) of SK-BR-3 cells examined using various concentrations of ErbB2 primary antibody according to the present disclosure.

[0104] [Figure 88] FIG. 88 is an exemplary embodiment of a fluorescence histogram of SK-BR-3 cells examined using conventional Fluor (680LT) followed by the addition of Plasmon-Fluor-680LT (primary antibody at a 103 dilution) in accordance with the present disclosure.

[0105] [Figure 89] FIG. 89 is an exemplary embodiment of a histogram showing the amount of fluorescence of SK-BR-3 cells before (top) and after (bottom) addition of Plasmon-Fluor-680LT according to the present disclosure.

[0106] [Figure 90] FIG. 90 is an exemplary embodiment of a plot showing mean fluorescence intensity obtained from flow cytometry at various primary antibody concentrations according to the present disclosure.

[0107] [Figure 91] FIG. 91 is an exemplary embodiment of a schematic diagram showing bone marrow-derived dendritic cells (BMDCs) treated with an immunostimulant [lipopolysaccharide (LPS)] according to the present disclosure.

[0108] [Figure 92] FIG. 92 is an exemplary embodiment of an exemplary embodiment of two schemes for using plasmon-fluor labeled antibodies in labeling target antigens on cells according to the present disclosure.

[0109] [Figure 93] FIG. 93 is an exemplary embodiment of fluorescence intensity distributions corresponding to naive (control) and LPS-stimulated BMDCs obtained using conventional Fluor (680LT) in accordance with the present disclosure.

[0110] [Figure 94] FIG. 94 is an exemplary embodiment of fluorescence intensity distributions corresponding to naive (control) and LPS-stimulated BMDCs obtained using Plasmon-Fluor-680LT according to the present disclosure.

[0111] [Figure 95]Figure 95A is an exemplary embodiment of a pseudocolor plot showing side scatter versus CD80 fluorescence of a BMDC population without LPS stimulation (left: naive) and after treatment with 0.05 μg / ml LPS (right) using conventional immunofluorescence staining in accordance with the present disclosure. Figure 95B is an exemplary embodiment of a pseudocolor plot showing side scatter versus CD80 fluorescence of a BMDC population without LPS stimulation (left: naive) and after treatment with 0.05 μg / ml LPS (right) using Plasmon-Fluor-680LT in accordance with the present disclosure.

[0112] [Figure 96] FIG. 96 is an exemplary embodiment of a plot showing the mean fluorescence intensity (corresponding to the expression level of CD80) of BMDCs after stimulation with various amounts of LPS, according to the present disclosure.

[0113] [Figure 97] Figure 97A is an exemplary embodiment of a plot showing the mean fluorescence (corresponding to the expression level of CD80) of BMDCs examined using conventional immunofluorescence staining after stimulation with various amounts of LPS, in accordance with the present disclosure. Figure 97B is an exemplary embodiment of a plot showing the mean fluorescence (corresponding to the expression level of CD80) of BMDCs examined using Plasmon-Fluor-680LT after stimulation with various amounts of LPS, in accordance with the present disclosure.

[0114] [Figure 98] FIG. 98 is an exemplary embodiment of secretion levels of pro-inflammatory cytokines (TNF-α and IL-12) according to the present disclosure.

[0115] [Figure 99] FIG. 99 is an exemplary embodiment of individual data points (absorbance and concentration), mean concentration and standard deviation of ELISA results corresponding to inflammatory cytokines secreted after LPS stimulation according to the present disclosure.

[0116] [Figure 100]Figure 100(A-C) are exemplary embodiments of plots showing IL-6 dose-dependent fluorescence intensity from p-FLISA, according to the present disclosure. Figures 100A, 100B, and 100C are illustrative of experiments performed independently on various days using different batches of Plasmon-Fluor-800CW.

[0117] [Figure 101] Figure 101 (A-B) is an exemplary embodiment of a bead-based mouse TNF-α standard curve obtained after applying Plasmon-Fluor-Cy3 according to the present disclosure. Figures 101A and 101B illustrate independently performed experiments for different batches of Plasmon-Fluor-Cy3.

[0118] [Figure 102] Figure 102 (A-B) is an exemplary embodiment of a bead-based mouse IL-6 standard curve obtained after applying Plasmon-Fluor-Cy3 according to the present disclosure. Figures 102A and 102B illustrate independently performed experiments for different batches of Plasmon-Fluor-Cy3.

[0119] [Figure 103] Figure 103A is another exemplary embodiment of fluorescence intensity corresponding to the concentrations of various urinary biomarkers before addition of Plasmon-Fluor-800CW (a typical assay using a conventional fluorophore) according to the present disclosure. Figure 103B is another exemplary embodiment of fluorescence intensity corresponding to the concentrations of various urinary biomarkers after addition of Plasmon-Fluor-800CW (a typical assay using a conventional fluorophore) according to the present disclosure.

[0120] [Figure 104]Figure 104A is another exemplary embodiment of a confocal laser scanning microscopy (CLSM) image of ErbB2-stained breast cancer cells (SK-BR-3) obtained using a conventional immunocytochemistry procedure (cells are sequentially labeled with biotinylated primary antibody and streptavidin-Fluor (800CW)) at various dilutions of the ERbB2 primary antibody, according to the present disclosure. Figure 104B is another exemplary embodiment of a confocal laser scanning microscopy (CLSM) image of ErbB2-stained breast cancer cells (SK-BR-3) after the addition of Plasmon-Fluor-800CW at various dilutions of the ERbB2 primary antibody, according to the present disclosure.

[0121] [Figure 105] Figure 105A is yet another exemplary embodiment of a confocal laser scanning microscopy (CLSM) image of ErbB2-stained breast cancer cells (SK-BR-3) obtained using a conventional immunocytochemistry procedure (cells are sequentially labeled with biotinylated primary antibody and streptavidin-Fluor (800CW)) at various dilutions of the ERbB2 primary antibody, according to the present disclosure. Figure 105B is yet another exemplary embodiment of a confocal laser scanning microscopy (CLSM) image of ErbB2-stained breast cancer cells (SK-BR-3) after the addition of Plasmon-Fluor-800CW at various dilutions of the ERbB2 primary antibody, according to the present disclosure.

[0122] [Figure 106] Figure 106A is another exemplary embodiment of a histogram showing the amount of fluorescence of SK-BR-3 cells before (top) and after (bottom) the addition of Plasmon-Fluor-680LT in accordance with the present disclosure. Figure 106B is another exemplary embodiment of a plot showing the mean fluorescence intensity obtained from flow cytometry at various primary antibody concentrations in accordance with the present disclosure.

[0123] [Figure 107]Figure 107A is yet another exemplary embodiment of a histogram showing the amount of fluorescence of SK-BR-3 cells before (top) and after (bottom) the addition of Plasmon-Fluor-680LT in accordance with the present disclosure. Figure 107B is yet another exemplary embodiment of a plot showing the mean fluorescence intensity obtained from flow cytometry at various primary antibody concentrations in accordance with the present disclosure.

[0124] [Figure 108] Figure 108A is another exemplary embodiment of a fluorescence intensity distribution corresponding to naive (control) and LPS-stimulated BMDCs obtained using conventional Fluor (680LT) in accordance with the present disclosure. Figure 108B is another exemplary embodiment of a fluorescence intensity distribution corresponding to naive (control) and LPS-stimulated BMDCs obtained using Plasmon-Fluor-680LT in accordance with the present disclosure. Figure 108C is another exemplary embodiment of a plot showing the mean fluorescence intensity (corresponding to the expression level of CD80) of BMDCs after stimulation with various amounts of LPS in accordance with the present disclosure.

[0125] [Figure 109] Figure 109A is yet another exemplary embodiment of fluorescence intensity distributions corresponding to naive (control) and LPS-stimulated BMDCs obtained using conventional Fluor (680LT) in accordance with the present disclosure. Figure 109B is yet another exemplary embodiment of fluorescence intensity distributions corresponding to naive (control) and LPS-stimulated BMDCs obtained using Plasmon-Fluor-680LT in accordance with the present disclosure. Figure 109C is yet another exemplary embodiment of a plot showing the mean fluorescence intensity (corresponding to the expression level of CD80) of BMDCs after stimulation with various amounts of LPS in accordance with the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0126] The present disclosure is based, at least in part, on the discovery that fluorescent plasmonic nanostructures can be tuned to match the wavelength of a conjugated fluorophore, resulting in at least a 500-fold enhancement in fluorescence intensity.

[0127] The present disclosure is directed to ultrabright fluorescent nanoconstructs specifically designed for use in the biological detection and quantification of target analytes. As an example of their extreme potency, plasmon-fluor conjugated to a standard targeting agent (e.g., streptavidin) is at least 500 times brighter than the same standard targeting agent linked to a fluorescent molecule commonly used in microplate-based fluorescence-linked immunosorbent assays. This results in significant improvements in assay performance due to both sensitivity (an order of magnitude or more improvement in the detection limit) and dynamic range.

[0128] Design Advantages of the Present Disclosure Advantages of the design disclosed herein over previous approaches include, but are not limited to: (1) plasmonic-fluors are significantly more solution-phase useful than substrates decorated with plasmonic species; (2) straightforward wet-chemical synthesis compared to Cu-Ag NP alloys, lithographically generated structures, or vapor deposition or layer-by-layer synthesis; (3) high degree of control over particle homogeneity / stability / synthesis, which is important for immunoassays: (a) aggregation is generally a major issue with nanoparticles and can lead to serious artifacts; (b) high nonspecific background is also a recognized problem; and (4) spacers between the fluorophore and plasmonic nanostructure core using MTPMS / APTMS / TMPS are used to achieve precise thickness on the nanometer scale. (5) the silane-based spacer layer is easily functionalized; (6) the improvement in assay performance is greater than previous methods; (7) the increase in fluorescence per dye molecule (on average) is greater than previously reported for configurations suitable for immunoassay applications; and (8) the larger particles used in this disclosure can be loaded with more dye molecules than other designs (i.e., more dye and enhanced total conjugated dye means ultrabright constructs).

[0129] According to the present disclosure, plasmonic enhancement improves the quantum yield of the conjugated dye (which is an important factor in the "brightness" generated by the dye) and reduces its fluorescence lifetime. Thus, a higher enhancement factor (increased relative brightness) can be achieved for dyes with low quantum yields and / or long fluorescence lifetimes than for dyes that already have high quantum yields and short fluorescence lifetimes.

[0130] The fluorescent nanoconstruct particles disclosed herein are at least 500 times brighter than the fluorescent species to which they are attached when these non-plasmon-enhanced fluorescent species are measured in free solution. A brightness metric or test is used that simply compares the fluorescence intensity of the plasmon-fluor with that of the fluorescent species / agent alone. This brightness metric is independent of the functional layer and / or biorecognition element used in the nanoconstruct. Such a test for "relative brightness" is illustrated, for example, in Figures 1, 2, and 3. In this test, fluorescence intensity is plotted as a function of fluorescent species concentration for identical excitation and detection conditions. The ratio of the slopes indicates the relative brightness of the fluorescent species. As disclosed herein below, data collected for multiple PFs at various wavelengths compares the relative brightness to their conjugated fluorophores.

[0131] In some embodiments, the nanoconstruct comprises a fluorescent agent that is at least about 5 times, at least about 10 times, at least about 50 times, at least about 100 times, at least about 500 times, at least about 1,000 times, at least about 2,000 times, at least about 3,000 times, at least about 4,000 times, at least about 5,000 times, at least about 6,000 times, or at least about 7,000 times brighter than the free fluorescent species of the fluorescent agent.

[0132] Features of the present disclosure include: (1) Plasmonic nanostructures that act as nanostructures across a range of optical wavelengths, with a local maximum defined by the particle's localized surface plasmon resonance (LSPR) wavelength. Plasmonic particles can have one or more LSPR wavelengths. The plasmonic particles "pull" at light of wavelengths corresponding to the LSPR wavelength, effectively focusing the light and enhancing the electromagnetic field near the particle's surface.

[0133] (2) A fluorescent species (e.g., an organic fluorophore) that is excited by wavelengths of light near at least one of the particle's LSPR wavelengths such that it is present in an enhanced EM field while not being close enough to cause what is known as "metal-induced quenching," and that is maintained near the surface of the plasmonic particle. Optimally, the separation between the plasmonic nanostructure and the fluorescent species ranges from about 2 nm to about 10 nm. In some embodiments, this separation distance is the spacer thickness. In other embodiments, when the fluorescent species is conjugated to a functional layer, as in Figure 4, this separation distance is the sum of the spacer thickness and the average spacing provided by the functional layer between the fluorophore and the spacer surface.

[0134] (3) A spacer layer that provides a barrier to prevent metal-induced quenching and to which fluorophores can be immobilized (either directly or via bonding to carrier molecules) to maintain an optimal distance from the plasmonic particle surface. The spacer material ideally contains functional groups that allow for covalent conjugation of fluorophores and / or biorecognition elements at the surface distal to the plasmonic particle surface.

[0135] (4) Several purposes: stabilization of the nanoconstruct from aggregation and nonspecific binding, attachment points for biorecognition elements; and a functional layer that can also serve as a carrier for fluorophores.

[0136] (5) A biorecognition element capable of using plasmon-fluor for specific detection of the target of the biorecognition element (e.g., the target antigen when the biorecognition element is an antibody or aptamer, biotin when the biorecognition element is streptavidin, or an oligonucleotide when the biorecognition element is a complementary oligonucleotide).

[0137] It is currently believed that particles with the composition and performance characteristics of the fluorescent nanoconstructs described herein never exist. Many previous attempts to generate plasmon-enhanced fluorescent nanoconstructs in the solution phase have achieved "brightness enhancements" on the order of about 10-fold.

[0138] Fluorescent nanoconstructs The fluorescent nanoconstructs disclosed herein overcome the above challenges and provide a path to broad application of these fluorescent nanoconstructs in immunoassays and other biological assays. As used herein, the term "fluorescent nanoconstruct" also refers to plasmon-fluor (PF). In one example, with respect to the detection of biomarkers related to kidney function, the fluorescent nanoconstructs significantly enhance the ability to resolve low-level kidney function parameters (biomarkers) and provide comprehensive kidney disease information. Notably, the superior performance of multiplexed microarrays results from the very simple addition of nanostructures to the assay prior to detection using standard techniques. Furthermore, this technique is inexpensive and provides an easily implemented approach for enhancing fluorescence. Such easily deployable techniques are seamlessly applied to a wide range of platforms in diagnostics, proteomics, and genetics to address the unmet need for brighter signal intensity.

[0139] In one aspect, fluorescent nanoconstructs are disclosed herein, which generally comprise a plasmonic nanostructure, a polymer, a biorecognition element, and a fluorescent agent. In some embodiments, the fluorescent nanoconstruct comprises a plasmonic nanostructure having at least one localized surface plasmon resonance wavelength (λLSPR), at least one spacer coating, at least one fluorescent agent having a maximum excitation wavelength (λEX), and at least one biorecognition element.

[0140] The fluorescent nanoconstructs disclosed herein are useful for enhancing the bioanalytical parameters (sensitivity, LOD, and dynamic range) of fluorescent immunoassays performed in microplate, membrane, antibody microarray, and bead-based formats, among many other formats. In some embodiments, the microplate is in the form of a standard 6-well, 12-well, 24-well, 48-well, 96-well, 384-well, or 1536-well plate. In other embodiments, the immunoassay format is on a glass slide, nitrocellulose or PVDF membrane, latex microbeads, or other formats known in the art. In some aspects, the assay or analysis format is solution-phase. In other embodiments, the assay is applied to cells or tissues. In some embodiments, the fluorescent nanoconstructs provide a 10-fold, 50-fold, 100-fold, 200-fold, 250-fold, 500-fold, 1000-fold, or even 10,000-fold enhancement in fluorescence intensity compared to a biorecognition element labeled with a fluorescent agent without plasmonic enhancement.

[0141] Most existing plasmon-enhanced fluorescence techniques require fluorescence-based bioassays to be performed on prefabricated plasmon substrates, typically glass slides coated with metal nanostructures instead of standard materials, or sometimes on non-interchangeable biological analytical platforms (e.g., 96-well plates, nitrocellulose membranes, or microbeads), which significantly limits the broad applicability of this technique. More importantly, the need for dedicated substrates limits cross-platform and cross-laboratory compatibility and seamless integration with widely used biological analytical procedures, which is a major drawback of conventional plasmon-enhanced fluorescence techniques. The present disclosure develops a "non-invasive" (no modification of current assay protocols) ultra-bright fluorescence technique based on plasmon-fluor, in which plasmon-fluor is simply added to microtiter wells (or microarrays, microbeads, or cell surfaces) in place of conventional fluor.

[0142] Customizable Plasmon-Fluor (PF) for maximum fluorescence enhancement According to the present disclosure, the optical properties of plasmon-fluorescence (e.g., the LSPR wavelength of metallic nanostructures, which plays a key role in the final enhancement efficiency) are easily tuned and optimized for a given fluorescent emitter (organic dye, quantum dot, or upconversion nanoparticle) through rational selection of the nanostructure size, shape, and composition. This is in marked contrast to conventional plasmonic substrates (e.g., metallic nanoislands), which offer poor control over the LSPR wavelength and are usually limited to suboptimal "one size fits all" approaches.

[0143] [Table 1-1] [Table 1-2]

[0144] High stability, performance and affordability The techniques disclosed herein for enhancing biological assays are a cost-effective solution for improving biological assay performance, with plasmon-fluor cost estimates for one run of a 96-well microtiter plate comparable to current industry standards and substantially cheaper than the specialized substrates mentioned above (e.g., glass slides coated with metal island films). The high stability of metallic nanostructures further ensures the integrity and functionality of plasmon-fluor under typical storage / transport / handling conditions used in biological assays. In general, plasmon-fluor can be stored and handled similarly to handling fluorescently labeled biorecognition elements. Collectively, the enhanced signal-to-noise ratios achieved by the techniques described herein significantly improve assay sensitivity, relax stringent instrument requirements (such as low background noise and high sensitivity), reduce required reagent volumes, and / or significantly shorten total assay time, enabling these assays to be performed in a wide range of research and clinical diagnostic settings with minimal effort or expense and significant improvements in assay performance.

[0145] In assays where fluorescence detection is already used as a readout, improving fluorescence intensity by using plasmon fluorescers instead of current gold-standard fluorophores results in an improvement in the lower limit of detection (LLOD) of the biological assay. In some embodiments, the LLOD is reduced by at least 2-fold, 5-fold, 10-fold, 20-fold, 40-fold, 50-fold, 100-fold, 500-fold, or even 1,000-fold. Furthermore, this improves the dynamic range of detection. In some embodiments, the improvement in dynamic range is greater than 2-fold, 5-fold, 10-fold, 20-fold, 40-fold, 50-fold, 100-fold, 500-fold, or even 1,000-fold. In assays where fluorescence detection is not already used as the readout but where the readout is chemiluminescent or colorimetric, for example, in a chemiluminescent / colorimetric ELISA or Western blot, switching to fluorescence detection and using plasmon fluorescers with appropriate detection equipment will result in at least comparable performance of the LLOD and dynamic range of the bioassay compared to the gold-standard reporter method for the assay. Improvements in bioanalytical parameters have been found to be consistent across various assay formats, target biomarkers, and fluorophores. Importantly, this method can be implemented using existing bioassays with minimal modification of standard operating procedures and no additional operator training. In some embodiments, the only modification from an existing assay protocol is the addition of a fluorescent nanoconstruct in place of an existing fluorescent reporter molecule. In some embodiments, the only modification from an existing assay protocol is the addition of a fluorescent nanoconstruct in place of detecting fluorescence using an existing reporter molecule and appropriate detection equipment.

[0146] As part of the accurate validation of this technology, urine samples from patients with kidney disease and healthy volunteers were analyzed. In contrast to unenhanced fluorescent immunoassays and ELISAs, plasmon-enhanced fluorescent immunoassays enabled the detection and quantification of low-abundance biomarkers, both in patients and healthy volunteers. The added sensitivity of the plasmon-enhanced assays allows for easy quantification of low-abundance biomarkers, providing physiological and pathological information often missed by conventional immunoassays.

[0147] Plasmonic nanostructures The nanoconstructs described herein include a plasmonic nanostructure core. The plasmonic nanostructures used herein provide plasmonic enhancement to the fluorescence signal and are selected based on a number of criteria (see, for example, Table 2). Plasmonic nanostructures can include any material with surface plasmons that can resonate at a suitable wavelength of light, such as gold (Au), silver (Ag), copper (Cu), or a combination thereof. Suitable examples of plasmonic nanostructures include, but are not limited to, nanorods, nanocubes, nanospheres, bimetallic nanostructures (e.g., Au@Ag core-shell nanocubes), nanostructures with sharp tips (e.g., nanostars), hollow nanostructures such as nanocages and nanorattles, nanobipyramids, nanoplates, self-assembled nanostructures, and nanoraspberries. In some embodiments, 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) canocuboids, nanostructures with sharp tips, nanostars, hollow nanostructures, nanocages, nanorattle, nanobipyramids, nanoplates, self-assembled nanostructures, nanoraspberries, and combinations thereof.

[0148] One criterion for selecting a plasmonic nanostructure for use in a fluorescent nanoconstruct is the LSPR wavelength. Different plasmonic nanostructures have different LSPR wavelengths, as illustrated in Figure 5. Even the same plasmonic nanostructure can have multiple LSPR wavelengths corresponding to different resonance modes of the surface plasmon. The specific LSPR wavelength optimal for fluorescence enhancement of a fluorophore is based on the excitation spectrum of that fluorophore. Specifically, it is important that there is overlap between the LSPR wavelength and excitation spectrum of the fluorophore. Generally, a greater degree of overlap results in better enhancement. An ideal situation for fluorescence enhancement occurs when the LSPR wavelength, fluorophore excitation maximum, and wavelength of light used for excitation are the same. This allows the fluorescent nanoconstruct to be selectively tuned to match the fluorophore used for enhancement. In some embodiments, the LSPR wavelength is between about 200 and about 1200 nm, between about 250 and about 950 nm, between about 300 and about 850 nm, between about 350 and about 800 nm, or between about 400 and about 750 nm. In still other embodiments, the LSPR wavelength is about (mean ± 25 nm) 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm, 1050 nm, 1100 nm, 1150 nm, or 1200 nm.

[0149] In some embodiments, fluorescent nanoconstructs based on gold nanorods (AuNR) can have an LSPR wavelength between about 600 and 1200 nm. As another example, plasmonic fluorescers with cores of either silver nanocubes, AuNR@Ag cuboids, or Au@Ag cubes can have an LSPR wavelength between about 400 nm and about 600 nm.

[0150] In some embodiments, the plasmonic nanostructure has an LSPR wavelength between about 400 and about 1,000 nm. In some embodiments, the plasmonic nanostructure serving as the plasmonic core of the fluorescent nanoconstruct is an Au@Ag cuboid. In some embodiments, the plasmonic nanostructure is an Au nanorod (AuNR). In some embodiments, the plasmonic nanostructure is a silver-coated gold nanorod (AuNR@Ag). In some embodiments, the plasmonic nanostructure is one of any other number of plasmonic structures.

[0151] In some embodiments of the fluorescent nanoconstruct, the plasmonic nanostructure comprises gold nanorods (AuNR) or silver-coated gold nanorods (AuNR@Ag). The spacer coating comprises a stable silane network containing reactive groups that can be functionalized. The biorecognition element comprises biotin, streptavidin, an antibody, or any combination thereof.

[0152] [Table 2]

[0153] Plasmonic nanostructure size The size of the plasmonic nanostructure forming the core of the plasmonic fluor can be any size suitable for enhancing or amplifying the fluorescence intensity of the conjugated fluorophore. In some embodiments, at least one dimension of the plasmonic nanostructure is at least 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, or 200 nm. In some embodiments, the size of the plasmonic nanostructure and its LSPR wavelength are adjusted so that the size of the nanostructure has an LSPR wavelength that overlaps with the maximum of the excitation wavelength maximum of the fluorophore.

[0154] Wavelength Match As described herein, the size, shape, and composition of a plasmonic nanostructure can be tailored to have an LSPR wavelength that matches the maximum of the excitation wavelength of a fluorophore. Furthermore, the LSPR maximum / maximum shifts after coating with a spacer layer and / or functional layer, and the overlap / matching described below must be in accordance with the plasmonic nanostructure core coated with the spacer and / or functional layer. Wavelength matching can be a significant overlap between the LSPR wavelength and the excitation spectrum of a fluorophore; a significant coincidence between the LSPR wavelength and the maximum excitation wavelength of a fluorophore, indicating LSPR / fluorophore excitation maxima coincidence and overlap [see, e.g., Figures 5 and 6(A-B)]; or the extinction spectrum of the nanostructure in solution exhibits a significant overlap with the extinction spectrum and / or absorption maximum of the fluorophore in solution. Excitation Maximum Wavelength. The overlap between the LSPR wavelength and the excitation maximum wavelength of the conjugated fluorophore can be 100% overlap, or the maximum LSPR wavelength can be 100% coincidence or overlap between the maximum excitation maximum wavelength of the fluorophore. In some embodiments, the coincidence between the LSPR wavelength and the excitation maximum wavelength of the conjugated fluorophore results in at least 500-fold greater fluorescence intensity of a fluorescent nanoconstruct conjugated with 20-2000 fluorophores compared to the free fluorophores in solution, when determined under similar excitation and detection conditions.

[0155] The maximum LSPR wavelength of gold nanorods (AuNRs) can be easily tuned to match the excitation maximum wavelength of a fluorophore between 600 nm and >1200 nm. The maximum LSPR wavelength of silver cubes, AuNR@Ag cuboids, and Au@Ag cuboids can be easily tuned to match the excitation maximum wavelength of a fluorophore between 400 nm and 600 nm. Therefore, while these were used as exemplary materials, any plasmonic nanostructure that can be tuned to have an LSPR wavelength that matches the excitation maximum wavelength of a particular fluorophore (e.g., any visible or IR fluorescent dye) can be used in accordance with the methods described herein.

[0156] The size, shape, and material of the plasmonic nanostructure are tailored to match the LSPR wavelength to the longest excitation maximum wavelength of the fluorophore. As another example, cuboids having at least one dimension between about 60 nm and about 130 nm have been found to be sufficient for tunable matching to wavelengths <600 nm. As another example, gold nanorods having lengths between about 30 nm and about 130 nm have been found to be sufficient for tunable matching to wavelengths >600 nm. As an example, wavelengths can be considered matched if the excitation maximum wavelength (λEX) of the fluorophore (PF) is within about 100 nm of the LSPR wavelength (λLSPR) of the plasmonic fluor (PF).

[0157] In some embodiments, the absolute value of Δ, i.e., the difference between at least one λLSPR and λEX, is 100 nm (i.e., ±100 nm). In some embodiments, the absolute value of Δ is less than about 75 nm. In some embodiments, the absolute value of Δ is 50 nm (i.e., ±50 nm). While smaller absolute values ​​of Δ are preferred (i.e., values ​​closer to zero), LSPR absorption peaks are typically very broad (>50 nm, or even >100 nm half-width), which means that there can be sufficient overlap in the extinction spectra of plasmonic fluores and fluorophores to result in significant fluorescence enhancement even when the maxima of both spectra (λLSPR and λEX, respectively) do not coincide. Windows for plasmonic nanostructure and dye pairs have also been disclosed (e.g., there are several fluorescent dyes (fluorophores) that can absorb and emit in regions similar to fluorescein, Cy3, Cy5, 680LT, and 800CW).

[0158] Spacer Coating As described herein, spacer coatings can be used to coat plasmonic nanostructures to reduce or prevent quenching by maintaining fluorophores at a sufficient distance, on average, from the surface of the plasmonic nanostructure (e.g., at least about 0.5 nm to 4 nm from the plasmonic nanostructure). The spacer coating can be any material capable of coating the plasmonic nanostructure and can be controlled to have a thickness of 0.5 to 100 nm. In some embodiments, the spacer can be functionalized with a fluorophore.

[0159] In some aspects, the fluorescent nanoconstruct further comprises a spacer in the form of a coating on the plasmonic nanostructure. In some embodiments, the spacer is a dielectric material. In some embodiments, the thickness of the coating can be adjusted to achieve various amounts of fluorescence enhancement of the fluorescent signal. In some embodiments, the coating thickness (d) is about 0.5 nm to about 100 nm. In still other embodiments, the coating thickness is about 2 nm, 3 nm, 4 nm, 5 nm, 8 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, or 100 nm. As used herein, "about" refers to ±25%. In some embodiments, the coating thickness is controlled by increasing the concentration of the monomer during preparation. In some embodiments, the spacer coating has a thickness of at least 0.5 nm, at least 1 nm, at least 2 nm, at least 3 nm, at least 4 nm, or at least 5 nm, hi some embodiments, the variation in thickness of the spacer coating across a single fluorescent nanoconstruct is less than about 2 nm, 3 nm, 4 nm, or 5 nm.

[0160] In some embodiments, the coating may comprise any polymer or mixture of polymers that can be uniformly deposited over the plasmonic nanostructure and controlled to have the thickness described above. Examples of polymers for use as spacers include, but are not limited to, proteins (e.g., BSA), silanes, and polyethylene glycol. Preferably, the coating is MPTMS, APTMS, TMPS, or a combination thereof. In some embodiments, the spacer is a siloxane network. Preferably, the coating can be applied in solution. In some embodiments, the spacer coating is covalently attached to the plasmonic nanostructure. Furthermore, in some embodiments, inorganic coatings such as silica, alumina, and / or zinc oxide are used. In some embodiments, the spacer is a rigid polymer network. In some embodiments, the spacer coating contains functional groups for covalently binding other molecules, such as amines, aldehydes, carboxylic acids, sulfhydryls, ketones, and moieties compatible with click chemistry. In some embodiments, the spacer coating is a polymer coating comprising a functionalizable dielectric rigid polymer network.

[0161] In some embodiments, the spacer is functionally active and contains any number of reactive groups and mixtures thereof. In some embodiments, the spacer is initiated with a mercapto-containing moiety that forms a reactive layer on the gold / silver surface, and the siloxane network is constructed from this initiation layer using a mixture of functional silanes. The advantages of silanes are: 1) wet chemistry compatibility; 2) a variety of functional groups are available for further modification and tuning of particle characteristics [e.g., PEG, amino, epoxy, mercapto, vinyl, click chemistry moieties (TCO, azide), PEG-biotin, aldehyde, fluorophore, and amino acid]; and 3) precise control of spacer thickness from 0.5 nm to 100 nm.

[0162] [Table 3]

[0163] [Table 4] In some embodiments, the spacer coating layer or functional layer serves as a scaffold for the light emitter (fluorophore) and biorecognition element (e.g., biotin, streptavidin, antibody, nucleic acid). In some embodiments, the fluorophore is attached to the functional layer, in which case the functional layer also serves as an additional spacer between the fluorophore and the plasmonic nanostructure surface, even in the presence of a separate spacer coating layer. In some embodiments, the spacer coating layer or functional layer serves as a stabilizer to prevent aggregation of the fluorescent nanoconstruct. The functional layer also helps minimize nonspecific binding of the fluorescent nanoconstruct to the bioassay surface. In some aspects, the spacer coating layer or functional layer is a protein. Specific examples include, but are not limited to, albumin, lysozyme, protein A, and hemoglobin. In some aspects, the protein on the fluorescent nanoconstruct is bovine serum albumin (BSA), human serum albumin (HSA), or a combination thereof. In some aspects, the protein is BSA.

[0164] functional base layer In some aspects, the spacer coating is a functional layer or a fluorescent nanoconstruct, and can further include a functional layer coating the spacer layer. In some aspects, the functional layer is a polymer. Any polymer or combination of polymers that can adhere the surface of the spacer coating to the surface of the nanostructure or that can be bonded to the surface can be used as the functional layer. In some aspects, the polymer contains functional groups for covalently binding to other molecules, such as amines, aldehydes, carboxylic acids, sulfhydryls, ketones, and moieties compatible with click chemistry. In some embodiments, the functional layer comprises a polypeptide. In some embodiments, the functional layer is an albumin protein or a homolog thereof. In some embodiments, the functional layer is adsorbed to the spacer layer by hydrophobic or electrostatic interactions, or a combination thereof. In some embodiments, the functional layer is covalently linked to the spacer layer. In some embodiments, the functional layer is the same material used for "blocking" in an immunoassay. For example, in plate-based immunoassays, BSA is used to block nonspecific binding to the surface, and BSA is used as the material for the functional layer.

[0165] biorecognition element As described herein, the fluorescent nanoconstructs include a biorecognition element (see, for example, Table 5). The biorecognition element targets a specific analyte or species. For example, the biorecognition element can be an antibody when the target is an antigen, or the biorecognition element can be streptavidin.

[0166] In some embodiments, the biorecognition element is selected from the group consisting of biotin, streptavidin, an antibody (or functional fragment thereof), an oligo (DNA, PNA, etc.), an aptamer, a "click" moiety (e.g., tetrazine), a molecularly imprinted polymer ("artificial antibody"), digoxigenin, a peptide tag, a protein tag, and combinations thereof. In some embodiments, the target is selected from the group consisting of streptavidin, biotin, a target antigen, a complementary oligo (DNA, RNA), a target analyte, a pair-forming complementary "click" moiety, a DIG-binding protein or an anti-digoxigenin antibody, and combinations thereof.

[0167] In some embodiments, biorecognition elements, such as antibodies, streptavidin, aptamers, and nucleic acids, are attached to the spacer layer or functional group layer via many of the same chemistries used to attach fluorophores. Furthermore, in some embodiments, biotinylated plasmon-fluors are directly conjugated to streptavidin, which can be further conjugated to biotinylated antibodies. In some embodiments, the biorecognition elements are attached to PFs using flexible linkers. In some embodiments, the flexible linker is PEGx, where x is 2 to 36. In some embodiments, the fluorescent nanoconstruct comprises a plasmonic nanostructure having at least one localized surface plasmon resonance (LSPR) wavelength (λLSPR) and a spacer comprising a first material; at least five fluorescent organic dyes having excitation maxima wavelengths (λEX); and a biological recognition element, wherein the plasmonic nanostructure is substantially covered by the spacer having a thickness between 0.5 and 20 nm, the fluorescent species are attached to a surface of the spacer distal to the surface of the plasmonic nanostructure, the spacer is substantially covered by a functional layer, the biological recognition element is attached to the functional layer, the difference between the LSPR wavelength and the excitation maxima wavelength of the fluorescent organic dyes is less than 75 nm, and each fluorescent organic dye is at least 10 times brighter in aqueous solution than the unconjugated fluorescent species under typical illumination and detection conditions.

[0168] [Table 5]

[0169] fluorescent agent The fluorescent agent is selected based on various criteria. As discussed herein, the terms fluorescent agent, fluorescent species, fluorophore, and fluorescent dye are used interchangeably. One selection criterion is the wavelength of the fluorescent agent's fluorescence excitation maximum. Another selection criterion is the ease of attachment of the fluorescent agent to the spacer coating of the functional layer in the fluorescent nanoconstruct. In some embodiments, the fluorescent agent is either a UV, visible, near-infrared (NIR), or infrared (IR) organic fluorophore. In some embodiments, the fluorescent agent is fluorescein, Cy3, Cy5, 680LT, 800CW, acridine, acridone, anthracene, anthracyclines, anthraquinone, azaazulene, azoazulene, benzene, benzimidazole, benzofuran, benzoindocarbocyanine, benzoindole, benzothiophene, carbazole, coumarin, cyanine, dibenzofuran, dibenzothiophene, dipyrrolo dyes, flavone, fluorescein, imidazole, indocarbocyanine, indocyanine, indole, iso The compound is selected from the group consisting of indole, isoquinoline, naphthacenedione, naphthalene, naphthoquinone, phenanthrene, phenanthridine, phenanthridines, phenoselenazine, phenothiazine, phenoxazine, phenylxanthene, polyfluorobenzene, purine, pyrazine, pyrazole, pyridine, pyrimidone, pyrrole, quinoline, quinolone, rhodamine, squaraine, tetracene, thiophene, triphenylmethane dye, xanthene, xanthone, and derivatives thereof.

[0170] Furthermore, the fluorescent nanoconstructs disclosed herein are suitable for enhancing fluorescent signals from a wide variety of different fluorescent sources or species. In addition to the fluorescent agents and assays disclosed elsewhere herein, in some embodiments, the fluorescent nanoconstructs enhance fluorescent signals from quantum dots and upconversion nanoparticles. In some embodiments, fluorescent molecules are attached via standard chemistries: succinimidyl esters, NHS-esters, TFP esters, or isothiocyanates to primary amines; maleimides to mercapto groups; click chemistry, either directly to functionalized silanes (e.g., tetrazine-linked fluorophores to TCO-PEG-triethoxysilane) or by first functionalizing another reactive group with the click moiety; or hydrazides or hydroxylamines to aldehydes or ketones. Furthermore, in some embodiments, fluorophores are first conjugated to functional layer molecules, such as proteins, which are then attached to the spacer layer.

[0171] In some embodiments, the fluorescent species is an organic dye. In some embodiments, the organic dye is present at a coating density of 5-2000 fluorophores per plasmon-fluor. In some embodiments, the fluorescent species is covalently attached to the spacer layer. In some embodiments, the fluorescent species is covalently attached to the functional group layer.

[0172] [Table 6]

[0173] Plasmon-Fluor (PF) Exemplary Embodiments The plasmon-fluor (PF) variants described below differ in the arrangement of the PF components, but all include a plasmon nanostructure coated on a spacer layer with a fluorophore held at a specific distance from the plasmon nanostructure surface, a biological recognition element bound somewhere to the PF, and a coupling site between the fluorophore and the plasmon nanostructure defined by the overlap of at least one of the LSPR wavelengths of the PF with the excitation maximum of the bound fluorophore. In some cases, a layer called a "functional layer" is present, which can serve several purposes: to bind molecules (fluorophores, biological recognition elements) and to stabilize the structure against aggregation and nonspecific binding.

[0174] In some embodiments, a functional layer is present on the PF. Specifically, disclosed are plasmonic nanostructures having at least one localized surface plasmon resonance wavelength (λ LSPR), a spacer material of a particular thickness (d) that substantially covers the surface of the plasmonic nanostructure, a fluorophore conjugated to the spacer material, a functional layer material that substantially covers the spacer material, and a biorecognition element conjugated to the functional layer material.

[0175] In some embodiments, the method comprises coating the plasmonic nanostructure with at least one spacer coating, optionally coating the at least one spacer coating with a functional layer, conjugating a fluorescent agent to one of the at least one spacer coating or the functional layer, and conjugating a biorecognition element to one of the at least one spacer coating or the functional layer.

[0176] In some embodiments, coating the plasmonic nanostructure with at least one spacer coating comprises applying an initial layer on the plasmonic nanostructure core and applying a polysiloxane coating over the initial layer. In some embodiments, the initial layer comprises 3-mercaptopropyltrimethoxysilane (MPTMS). In some embodiments, the polysiloxane coating comprises trimethoxypropylsilane (TMPS) and 3-aminopropyltrimethoxysilane (APTMS).

[0177] In some embodiments, a plasmonic nanostructure is disclosed having a localized surface plasmon resonance wavelength (λLSPR), a spacer material of a specific thickness (d) that substantially covers the surface of the plasmonic nanostructure, a fluorophore conjugated to the spacer material, and a biorecognition element conjugated to the spacer material. In some embodiments, a fluorescent nanoconstruct is disclosed that includes a plasmonic nanostructure having a localized surface plasmon resonance (LSPR) wavelength (λLSPR) and a spacer comprising a first material; a fluorescent species; and a biorecognition element, wherein the nanostructure is substantially covered by the spacer having a thickness (d), the spacer is conjugated to a fluorescent species, the biorecognition element is bound to the spacer, and the fluorescent species has an excitation maximum wavelength (λEX), where the difference between the LSPR wavelength and the excitation maximum wavelength is |Δ|.

[0178] In some embodiments, a plasmonic nanostructure is disclosed having a localized surface plasmon resonance wavelength (λLSPR), a spacer material of a specific thickness (d) that substantially covers the surface of the plasmonic nanostructure, a fluorophore conjugated to the spacer material, a biorecognition element conjugated to the spacer, and a functional layer material that substantially covers the spacer. In some embodiments, a fluorescent nanoconstruct is disclosed that includes a plasmonic nanostructure having a localized surface plasmon resonance (LSPR) wavelength (λLSPR) and a spacer comprising a first material; a fluorescent species; a biorecognition element; and a functional layer comprising a second material, wherein the nanostructure is substantially covered by the spacer having a thickness (d), the spacer is conjugated to a fluorescent species, the biorecognition element is bound to the spacer, the functional layer material is bound to the spacer, and the fluorescent species has an excitation maximum wavelength (λEX), and the difference between the LSPR wavelength and the excitation maximum wavelength is |Δ|.

[0179] In some embodiments, a plasmonic nanostructure is disclosed having a localized surface plasmon resonance wavelength (λLSPR), a spacer material of a specific thickness (d) that substantially covers the surface of the plasmonic nanostructure, a functional layer material that substantially covers the spacer material, a fluorophore conjugated to the functional layer material, and a biorecognition element conjugated to the functional layer material. In some embodiments, the fluorophore is attached to the functional layer with the biorecognition element attached directly to the spacer. In some embodiments, the fluorescent nanoconstruct comprises a plasmonic nanostructure having a localized surface plasmon resonance (LSPR) wavelength (λLSPR) and a spacer comprising a first material; a fluorescent species; a functional layer comprising a second material; and a biological recognition element, wherein the nanostructure is substantially covered by the spacer having a thickness (d), the spacer is substantially covered by the functional layer, the fluorescent species is bound to the functional layer, the biological recognition element is bound to the functional layer, the fluorescent species has an excitation maximum wavelength (λEX), and the difference between the LSPR wavelength and the excitation maximum wavelength is |Δ|.

[0180] In some embodiments, the fluorescent nanoconstruct has a zeta potential in water at pH 7 with an absolute value greater than about 20 mV, or about 25 mV, or about 30 mV, or about 35 mV, or about 40 mV, or about 45 mV.

[0181] In some embodiments, the brightness of the plasmon-fluor is at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1,000 times, at least 2,000 times, at least 3,000 times, at least 4,000 times, at least 5,000 times, and even at least 10,000 times brighter than the fluorescent species alone under the same typical illumination and detection conditions. In some embodiments, the brightness of each fluorescent species bound to the plasmon-fluor is, on average, at least 10 times, at least 20 times, at least 30 times, at least 40 times, or at least 50 times brighter than the free fluorescent species under the same typical illumination and detection conditions.

[0182] Assays Suitable for Use with Fluorescent Nanoconstructs The fluorescent nanoconstructs disclosed herein are suitable for use with any assay that uses or can use fluorescence for the detection and / or quantification of an analyte. Examples of assays suitable for use herein include, but are not limited to, antibody / protein microarrays, bead / suspension assays, biochip assays, capillary / sensor assays, cell assays, tissue assays, DNA / RNA microarrays, polymerase chain reaction (PCR)-based assays, glycan / lectin arrays, immunoassays, enzyme-linked immunosorbent assays (ELISAs), microfluidic chips, and membrane-based assays.

[0183] Disclosed herein are methods for improving the performance of biological assays. The methods generally involve using a fluorescent nanoconstruct described elsewhere herein as a reporter molecule in a biological assay, wherein the fluorescent nanoconstruct is targeted to a specific analyte or species by a biorecognition element, the fluorescent signal is detected using a fluorophore or any method known in the art for detecting a fluorescent signal, and the concentration of the analyte is proportional to the fluorescent signal. In some embodiments, the biorecognition element is directly targeted to the specific analyte of interest (e.g., the biorecognition element is a primary antibody to the analyte or a complementary oligonucleotide to a specific target oligonucleotide). In some embodiments, the biorecognition element is targeted to a moiety on another molecule that specifically binds to the target analyte (e.g., the biorecognition element is a secondary antibody that recognizes a primary antibody bound to the target analyte, or the biorecognition element is streptavidin that recognizes a biotinylated primary detection antibody).

[0184] Because of the larger fluorescent signal produced by the use of fluorescent nanoconstructs compared to standard fluorophores, the lower limit of detection of a fluorescent assay is significantly improved (i.e., the LOD is lower, allowing for detection of less concentrated samples) compared to the lower limit of detection achievable using current standard fluorescent reporter molecules. In some embodiments, the lower limit of detection of an assay using fluorescent nanoconstructs is lower than the lower limit of detection of the same assay using current standard fluorescent reporter molecules. In some embodiments, the LOD is improved by 2x (i.e., the LOD is half the LOD of the same assay using current standard fluorescent reporter molecules, i.e., twice the detection sensitivity). In some embodiments, the LOD is at least 2x better, at least 3x better, at least 4x better, at least 5x better, at least 10x better, at least 25x better, at least 50x better, at least 100x better, at least 500x better, at least 1000x better, at least 5000x better, or even at least 10,000x better than the same assay using the current standard fluorescent reporter molecule.

[0185] [Table 7]

[0186] Manufacturing method In some embodiments, the plasmon-fluor synthesis is as follows: (1) coating the plasmon nanostructure with a spacer layer; (3) conjugating a fluorescent species to the spacer layer; (4) coating the nanoconstruct resulting from (3) with a functional layer; and (5) conjugating a biorecognition element to the functional layer. This synthesis also includes several variations and other embodiments.

[0187] Variant 1: Biorecognition element conjugated to a functional layer: (1) Starting with a plasmonic nanostructure; (2) Coating the plasmonic nanostructure with a spacer layer (the spacer layer can be a mixture of MPTMS, APTMS, and TMPS, or alternative silanes with different functional moieties (e.g., aldehydes or tetrazines) for binding fluorescent species (e.g., via hydrazine or TCO, respectively) can be used); (3) Conjugating a fluorescent species to the spacer layer (the fluorescent species is covalently attached to an amine (via NHS or TFP ester), or alternative silanes with different functional moieties (e.g., via aldehydes or tetrazines) for binding fluorescent species (e.g., via hydrazine or TCO, respectively) can be used); (4) Conjugating a biorecognition element to the functional layer: (a) The biorecognition element is (b) using click chemistry, for example, to react NHS-PEG-TCO with BSA and NHS-PEGy-tetrazine with streptavidin or an antibody to coat the nanoconstruct with BSA-TCO, and then, after the following step (5), to directly bind streptavidin or an antibody to BSA by mixing in the tetrazine-biorecognition element; (5) in the functional layer step, a mixture of biotinylated BSA (or other suitable protein) and native BSA (or other suitable protein) is used to coat the nanoconstruct from (3) with the functional layer in (4).

[0188] Variant 2: Fluor-biorecognition element conjugated to a functional layer: (1) start with a plasmonic nanostructure; (2) coat the plasmonic nanostructure with a spacer layer, (3) conjugate a biorecognition element and a fluorophore to the functional layer; and (4) coat the particle from (2) with the functional layer from (3).

[0189] In some embodiments, biotinylated PF is used as a building block and added to other biorecognition elements.Biotin can be used as a biorecognition element, but it can also be used to connect to additional biorecognition elements (for example, streptavidin).Therefore, there is an additional step in which streptavidin is conjugated to the biotinylated PF nanoconstruct.Similarly, this streptavidin-conjugated PF can be used to bind biotinylated antibody.In this case, there is yet another step in which biotinylated antibody is conjugated to the PF that streptavidin is conjugated to.

[0190] In some embodiments, the PF is further modified by attaching a linear or branched hydrophilic polymer to the functional layer, streptavidin, or antibody. In some embodiments, the hydrophilic polymer is PEG.

[0191] How to use Plasmon-Fluors are designed to enhance the performance of fluorescence-based biological assays. Specifically, they are used as reporter molecules; upon excitation at an appropriate wavelength, the fluorescent signal generated by the Plasmon-Fluor correlates with the concentration of the target analyte. PFs can be used in several different assay types and formats. The most obvious use of PFs is as a reporter molecule in immunoassays. In this case, a primary detection antibody is used to detect the target analyte, and PFs are used to report on the concentration of the detection antibody present, which is proportional to the amount of target analyte. This can be done by binding a detection antibody directly to the PF (where the biorecognition element is the detection antibody) and using the resulting construct to bind to the target analyte; by binding a streptavidin-conjugated PF (where the biorecognition element is streptavidin) to a biotinylated detection antibody that is already bound to the target analyte; by binding a biotinylated PF (where the biorecognition element is biotin) to streptavidin that is bound to a biotinylated detection antibody that is bound to the target analyte; or by binding a PF conjugated to a secondary antibody (where the biorecognition element is the secondary antibody) directed against the detection antibody that is bound to the target analyte.

[0192] Additionally, PF can be used to detect target nucleic acid sequences by either 1) using a complementary nucleic acid sequence as the biorecognition element, or 2) using a biotinylated nucleic acid sequence that first binds to the target and is then detected by streptavidin-linked PF (the biorecognition element is streptavidin).

[0193] In some embodiments, a method of detecting an analyte includes providing a plasmon-fluor, wherein at least one biorecognition element is targeted to the analyte (either directly or by the means described above); exciting the plasmon-fluor with an appropriate excitation wavelength; and detecting emitted light, wherein the amount of light detected is proportional to the concentration of the analyte.

[0194] In some embodiments, the assay comprises an immune targeting-based assay selected from the group consisting of: FLISA, FACS, flow cytometry, Western blot, protein microarray, bead-based multiplex immunoassay (e.g., Luminex), immunohistochemistry, immunocytochemistry, lateral flow assay, microfluidics, ELISPOT, fluorescence microscopy, FLIM, dot blot, single-cell Western, in-cell Western, competitive immunoassay, digital immunoassay, ImmunoCAP assay, protein simple ELLA assay, and combinations thereof. In some embodiments, the assay comprises a nucleic acid-based assay selected from the group consisting of Northern blot, microarray, next-generation sequencing, RNA-seq, FISH, EMSA, and combinations thereof.

[0195] kit Similarly, kits are provided. Such kits can include the agents or compositions described herein and, in certain embodiments, instructions for use. Such kits can facilitate the performance of the methods described herein. When supplied as a kit, the various components of the composition can be packaged in separate containers and mixed immediately before use. Components include, but are not limited to, assays or fluorescent nanoconstructs or components thereof, such as fluorophores, plasmonic nanostructures, coating or spacer reagents, polymers, biotin, streptavidin, antibodies, proteins, binders, or linkers. If desired, such packaging of components can be provided individually in a pack or dispenser device that can contain one or more unit dosage forms containing the composition. The pack can, for example, include metal or plastic foils, such as blister packs. In certain cases, such packaging of components can also allow for long-term storage without losing the activity of the components individually.

[0196] In some embodiments, the kit contains reagents in separate containers, such as sterile water or saline, added to the individually packaged components. For example, sealed glass ampoules can contain the components in separate ampoules, sterile water, sterile saline, or sterile materials, each packaged under a neutral, non-reactive gas such as nitrogen. The ampoules can be made of any suitable material, such as glass, organic polymers such as polycarbonate or polystyrene, ceramic, metal, or any other material typically used to hold reagents. Other examples of suitable containers include bottles made from materials similar to ampoules, and envelopes that can be lined with foil, such as aluminum or alloys. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. The container may have a sterile access port, such as a bottle with a stopper that can be punctured by a hypodermic needle. Other containers may have two compartments separated by an easily removable membrane that allows the components to mix upon removal. The removable membrane may be glass, plastic, rubber, or the like.

[0197] In certain embodiments, the kit is provided with instructional documentation. The instructions may be printed on paper or other substrate and / or provided as an electronically readable medium or video. The detailed instructions do not have to be physically associated with the kit. Instead, the user may be directed to an internet website designated by the kit manufacturer or distributor.

[0198] In some embodiments, the kit comprises a fluorescent nanoconstruct; or a plasmonic nanostructure, a spacer material, a biorecognition element, and at least one fluorescent agent. In some embodiments, the fluorescent nanoconstruct, or the combination of a plasmonic nanostructure, a spacer material, a biorecognition element, and at least one fluorescent agent, can have at least 500 times higher fluorescence intensity than the at least one fluorescent agent alone. In some embodiments, the kit comprises a liquid suspension of PF. In some embodiments, the kit comprises a frozen solution of PF. In some embodiments, the kit comprises a lyophilized solution of PF. In some embodiments, the kit comprises streptavidin-conjugated PF and user instructions for conjugating the streptavidin-conjugated PF to a biotinylated primary antibody and purifying the primary antibody-conjugated PF.

[0199] Exemplary embodiments of fluorescent nanoconstructs and methods for using the same are described in detail above. The fluorescent nanoconstructs and methods described herein are not limited to the specific embodiments described, but rather the components of the devices, systems, kits, and / or steps of the methods can be used independently and separately from other components and / or steps described herein. For example, the methods can also be used in combination with other polymers, nanostructures, and biological assays, and are not limited to being performed using only the devices, systems, and methods described herein. Rather, exemplary embodiments can be implemented and utilized in conjunction with numerous other systems.

[0200] Although specific features and applications of various embodiments of the present disclosure may be shown in some drawings and not in others, this is for convenience only. In accordance with the principles of the present disclosure, any feature illustrated herein may be referenced and / or claimed in combination with any other feature.

[0201] In the following specification and claims, reference will be made to a number of terms that will be defined to have the following meanings: The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements. "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and includes instances when said event occurs and instances when said event does not occur.

[0202] Throughout this specification and the claims, approximation language, as used herein, may be applied to modify any quantitative expression that can be varied to any possible extent without resulting in a change in the relevant basic function. Thus, values ​​modified by terms such as "about," "approximately," and "substantially" are not limited to the exact value specified. In at least some instances, approximation language may correspond to the precision of an instrument for measuring the value. Here, and throughout this specification and the claims, range limitations may be combined and / or interconverted. Such ranges are specified and include all subranges contained therein, unless the context or language indicates otherwise. [Example]

[0203] The following examples describe compositions and methods of making and using plasmon-fluors (PFs) to maximize fluorescence enhancement. [Example]

[0204] In the design and synthesis of fluorescent nanoconstructs, two factors must be carefully considered: (i) the bound fluorophore must be far enough from the plasmonic nanostructure surface to avoid metal-induced fluorescence quenching; and (ii) the fluorophore must be close enough to the plasmonic nanostructure surface to benefit from an enhanced electromagnetic field that decays rapidly with increasing distance from the plasmonic nanostructure surface. The transient nature of the electromagnetic field enhancement at the surface of the plasmonic nanostructure is known to result in a strongly distance-dependent enhancement of fluorescence at the surface of the plasmonic nanostructure. When the fluorophore comes into direct contact (or closest proximity) with the plasmonic nanostructure, nonradiative energy transfer between the fluorophore and the metal surface results in fluorescence quenching. On the other hand, increasing the distance between the fluorophore and the metal nanostructure results in a decrease in enhancement due to the attenuation of the electromagnetic field from the nanostructure surface. In summary, the optimal distance between the metal surface and the fluorophore is one of the key aspects of the nanostructure to ensure maximum enhancement. The optimal spacer thickness (d) is <10 nm. More specifically, for maximum enhancement, the spacer thickness should be between 1 and 10 nm when the fluorophore is directly attached to the spacer layer, and between 0.5 and 5 nm when the fluorophore is attached to a functional layer.

[0205] To achieve an optimal distance between the surface plasmonic nanostructures and the fluorophore, a polysiloxane copolymer layer was formed on the surface of the plasmonic nanostructures as a spacer layer. 3-Mercaptopropyltrimethoxysilane (MPTMS) was used to bond to the plasmonic nanostructure surface to create an initiation layer for the spacer. Hydrolytically unstable trimethoxypropylsilane (TMPS) and 3-aminopropyltrimethoxysilane (APTMS) were copolymerized onto the plasmonic nanostructures via the initiation layer. The formation of the spacer layer resulted in a red shift in the longitudinal LSPR wavelength of the plasmonic nanostructures due to an increase in the refractive index of the medium surrounding the nanostructures.

[0206] Transmission electron microscopy and atomic force microscopy (AFM) imaging confirmed the successful formation of the spacer layer on the plasmonic nanostructures. AFM height profiles of AuNRs before and after polymerization revealed that the spacer layer was approximately 3 nm thick. Fluorescence enhancement of nanostructures with and without dielectric spacers was investigated by attaching the nanostructures to substrates coated with streptavidin-800CW. The transient fluorescence enhancement factors of nanostructures of the same density (confirmed by scanning electron microscopy imaging) with and without the polymer spacer layer were found to be approximately 1000 and 200, respectively, highlighting the importance of the spacer layer for the large fluorescence enhancement (Figure 7A). Note that these particles have fluorophores attached to BSA, providing some space. If the fluorophores were directly attached to the AuNRs, there would be little fluorescence. The observed bright emission is caused by both excitation enhancement (EM field enhancement) and changes in emissivity. For a more detailed examination of the enhancement rate, we determined the excited-state lifetimes of 800CW-BSA dispersed in solution and nanostructures composed of 800CW-BSA. The fluorescence lifetime of 800CW-BSA adsorbed on AuNRs was four times shorter (0.18 ns) than that of free 800CW-BSA (0.75 ns) (Figure 7B). This large decrease in fluorescence lifetime directly contributes to an increase in the emission rate of the fluorophore. [Example]

[0207] Assay validation Following the synthesis of fluorescent nanoconstructs, the application of these novel materials and methods was validated in several bioanalytical techniques to enhance weak fluorescent signals and related bioanalytical parameters. To compare the enhancement of assay parameters to the commercially available assays described below, the assays were performed according to the supplier's specifications, and Plasmon-Fluor was added at a concentration <10x that of the gold-standard reporter molecule. It is possible that even higher performance can be achieved by optimizing reagents, incubation times, and concentrations.

[0208] Plasmon-Fluor acts as an ultrabright fluorescent probe in the final step of a bioassay, enhancing weak fluorescence and signal-to-noise ratios (SNR) without requiring any changes or modifications to existing bioassay protocols (i.e., a "non-invasive" method). The ultrabrightness of Plasmon-Fluor is due to the presence of a metallic core, which acts as an antenna to strongly enhance the fluorescence emission of surface fluorophores. The enhanced emission of fluorophores in the vicinity of the metallic nanostructure is due to the enhanced electromagnetic field at the surface of the plasmonic nanostructure (localized excitation region) and the reduced fluorescence lifetime due to coupling between the excited fluorophore and the nanostructure's surface plasmons. Plasmon-Fluor is highly versatile and versatile, seamlessly integrating with a variety of existing fluorescence-based bioanalysis techniques.

[0209] This disclosure tested the application of Plasmon-Fluor as a fluorescence enhancer in fluorophore-linked immunosorbent assay (FLISA). A typical sandwich FLISA involves the following main steps: (i) capture of a target antigen by an immobilized antibody; (ii) binding of a biotinylated detection antibody to the captured antigen; and (iii) binding of fluorescently labeled streptavidin. As shown herein, adding biotinylated Plasmon-Fluor after the last step (i.e., binding of fluorescently labeled streptavidin) resulted in a large increase in fluorescence intensity and a significant improvement in the limit of detection (LOD). The addition of biotinylated Plasmon-Fluor allows for a direct comparison of assay improvement compared to the current fluorescently labeled reporter standard (fluorescently labeled streptavidin). This method of adding biotinylated PF to a sample already interrogated with streptavidin still allows users to interrogate samples over a very high dynamic range of target analyte concentrations when the reading device cannot sufficiently attenuate the signal (i.e., high concentrations of analyte using PF result in fluorescence that saturates the detector, but are readable using standard fluorophore-labeled streptavidin, in which case the streptavidin can be conjugated to one or more fluorophores). To achieve a high dynamic range, users first add fluorescently labeled streptavidin and measure the fluorescence obtained from the assay, then add biotinylated PF and reread the fluorescence obtained from the assay. This is particularly attractive for plate-based and bead-based assays. In fact, end users may prefer to directly use streptavidin-conjugated antibody PF or detection-antibody PF instead of first adding fluorescently labeled streptavidin, performing a read, and then re-interrogating with biotinylated PF. According to the present disclosure, FLISA was performed in a heterogeneous solid-phase format by using 96-well microtiter plates as the sampling platform, a standard assay format widely used in biomedical research and clinical diagnostics.

[0210] The first application explored was a fluorescent immunoassay performed in 96-well plates using human IL-6 as a model target. Preliminary results showed that simply adding the fluorescent nanoconstruct as the final step in the assay improved fluorescence intensity by up to 2000-fold. In some embodiments, due to the significant improvement in fluorescence intensity, the assay sensitivity decreased by five orders of magnitude to 3 fg / ml, which is three orders of magnitude lower than what is achievable using the current gold-standard ELISA assay, even when using the same antibody and standard analyte.

[0211] As a final signal enhancer, the nanostructures were added following the conventional fluorescent tag, 800CW-streptavidin. To examine the enhanced sensitivity and LOD, serial dilutions of known concentrations of IL-6 (6 fg / mL to 6 ng / mL) in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) were used as standards. Fluorescence images obtained after applying the nanostructures revealed a 2,000-fold enhancement in fluorescence intensity compared to conventional FLISA. Specifically, the fluorescent signal from conventional Fluor (800CW) was detectable only at the two highest concentrations (6 and 0.6 ng / mL). On the other hand, the fluorescent signal from the fluorescent nanoconstructs was detectable even at concentrations as low as 6 fg / mL. The lower limit of detection (LLOD = mean value of blank + 3σ) of the unenhanced and plasmon-enhanced IL-6 assays was determined to be 600 pg / mL and 6 fg / mL, respectively, corresponding to a 105-fold improvement in LOD after the addition of the fluorescent nanoconstruct. Surprisingly, the LOD of the plasmon-enhanced assay was found to be 1000-fold lower than that of the supplier-specified enzyme-linked immunosorbent assay (ELISA), which involves enzymatic amplification of the colorimetric signal. Even more surprisingly, the plasmon-enhanced assay exhibited a dynamic range of seven orders of magnitude, which is more than four orders of magnitude higher than that of the ELISA. Essentially, nanostructures offer the potential to significantly improve the bioanalytical parameters [LLOD, lower limit of quantification (LLOQ = mean value of blank + 10σ), dynamic range] of commercially available immunoassay kits without requiring either tedious repetitive steps or specialized or expensive equipment.

[0212] The second application explored was signal enhancement in protein microarrays. For this purpose, a human kidney biomarker microarray was utilized on a 3D microporous nitrocellulose membrane. By simply adding fluorescent nanoconstructs, all 38 protein biomarkers in human patient urine samples were visualized in one simple test, compared to the 15 biomarkers revealed in an assay using fluorescently labeled streptavidin.

[0213] In yet another embodiment, we investigated the applicability of fluorescent nanoconstructs in enhancing the sensitivity of immunomicroarrays. Using an antibody microarray for biomarkers of human kidney disease (R&D systems, Inc. ARY019) as a representative example, we tested the performance of fluorescent nanoconstructs in a spatially multiplexed, high-throughput biosensing platform. The microarray consisted of 38 capture antibodies corresponding to human kidney protein biomarkers printed in duplicate on a three-dimensional nitrocellulose membrane. Biotinylated IgG was printed in duplicate as a reference (positive control). Duplicate spots of PBS were printed as a negative control. Human urine samples from kidney disease patients were diluted 2-fold using blocking buffer and added to the array. The captured biomarker proteins were then exposed to a biotinylated detection antibody cocktail, followed by exposure to 800CW-streptavidin. This step concludes the conventional microarray procedure, and at this point, biomarker concentrations are (semi-)quantified by analyzing the fluorescence intensity corresponding to each analyte. For the plasmon-enhanced assay, a solution of biotinylated fluorescent nanoconstructs was added to the microarray, incubated for 30 minutes, and thoroughly washed to remove weakly bound nanostructures. This allows for a direct comparison of the gold-standard reporter method with the nanoconstructs, although in practice users may prefer to use streptavidin-conjugated PF instead of first adding fluorescently labeled streptavidin and then labeling it with biotinylated PF.

[0214] Fluorescence maps obtained using conventional Fluor and fluorescent nanoconstructs with a human urine sample illustrate the improvement in fluorescent signal. Initially, the brightness and SNR of the positive control were found to be enhanced 80-fold after the addition of the nanostructures. At the same time, no signal was detected from the negative control, indicating minimal nonspecific binding of the nanostructures to the nitrocellulose membrane, which is important for ensuring low background. With conventional Fluor, only 14 of the 38 targeted protein biomarkers were detectable, with the majority of them exhibiting weak intensity. After the addition of the nanostructures, the fluorescent signal intensity from each spot on the microarray increased significantly. SEM images of the nitrocellulose after the addition of the fluorescent nanoconstructs revealed a uniform distribution of AuNRs on the porous membrane without any signs of aggregation. The fluorescent signals corresponding to cystatin C, β2-microglobulin (β2M), serpin A3, and neutrophil gelatinase-associated lipocalin (NGAL) were found to be enhanced by up to 500-fold compared to those obtained using fluorescently labeled streptavidin. Furthermore, the nanoconstructs enabled the detection and quantification of all other targets that could not be detected by fluorescently labeled streptavidin. For example, kidney injury molecule-1 (KIM1), a specific biomarker for the early detection of acute kidney injury, could only be detected after the addition of the fluorescent nanoconstruct. [Example]

[0215] The ultrabright fluorescent nanoconstructs were also applied to cell imaging to reveal cell surface biomarkers. A breast cancer cell line was selected as a model and probed for overexpression of the biomarker ErbB2 using various dilutions of ErbB2 primary antibody followed by 800CW-conjugated streptavidin. This experiment demonstrates that the fluorescence intensity corresponding to ErbB2 was enhanced by up to 100-fold after the addition of the biotinylated fluorescent nanoconstruct (Figures 8A and 8B). Fluorescence microscopy images still revealed overexpression of ErbB2 even at a 105-fold dilution of the primary antibody (Figure 8A). In practice, most users will likely use PF conjugated to secondary or primary antibodies for cell- or tissue-based experiments, including flow cytometry, immunocytochemistry, and immunohistochemistry. By conjugating the PF to an antibody, users can more easily multiplex (i.e., detect multiple markers simultaneously by using specific antibody / PF pairs with unique fluorescent spectral signatures, a technique commonly used for these types of experiments using antibodies labeled with conventional fluorophores). [Example]

[0216] In yet another embodiment, the ability of fluorescent nanoconstructs to enhance the signal-to-noise ratio (SNR) in flow cytometry-based cell analysis was demonstrated [Figures 8(A-B) and 9(A-B)]. ErbB2 (human epidermal growth factor receptor 2)-positive epithelial breast cancer cells (SKBR3) were tested as a model cell line. The cell surface receptor ErbB2 was immunostained using a standard fluorescent probe followed by the addition of nanostructures. A conventional two-step staining procedure was performed by sequentially incubating formaldehyde-fixed SKBR3 single-cell suspensions with biotinylated anti-ErbB2 and streptavidin-fluorophore (streptavidin-680LT). The nanostructures were optimized for 680LT by changing the aspect ratio to adjust the longitudinal LSPR wavelength to 660 nm. After labeling with the streptavidin-fluorophore, the cells were further incubated in the nanostructure suspension for 1 hour. Before proceeding to flow cytometry, the enhancement of the fluorescent signal was tested and visually confirmed. Confocal laser scanning microscopy (CLSM) images of cells were obtained using conventional fluorophores and nanostructures. As specified above, anti-ErbB2 was diluted to various concentrations and then incubated with the cell suspension. Compared to conventional staining (i.e., streptavidin-fluorophore), a significantly brighter fluorescent signal was observed after adding the nanostructures, which was detectable even with a 100,000-fold diluted primary antibody [Figures 8(A-B) and 9(A-B)].

[0217] In flow cytometry experiments, 5,000 cells were analyzed using a Guava InCyte to obtain fluorescent signals [RED-R channel (excitation laser: 642 nm; filter: 662 / 15 nm)] in combination with forward scatter (FSC) and side scatter (SSC). Due to the nanoscale size (approximately 75 nm) of the nanostructures, binding of the nanostructures to the cell surface did not alter the intensity of forward or side scatter (data not shown). The fluorescent signal histogram demonstrates that the nanostructures were 60-fold more intense than the cells treated with streptavidin-fluor (Figure 9A). The fluorescent histogram also demonstrates that ErbB2 expression on the cell surface can be detected even at a 100,000-fold dilution of the primary antibody after addition of the nanostructures (Figure 9B and Figure 8b). In contrast, when streptavidin-fluor was used alone, no fluorescent signal was detectable at dilutions higher than 1,000-fold. The mean fluorescence values ​​obtained with various dilutions of primary antibody using Fluor and nanostructures demonstrate the promise of the novel nanoconstructs in detecting low-abundance targets on the cell surface (Figure 8B). [Example]

[0218] Alternative design for PF Figure 10 shows an exemplary embodiment in which a plasmonic nanostructure is first coated with a polymer (Step 1), which acts as a spacer between the fluorescent species and the surface of the plasmonic nanostructure. Next, at least one fluorescent species is conjugated to the polymer coating (Step 2), so that the fluorescent species is maintained, on average, at a distance of >0.5 nm from the surface of the plasmonic nanostructure. The plasmonic nanostructure and fluorescent species are selected so that there is a large overlap between the absorption spectrum of the plasmonic nanostructure and the excitation / absorption spectrum of the fluorescent species. The fluorescent nanocomposite resulting from Step 2 is at least 500 times brighter than the unbound individual fluorescent species (which are used to coat the fluorescent nanocomposite under suitable excitation and detection conditions). The fluorescent nanocomposite / nanoconstruct resulting from Step 2 is then coated with a functional polymer layer (Step 3), in this example, bovine serum albumin and biotinylated bovine serum albumin. The nanocomposite resulting from Step 3 is biotinylated plasmonic Fluor. The biotinylated Plasmon-Fluor can be conjugated to at least one streptavidin to generate a streptavidin-Plasmon-Fluor (step 4). Finally, this streptavidin-Plasmon-Fluor can be further modified with at least one biotinylated antibody (step 5) to generate an antibody-conjugated Plasmon-Fluor.

[0219] Figure 11 shows an exemplary embodiment in which a plasmonic nanostructure is first coated with a polymer (step 1), which acts as a spacer between the fluorescent species and the surface of the plasmonic nanostructure. Next, at least one fluorescent species is conjugated to the polymer coating (step 2), so that the fluorescent species is maintained, on average, at a distance of >0.5 nm from the surface of the plasmonic nanostructure. The plasmonic nanostructure and fluorescent species are selected so that there is a large overlap between the absorption spectrum of the plasmonic nanostructure and the excitation spectrum of the fluorescent species. The fluorescent nanocomposite / nanoconstruct resulting from step 2 is at least 500 times brighter than the unbound individual fluorescent species (with which the fluorescent nanocomposite / nanoconstruct is coated under suitable and identical excitation and detection conditions). The fluorescent nanocomposite from step 2 is then coated with a functional polymer layer, in this example, bovine serum albumin conjugated with a reactive moiety suitable for use in a click chemistry reaction, such as trans-cyclooctene (TCO) (step 3). The nanocomposite from step 3 can be conjugated to at least one antibody by reacting the antibody with a click chemistry compatible moiety, such as tetrazine, that is complementary to the moiety used in step 3, resulting in an antibody-plasmon-fluor.

[0220] Figure 12 shows an additional alternative design in which a biorecognition element, depicted herein as an antibody, is attached to a polymer spacer layer by a linker moiety, e.g., polyethylene glycol. Other non-limiting examples of biorecognition elements are streptavidin, oligonucleotides, or aptamers. It should be noted that elements from Figures 9-12 can be mixed and matched. For example, it is possible to have a polymer linker like that depicted here also used with BSA, as in Figures 9-11.

[0221] Silane-aldehyde can be used to link hydrazine-conjugated materials (PEG or fluorophores) to the spacer layer, in which case the silane-aldehyde is added during the formation of the spacer layer with TMPS / APTMS.

[0222] Figure 13 shows an example plasmonic nanostructure in a dielectric material matrix, which acts as a spacer layer / coating. The dielectric material matrix is ​​coated with a functional layer (blue clouds). A targeting agent (pink "y"-shaped, e.g., an antibody) is conjugated to the functional layer.

[0223] Figure 14 shows the extinction spectrum of Plasmon-Fluor (AuNR coated with Ag plasmonic nanostructures) conjugated to IRDye 800CW (excitation maximum = 784). The inset shows the LSPR maximum.

[0224] Figure 15 shows that the mismatch between the LSPR maximum of the plasmon-fluor (AuNR coated with Ag plasmonic nanostructures) and the excitation maximum of the IRDye 800CW indicates that the overall resulting plasmon-fluor brightness significantly affects the overlap between the LSPR maximum of the plasmon-fluor and the excitation maximum of the dye.

[0225] Figure 16 shows the extinction spectrum (excitation maximum = 550 nm) of Plasmon-Fluor (AuNR@Ag cuboid plasmonic nanostructures) conjugated to Cy3. The inset shows the maximum LSPR wavelength.

[0226] Figure 17 shows that the mismatch between the LSPR maximum of the plasmon-fluor (AuNR@Ag cuboid plasmonic nanostructure) and the excitation maximum of Cy3 does not significantly affect the overall brightness of the resulting plasmon-fluor, indicating that the overlap between the LSPR maximum of the plasmon-fluor and the excitation maximum of the dye is not significant. This is because all of the plasmon-fluors with AuNR@Ag cuboid plasmonic nanostructures have significant absorption in the region of the excitation maximum of Cy3 (i.e., all of these structures show significant overlap with the excitation spectrum of Cy3). Furthermore, there are multiple LSPR peaks for these nanostructures, and even when the LSPR peak with the highest amplitude is significantly different from the excitation maximum wavelength of Cy3, some peaks are present in the region of the excitation maximum of Cy3. Comparing the extinction spectra in Figures 14 and 15, it is clear that in the case of Plasmon-Fluor conjugated to IRDye 800CW (AuNR coated with Ag plasmonic nanostructures), the key parameter for the large enhancement is that the Plasmon-Fluor has a large absorption near the excitation maximum of the fluorescent dye, and the absorption spectrum of the Plasmon-Fluor shows a large overlap with the excitation spectrum of the dye.

[0227] Figure 18 shows plasmonic nanostructures (silver-coated gold nanorods) coated in a dielectric matrix of a specific thickness (green shell). Fluorophores (red stars) are directly attached to the outer surface of the dielectric matrix. Biorecognition elements (pink "y" shapes, e.g., antibodies) can be directly conjugated to a spacer, which can be coated with a functional layer material (blue cloud). [Example]

[0228] Plasmon-Fluor preparation procedure In some embodiments, the Plasmon-Fluor is prepared using streptavidin and / or antibody conjugated Plasmon-Fluor. One can stop after step 7 to obtain only BSA-biotin Fluor. These steps are as follows:

[0229] Step 1: Calibration. Create a 40 mL solution with an extinction of 2 at the LSPR maximum based on the extinction of the core plasmonic nanostructure.

[0230] Step 2: Interfacial Layer: In a fume hood, 40 μL of MPTMS was added to the solution of plasmonic nanostructures, which was then placed on an orbital shaker at 125 RPM for 1 hour.

[0231] Step 3: Spacer layer. In a fume hood, 160 μL of APTMS was added, the tube was inverted 10 times, 160 μL of TMPS was added, the tube was inverted 10 times, and the tube was placed on an orbital shaker for 4 hours (resulting in an M:A:T ratio of 1:4:4 for this volume).

[0232] Step 4: Purification of free monomer / polymer. The solution from step 3 is centrifuged. The spacer-coated plasmonic nanostructures are collected in a pellet. The supernatant is removed and replaced with 1 mM CTAC to remove free silane.

[0233] Step 5: Dye labeling. To a 4 mL volume of the above NP solution (extinction at the LSPR maximum is 20), add 250 μL of 10×PBS buffer (pH 7.4). Add 0.1-20 μL of NHS-ester conjugated dye molecules and allow to react at room temperature for 1 hour.

[0234] Step 6: Purification of free dye The solution of dye-labeled nanoparticles is centrifuged and the supernatant is removed.

[0235] Step 7: BSA / BSA-biotin coating. Resuspend the nanoparticles from step 6 (or a mixture of BSA-biotin and free BSA of varying biotin density) in a solution of 5 mg / mL BSA-biotin at pH > 6, mix thoroughly, and incubate overnight at 4°C in the dark. Purify the coated nanoparticles from the free BSA-biotin using centrifugation.

[0236] Step 8: Streptavidin coating. Resuspend the particles from step 7 in a solution of 10 mg / mL streptavidin at pH > 6 and shake for 2 hours. Remove free streptavidin by centrifugation.

[0237] Step 9: Antibody conjugate. Resuspend the particles from step 7 in a solution of 10 mg / mL biotinylated antibody at pH > 6 and shake for 2 hours. Remove free antibody by centrifugation.

[0238] For storage, resuspend in 1x PBS (pH 7.4) and store at 4C. [Example]

[0239] Nanostructure and dye combinations for use in plasmon-fluors They are generally classified as follows according to the laser excitation wavelength used. Those skilled in the art will recognize that any excitation source that can be used to excite conjugated fluorescent species can also be used to excite plasmon-fluor containing such species. It is important to note that after coating with a spacer and functional group layer, the LSPR wavelength of the optimal plasmon nanostructure is generally blue-shifted (i.e., lower in wavelength) compared to the optimal LSPR wavelength, since the LSPR is red-shifted. The resulting plasmon-fluor has an absorption maximum close to that of the dye, and a large overlap between the plasmon-fluor extinction spectrum and the excitation / absorption spectrum of the dye.

[0240] In some embodiments, when using a laser excitation wavelength of 488 nm, suitable dyes include Fluorescein / FITC / FAM, AlexaFluor488, Atto488, Bodipy, Cy2, and Oregon Green. In some embodiments, suitable plasmonic nanostructures include AuNR@Ag nanocuboids (constructed from gold nanorods coated with silver), characterized by length = 92 nm (variable depending on LSPR, but size and LSPR are strictly linked in these particular plasmonic nanostructures, unlike AuNR, where LSPR is a function of aspect ratio), width = 63 nm (see above), and LSPR = 460-510 nm.

[0241] In some embodiments, when using a laser excitation wavelength of 532 nm or 543 nm, suitable dyes include Cy3, AlexaFluor532, AlexaFluor543, AlexaFluor555, Atto532, Atto550, rhodamine / tetramethylrhodamine / rhodamine 6G / TAMRA / TRITC, and Cy3.5 (Cy3.5). In some embodiments, suitable plasmonic nanostructures include AuNR@Ag nanocuboids characterized by a length of 86 nm (variable depending on the LSPR, but size and LSPR are strictly linked in these particular particles, unlike AuNR, where LSPR is a function of aspect ratio), a width of 73 nm (see above), and an LSPR of 500-570 nm.

[0242] In some embodiments, when using a laser excitation wavelength of 633 nm, suitable dyes include Cy5, Cy5.5, Alexa fluor 633, Alexa fluor 647, Alexa fluor 660, and Atto 633. In some embodiments, suitable plasmonic nanostructures include AuNRs with an LSPR of 600-670 nm.

[0243] In some embodiments, when using a laser excitation wavelength of 784 nm, suitable dyes include IRDye 800CW (LI-COR), Cy7.5, CF770, CF790, CF800, CF820, Alex790, and DyLight800. In some embodiments, suitable plasmonic nanostructures include AuNRs with LSPR = 720-800 nm. [Example]

[0244] Using Plasmon-Fluor, the time required to complete a sandwich immunoassay (compared to standard ELISA) can be significantly reduced while maintaining detection sensitivity similar to or even better than that of ELISA, as shown in Figures 19-22. Figures 19-22 show a comparison between conventional ELISA and p-ELISA for human NGAL detection and measurement. Figure 19 shows a plot showing the standard curve (dose-dependent colorimetric signal) of a human NGAL ELISA, which takes 280 minutes to complete. Figure 20 shows a plot showing the dose-dependent fluorescence intensity of human NGAL from p-FLISA performed within 20 minutes. Compared to conventional ELISA, p-FLISA containing the ultrabright fluorescent nanoconstruct (Plasmon-Fluor-800CW) can be completed within a 10-fold shorter period while achieving a similar detection limit. Figure 21 shows NGAL concentrations in urine samples from kidney patients and healthy volunteers determined using p-FLISA, which was completed within 20 minutes. FIG. 22 is a plot showing the correlation between the concentrations of human NGAL determined using ELISA (280 minute assay) and p-FLISA (20 minute assay), demonstrating excellent quantitative correlation (R 2 = 0.984). In summary, the human NGAL detection assay can be completed in 20 minutes using Plasmon-Fluor, as opposed to the 280 minutes required for conventional ELISA (recommended by the supplier and validated by the experiments described herein). The 20-minute Plasmon-Fluor-based assay showed the same limit of detection as the 280-minute ELISA. [Example]

[0245] Ultrabright Plasmon-Fluor as a Cross-Platform Nanolabel for Femtomolar Concentration Detection of Biological Analytes As noted throughout this disclosure, the detection, imaging, and quantification of low-abundance biomolecules in biological fluids, cells, and tissues is fundamentally important yet remains a significant challenge in biomedical research and clinical diagnostics. Utilizing plasmon-enhanced fluorescence, Plasmon-Fluor-800CW exhibited a signal that was approximately 6,700 times brighter than streptavidin labeled with the corresponding near-infrared (NIR) fluorophore (800CW). It should be noted that fluorescently labeled streptavidin can be labeled with one or more fluorescent dyes.

[0246] Figure 23 illustrates the working principle of plasmon-fluor as an "additional" biolabel to enhance the fluorescence intensity and resulting signal-to-noise ratio of a fluorescence-based assay without changing the workflow of the existing assay. Figure 24 is an exemplary embodiment of enhancement of a general sandwich immunoassay using streptavidin-conjugated plasmon-fluor according to the present disclosure. Figure 25 is an exemplary embodiment of a general sandwich immunoassay using a plasmon-fluor conjugated secondary antibody, where the antibody conjugated to plasmon-fluor recognizes a detection antibody according to the present disclosure. Figure 26 is an exemplary embodiment of a general sandwich immunoassay using a plasmon-fluor conjugated primary antibody, where the antibody conjugated to plasmon-fluor recognizes an analyte according to the present disclosure. It should be noted that the above examples of detection and readout are compatible with other assay types in addition to just sandwich immunoassays. The same general detection scheme can be used when the antigen is bound to a surface (e.g., a cell surface, a membrane, a substrate). [Example]

[0247] Gold nanorods (AuNRs) are used as representative plasmonic nanostructures due to their easy tunability of longitudinal localized surface plasmon resonance (LSPR) wavelength and aspect ratio, and the large electromagnetic field enhancement at both ends [see Figure 27(A-B)]. Figure 27A shows a TEM image of gold nanorods (AuNRs) used as nanostructures in a Plasmon-Fluor-800CW. Figure 27B is a finite-difference time-domain (FDTD) simulation showing the distribution of electric field strength around the AuNR (the polarization of the incident beam is along the longitudinal axis of the AuNR). AuNRs (length 83.0 ± 8.0 nm; diameter 24.3 ± 1.8 nm) were modified with (3-mercaptopropyl)trimethoxysilane (MPTMS), which served as an interfacial layer for the copolymerization of two organosilane monomers, namely, (3-aminopropyl)trimethoxysilane (APTMS) and trimethoxypropylsilane (TMPS) (Figure 28). Figure 28 is a schematic diagram illustrating the steps involved in the formation of polymer spacers on AuNRs. In aqueous media, APTMS and TMPS undergo rapid hydrolysis and subsequent condensation around the MPTMS-modified AuNRs, resulting in an amorphous copolymer network (Figure 28). The siloxane copolymer serves as a spacer layer between the metal surface and the fluorophore to prevent fluorescence quenching. As evidenced by atomic force microscopy (AFM), this sol-gel approach allows for easy control of the spacer layer thickness down to 1 nm (Figures 29-31). Figure 29 is an AFM image illustrating the increase in diameter of the AuNR / polymer with increasing amounts of monomer (MPTMS, TMPS, and APTMS). Figure 30 shows the UV-visible spectra of AuNR under various polymerization conditions. Figure 31 is a plot showing the increase in diameter of AuNR (2x greater than the thickness of the polymer layer) under each polymerization condition, as measured from the AFM images.Through modification of AuNR with MPTMS and subsequent polymerization of APTMS / TMPS, the zeta potential of AuNR capped with cetyltrimethylammonium bromide (CTAB) decreased from +38.4 ± 2.3 mV to +29 ± 2.6 mV and +25.8 ± 1.9 mV, respectively, due to partial replacement of the positively charged capping agent (CTAB) with the less charged siloxane copolymer (Figure 32). Figure 32 shows the zeta potentials of AuNR, AuNR / MPTMS, AuNR / MPTMS / polysiloxane (AuNR / polymer), and Plasmon-Fluor-800CW (AuNR / polymer / BSA-biotin-800CW). Error bars correspond to standard deviation (n = 3 replicates). [Example]

[0248] The near-infrared (NIR) fluorophore 800CW and biotin were conjugated to BSA using carbodiimide coupling chemistry, achieving a protein / biotin / fluorophore ratio of 1:8.7:1.2. Due to its stronger affinity for avidin, biotin displaces avidin-bound HABA, resulting in a decrease in absorbance intensity. The absorbance values ​​at 780 nm and 280 nm were used to quantify the dye-to-BSA ratio. The BSA-biotin-800CW conjugate was then adsorbed onto polysiloxane-coated AuNRs via electrostatic, hydrophobic, and hydrogen-bonding interactions between BSA and the functional groups (-NH3+, -CH3, -OH) of the polysiloxane layer, forming Plasmon-Fluor-800CW. Upon formation, Plasmon-Fluor-800CW exhibited a negative charge (zeta potential of -46.9 ± 0.5 mV at pH = 10) due to the abundance of carboxylic acid groups in BSA, which has an isoelectric point of 4.7 (Figure 32). The LSPR wavelength of the AuNR exhibited a gradual red shift to 2.6 nm and 2.7 nm upon the formation of the polymer spacer layer and BSA-biotin-800CW adsorption, respectively [Figure 33(A-B)]. Figure 33(A-B) shows the PF-800CW TEM image and extinction spectrum.

[0249] After structural characterization of Plasmon-Fluor-800CW, the brightness of the fluorescent nanoconstruct was determined. The excited-state fluorescence lifetimes of free 800CW (conjugated to BSA) and Plasmon-Fluor-800CW were measured to be 0.74±0.01ns and 0.179±0.001ns, respectively, resulting in a 7-fold improvement in quantum yield (calculated herein from about 11% to about 79%). To further understand the brightness of Plasmon-Fluor-800CW, the number of fluorophores conjugated to a single AuNR was estimated. Plasmon-Fluor-800CW at a concentration of 76.2pM (approximately 0.63 quenching) contains about 16nM of 800CW (calculated herein). Therefore, it is estimated that about 210 fluorophores are conjugated to a single AuNR. Notably, the fluorescence intensity from 76.2 pM Plasmon-Fluor-800CW (containing 16 nM 800CW) was found to be equivalent to that from 544 nM 800CW (measured based on Figure 2). The difference in the slopes of the two curves indicates that a single Plasmon-Fluor-800CW is as bright as 6700 (±900) fluorophores. Therefore, it can be concluded that each 800CW is enhanced by approximately 30-fold due to the presence of the plasmonic nanostructure. Error bars represent the standard deviation (n = 3 replicates). This corresponds to approximately 30-fold enhancement per bound fluorophore. This result was obtained for Plasmon-Fluor in which 800CW was conjugated to a functional layer of BSA. Figure 2 shows the fluorescence intensity of conventional Fluor-800CW and Plasmon-Fluor-800CW at different molar concentrations, where the Plasmon-Fluor-800CW has 800CW directly attached to a spacer layer approximately 2-4 nm thick. The difference in slope for a plot of the fluorescence intensity of Plasmon-Fluor based AuNRs conjugated with 800CW versus free, unconjugated 800CW in solution as a function of the concentration of the fluorescent species indicates that the Plasmon-Fluor-800CW is approximately 20,000 times brighter than the free 800CW.These data were collected on an Azure Sapphire scanner using the same excitation and emission conditions as for the plasmonic fluorophores and free 800 CW (excitation at 784 nm and detection through a 37 nm wide bandpass filter centered at 832 nm). The observed strong emission can be attributed to the enhancement of the electromagnetic field (localized excitation region) at the surface of the plasmonic nanostructures [Figure 27(A-B)] and the reduction in fluorescence lifetime due to coupling between the excited fluorophore and the surface plasmons of the nanostructures.

[0250] The feasibility of using Plasmon-Fluor-800CW as an ultrabright fluorescent reporter was tested by binding the reporter to a substrate coated with streptavidin-800CW, as shown in Figure 34, a schematic (not to scale) depicting a model system based on the binding events performed in this study. Plasmon-Fluor-800CW binding resulted in an average 1200-fold (±40)-fold enhancement in ensemble fluorescence intensity compared to streptavidin-800CW, demonstrating specific binding of 800CW-streptavidin fluorescence intensity and then Plasmon-Fluor-800CW through biotin-streptavidin interactions, resulting in an average 1200-fold (±40)-fold enhancement in fluorescence intensity. Significant signal enhancement was achieved by using a relatively low concentration of Plasmon-Fluor (76 pM). It should be noted that in all of the diagnostic assays to further verify the plasmonic enhancement of fluorescence, we used "non-resonant" gold nanoparticles (AuNPs) with surface areas similar to the "resonant" AuNRs (7850 nm² / AuNPs; 8064 nm² / AuNRs) [see Figure 5(A-B)]. To illustrate the importance of overlap between the absorbance of the plasmonic nanostructure and the absorbance / excitation spectrum of the conjugated dye, we fabricated plasmonic-fluors using either gold spheres, AuNPs, or gold nanorods, AuNRs as the plasmonic nanostructure core, and 800CW as the conjugated fluorescent species bound to BSA, which was then adsorbed onto the spacer layer and biotin as the biorecognition element. Their individual extinction spectra, as well as the absorption / excitation and emission spectra of 800CW, are shown in the plot on the left. The resulting fluorescence of the same concentration of material excited at 784 nm is shown in the plot on the right. Although AuNPs exhibit somewhat higher fluorescence compared to Fluor and 800CW alone, they are still approximately 100 times less bright than Plasmon-Fluor with AuNRs as the core plasmonic nanostructure.Not surprisingly, AuNP-Plasmon-Fluor-800CW only enhanced the fluorescence intensity by 18-fold, which was approximately 70-fold lower than that obtained with AuNR-Plasmon-Fluor-800CW, confirming plasmon-enhanced fluorescence [Figure 5(A-B)].

[0251] Figure 1 shows that the difference in slope for a plot of the fluorescence intensity of Plasmon-Fluor based AuNR@Ag nanocuboids conjugated with Cy3 versus free, unconjugated Cy3 in solution as a function of concentration indicates that the Plasmon-Fluor Cy3 is approximately 10,000x brighter than the free Cy3. These Plasmon-Fluors had the dye conjugated directly to a polymer spacer layer approximately 2 nm thick. These data were collected on a BioTek Synergy H1 using the same excitation and emission conditions as for the Plasmon-Fluor and free Cy3 (excitation at 530 nm and detection at 570 nm).

[0252] Figures 35 and 36 show various volumes of core AuNR particles to enhance the 800CW generated by adjusting the amount of added seed. The most commonly used seed amount in the plasmonic nanoparticle literature is 48 μL. Plasmonic Fluor was generated from various AuNR core particles and 800CW, and after normalization to the same molar concentration, the fluorescence intensity was measured using an Azure Sapphire scanner with an excitation wavelength of 784 nm and detection through a bandpass filter centered at 832 nm and 37 nm wide. Larger AuNRs have significantly higher brightness (indicated by #) than the most commonly used AuNRs at the same LSPR wavelength.

[0253] Figure 37 shows the extinction spectrum of AuNR@Ag cuboids, which form the core particles for plasmon-fluors designed to enhance dyes with excitation maxima near 488 nm, such as FITC and AlexaFluor488.

[0254] Figure 3 shows that the difference in slope for the fluorescence intensity plot of plasmon-fluor based AuNR@Ag nanocuboids conjugated with FITC (see Figure 35) versus free, unconjugated FITC in solution as a function of concentration indicates that the plasmon-fluor FITC is approximately 16,667x brighter than free FITC. These plasmon-fluors had the dye conjugated directly to a polymer spacer layer approximately 2-4 nm thick. These data were collected on a BioTek Synergy H1 using the same excitation and emission conditions as for the plasmon-fluor and free FITC (excitation at 490 nm and detection at 530 nm). [Example]

[0255] Figure 38 shows plasmonic nanostructures suitable for enhancing fluorophores that can be excited at 488 nm (Au@Ag-490), 658 nm (AuNR-670), and 784 nm (AuNR-760). Common standard fluorophore excitation regimes corresponding to the corresponding plasmonic particles are highlighted. [Example]

[0256] Figure 39 (A-B) shows TEM images of AuNR@Ag nanocuboids (left) and Plasmon-Fluor-Cy3 (right), which consist of AuNR@Ag nanocuboids, a polymer shell, and a coating of BSA-biotin-Cy3. The coatings (functional and spacer layers) are approximately 6 nm thick. Figure 40 shows the extinction spectra of AuNR@Ag nanocuboids, AuNR@Ag nanocuboids coated with a polymer spacer, and Plasmon-Fluor-Cy3, revealing a successive red shift after each coating step. [Example]

[0257] The optimal distance between the metal surface and the fluorophore is important for maximizing fluorescence enhancement by balancing two opposing factors: electromagnetic field enhancement and nonradiative energy transfer. We investigated the fluorescence enhancement of Plasmon-Fluor-800CW with various thicknesses of dielectric spacers (MPTMS, APTMS, and TMPS) by attaching the dielectric spacer to a substrate coated with streptavidin-800CW. The ensemble fluorescence enhancement factor (defined as the ratio of the fluorescence intensity obtained after attaching Plasmon-Fluor to a surface coated with streptavidin to which the fluorophore is conjugated) of Plasmon-Fluor without a polymer spacer layer was found to be approximately 146 ± 81. The enhancement efficiency gradually improved to approximately 1200 (± 40) times as the spacer thickness increased (Figure 41). Note that the polymer thickness plotted here is not actually the distance between the attached Fluor and the metal surface. This figure shows a plasmon fluor with fluorescently labeled BSA. While this figure seems to indicate that the optimal spacer thickness is generally between 0.8 and 2.9 nm, this is not necessarily the case for plasmon fluors in which the fluorophore is directly attached to the spacer coating. In other words, when the fluorophore is attached to BSA, which acts as a functional layer, the BSA itself acts as a spacer between the fluorophore and the plasmon nanostructure, so the polymer thickness is not actually the distance between the attached fluorophore and the surface of the plasmon nanostructure. When using fluorophore-conjugated BSA, some fluorophores are located directly next to the spacer layer, while others are approximately 4 nm away from the spacer layer. Therefore, the average distance of the fluorophores from the surface of the plasmon nanostructure is estimated to be approximately 2 nm greater than the thickness of the spacer layer.Studies in U.S. Provisional Patent Application No. 62 / 590,877, filed November 27, 2017, entitled "Plasmonic Film as a Universal Fluorescent Enhancer," which is incorporated herein by reference in its entirety, found that the optimal spacing between the fluorophore and the plasmonic nanostructure surface is between 2 and 5 nm, consistent with the results presented here. Notably, colloidal solutions of plasmon-fluor exhibited stable fluorescent signals after storage in the dark at 4°C for one month (Figure 42). For further ease of storage, transportation, and handling, plasmon-fluor can be lyophilized and reconstituted as needed without significant degradation of the fluorescent signal (Figure 42). [Example]

[0258] Plasmon-Fluor-enhanced Fluorescence-Linked Immunosorbent Assay (p-FLISA) and Bead-Based Multiplex Assays Among the many applications of Plasmon-Fluor, we performed a plasmon-enhanced fluorophore-linked immunosorbent assay (p-FLISA) in a standard microtiter plate. Human interleukin-6 (IL-6), a proinflammatory cytokine, was used as a representative protein biomarker. Conventional FLISA requires a standard sandwich format of capture antibody, analyte (IL-6), and biotinylated detection antibody, followed by exposure to a streptavidin-fluorophore (800CW in this study) (Figure 43). Figure 43 is a schematic diagram illustrating the concepts of conventional FLISA (800CW) and Plasmon-Fluor-800CW-enhanced FLISA (p-FLISA) performed in a standard 96-well plate. The p-FLISA assay does not require any changes to the routine workflow other than the addition of Plasmon-Fluor as a new final step. In p-FLISA, Plasmon-Fluor-800CW is introduced after the final step as a signal enhancer (Figure 43). To assess the improvement in sensitivity and limit of detection (LOD), defined as the mean value of the blank + 3σ, serial dilutions of known concentrations of IL-6 (6 ng / ml to 6 μg / ml in 1% BSA buffered with phosphate-buffered saline (PBS)) were used as standards. The fluorescence signal obtained after application of the Plasmon-Fluor-800CW revealed an approximately 1440-fold enhancement in ensemble fluorescence intensity compared to conventional FLISA at the highest analyte concentration tested here (6 ng / ml) (Figures 44, 45, and 46). Figure 44 shows fluorescence intensity maps of human IL-6 FLISA and p-FLISA at various analyte concentrations. Figure 45 shows fluorescence intensity maps of human IL-6 FLISA and p-FLISA (with enlarged scale bars) and a photograph of the colorimetric signal of the "gold standard" human IL-6 ELISA. The LOD of conventional FLISA was calculated to be approximately 95 pg / ml (Figures 47, 48, and 46, polynomial fitting). Figure 47 shows a plot of human IL-6 dose-dependent fluorescence intensity from conventional FLISA. Figure 48 shows the LOD of conventional IL-6 FLISA. A standard curve was generated using polynomial fitting.Error bars represent standard deviations (n ​​= 2 replicates). Figure 46 shows individual data points, means, and standard deviations from human IL-6 FLISA, p-FLISA, and ELISA. Meanwhile, the fluorescent signal by p-FLISA was detectable down to 20 fg / ml (approximately 1 fM) [Figure 49 and Figure 46, 4-parameter logistic (4PL) fit], corresponding to a 4750-fold improvement in LOD compared to conventional FLISA. Figure 49 shows a plot of human IL-6 dose-dependent fluorescence intensity from p-FLISA. Compared to conventional FLISA, p-FLISA demonstrates a 4750-fold improvement in limit of detection (LOD) and a dynamic range of over three orders of magnitude. Notably, Plasmon-Fluor demonstrated extremely high specificity (vs. streptavidin) and low nonspecific binding to interfering biomolecules in bioassays [Figure 50(A-B)]. Figure 50A shows IL-6 dose-dependent fluorescence intensity from p-FLISA. Error bars correspond to standard deviation (n=2 replicates). Figure 50B shows nonspecific binding of Plasmon-Fluor-800CW. C: capture antibody; D: detection antibody; S: streptavidin; PF: Plasmon-Fluor; Blank: no Plasmon-Fluor. Compared to the blank, no signal was observed after applying Plasmon-Fluor-800CW to BSA, capture antibody, or capture and detection antibodies. *****P<0.0001 (one-way ANOVA with Tukey's post-hoc test). NS: not significant. A nonspecific signal at zero IL-6 concentration was present only when streptavidin was introduced, implying the excellent specificity of Plasmon-Fluor. Error bars represent standard deviation (n=3 replicates). This "BSA blocking" strategy of Plasmon-Fluor is important for increasing the signal-to-background ratio. Scanning electron microscope (SEM) images revealed an increase in the density of Plasmon-Fluor-800CW at the bottom of the microtiter wells with increasing IL-6 concentration (Figure 51). Figure 51 shows an SEM image of the bottom surface of a 96-well plate after IL-6 p-FLISA, revealing that the density of Plasmon-Fluor-800CW increases with increasing IL-6 concentration. A much lower density of Plasmon-Fluor was observed in blank wells incubated with 1% BSA, again indicating low non-specific binding of Plasmon-Fluor (FIG. 51).

[0259] Surprisingly, the LOD and lower limit of quantitation (LLOQ), defined as the mean value of the blank + 10σ, approximately 82 fg / ml, for p-FLISA were found to be 189-fold and 120-fold lower than those of the "gold standard" enzyme-linked immunosorbent assay (ELISA), which involves enzymatic amplification of the colorimetric signal (Figures 45, 52, and 46). Figure 52 is a plot showing the standard curve for human IL-6 ELISA. Compared to ELISA, p-FLISA demonstrated a 189-fold lower LOD and a dynamic range greater than two orders of magnitude. More importantly, p-FLISA demonstrated a five-order dynamic range (the ratio between the upper and lower limits of quantitation), which is more than two orders of magnitude greater than the dynamic range of ELISA. As a validation of assay performance, p-FLISA was used to test healthy human serum samples and IL-6-spiked serum. Serum samples were diluted 10-fold, so that only 10 μl of the original sample was required for each subject. The concentration of IL-6 in healthy individuals typically ranges from 0.2 to 7.8 pg / ml. Increased levels of IL-6 in serum may indicate systemic inflammation, metabolic stimulation, and physiological stimuli. Of note, only the latter technique, ELISA, FLISA, and p-FLISA, was able to determine IL-6 concentrations in healthy individuals, which were measured as 8.1 pg / ml, 1.8 pg / ml, and 2.8 pg / ml after dilution correction (Figure 53). Figure 53 shows the IL-6 concentration in human serum samples (diluted 10-fold) measured using p-FLISA. Error bars represent standard deviation (n = 3 replicates).

[0260] In addition to the microtiter plate format, we also explored the application of Plasmon-Fluor as an ultrabright reporter in microbead-based multiplex fluorescent immunoassays that utilize non-planar sampling surfaces. Using a Luminex assay as an example, this assay utilizes magnetic microbeads embedded with ratio-programmed fluorophores as barcodes for each unique analyte (Figure 54). Figure 54 is a schematic diagram illustrating the concept of using Plasmon-Fluor-Cy3 to enhance the sensitivity of bead-based immunoassays (e.g., Luminex assays). Antibody-conjugated microbeads capture the analytes to facilitate their detection in a typical sandwich format, which is subsequently probed with streptavidin conjugated with phycoerythrin (PE), a highly fluorescent protein isolated from red algae or cyanobacteria. However, the PE used in Luminex assays is structurally unstable and prone to photobleaching. Here, a highly stable fluorophore with absorption and emission at 554 nm and 568 nm, respectively, similar to PE, was used as a substitute. As discussed above, it is crucial to select a plasmonic nanostructure with an LSPR wavelength that matches the excitation maximum wavelength of the fluorophore. To this end, we fabricated Plasmon-Fluor-Cy3 [Figures 55(A-B), 39(A-B), and 40] using AuNR@Ag nanocuboids with an LSPR wavelength of 520 nm. Figures 55(A-B) show TEM images of Plasmon-Fluor-Cy3 using AuNR@Ag as the plasmonic nanostructure, with a spacer coating thickness of approximately 6 nm. Figure 39 (A-B) shows TEM images of AuNR@Ag nanocuboids (left) and Plasmon-Fluor-Cy3 (right), which consist of AuNR@Ag nanocuboids, a polymer shell, and a coating of BSA-biotin-Cy3. The coating is approximately 6 nm thick.Figure 40 shows the extinction spectra of AuNR@Ag nanocuboids, AuNR@Ag nanocuboids coated with a polymer spacer, and Plasmon-Fluor-Cy3, revealing a continuous red shift after each coating step. Notably, the synthesized Plasmon-Fluor-Cy3 exhibits considerable brightness, and individual nanoconstructs can be easily identified under a standard epifluorescence microscope [Figure 56(A-C)]. Figure 56A shows a fluorescence microscope image of an individual Plasmon-Fluor-Cy3. Figure 56B shows the corresponding SEM image of an individual Plasmon-Fluor-Cy3 shown in Figure 56A. Figure 56C is an enlarged SEM image of a single Plasmon-Fluor-Cy3 (single nanocuboid) corresponding to the square shown in Figures 56A and 56B. These fluorescence images were obtained using a non-laser epifluorescence microscope, which is widely available in standard laboratories.

[0261] A Luminex assay was customized to simultaneously detect murine IL-6 and murine tumor necrosis factor-α (TNF-α), key proinflammatory cytokines involved in cell signaling and immune modulation. Microbeads were incubated with a mixture of serial dilutions of TNF-α and IL-6, followed by a detection antibody cocktail: streptavidin-Cy3 and biotinylated Plasmon-Fluor-Cy3 (Figure 54). The beads were then read using a dual-laser flow-based instrument (Luminex 200): the classification laser (635 nm) reads the barcode on each bead, and the reporter laser (532 nm) measures the intensity of Cy3 fluorescence, which is directly proportional to the amount of bound analyte (Figure 54). SEM images of the microbeads show uniform binding of Plasmon-Fluor-Cy3 with no signs of aggregation (Figure 57). Figure 57 shows SEM images of microbeads before and after interrogation with Plasmon-Fluor-Cy3. Plasmon-Fluor-Cy3 conjugation did not change the size and shape of the beads [Figure 58(A-B)] or the optical barcode signal [Figure 59(A-D)]. Figures 58(A-B) show bright-field and fluorescence images of Luminex microbeads before (Figure 58A) and after (Figure 58B) interrogation with Plasmon-Fluor-Cy3. Figures 59(A-D) show fluorescence images of Luminex microbeads after staining with Plasmon-Fluor-Cy3, showing the barcodes on the microbeads (excited with a 633 nm laser) of various emission intensities (Figure 59A) and the fluorescence of the conjugated Cy3 (excited with a 543 nm laser) (Figure 59B). A bright-field image of the microbeads (Figure 59C). Brightfield and fluorescence merged image (Figure 59D). Scale bar corresponds to 50 μm. A significant enhancement in microbead fluorescence intensity was observed after conjugation of Plasmon-Fluor-Cy3 (Figure 60). Figure 60 shows fluorescence images of microbeads before and after interrogation with Plasmon-Fluor-Cy3.The LODs for the plasmon-enhanced mouse IL-6 and TNF-α assays were determined to be 56.6 fg / ml (2.7 fM) and 7.5 fg / ml (0.3 fM), respectively (Figures 61, 62, and 63). Figure 61 shows the mouse IL-6 standard curves obtained before (left) and after (right) application of Plasmon-Fluor-Cy3. Figure 62 shows the mouse TNF-α standard curves obtained before (left) and after (right) application of Plasmon-Fluor-Cy3. All standard curves were performed at least three times independently on different days with different doses of Plasmon-Fluor. Compared to their non-enhanced counterparts [Figures 61, 62, 63, and 64(A-B)], the plasmon-enhanced assays showed 143- and 814-fold lower LODs for mouse IL-6 and mouse TNF-α, respectively. Figure 63 shows individual data points, means, and standard deviations from Luminex assays for mouse IL-6, Luminex assays for plasmon-fluor-Cy3-enhanced mouse IL-6, Luminex assays for mouse TNF-α, and Luminex assays for plasmon-fluor-Cy3-enhanced mouse TNF-α. Figure 64A is a plot showing the LOD of the unenhanced bead-based Luminex fluorescent immunoassay for mouse IL-6. Figure 64B is a plot showing the LOD of the unenhanced bead-based Luminex fluorescent immunoassay for TNF-alpha. Curves were generated using polynomial fitting. Error bars represent standard deviation (n=2 replicates). Notably, the supplier-specified LODs (using PE-streptavidin) for mouse IL-6 (2.3 pg / ml) and mouse TNF-α (1.47 pg / ml) were found to be 41- and 196-fold inferior to the plasmon-enhanced Luminex assay. Essentially, Plasmon-Fluor serves as a powerful platform technology that enhances the bioanalytical parameters (LOD, LLOQ, dynamic range) of various existing immunoassays without requiring any cumbersome steps or specialized equipment. [Example]

[0262] Plasmon-Fluor-Enhanced High-Throughput Multiplexed Proteomics Array: Biomolecular (micro)arrays based on fluorescence readout are important clinical and research tools, especially for simple, high-throughput, and rapid proteomic and genetic analyses, which allow the miniaturization of thousands of assays onto a single small piece on an analytical substrate. Despite advantages such as high multiplexing, rapid screening, and small sample volume, the method suffers from low sensitivity (even inferior to ELISA), which hinders its widespread application.

[0263] The applicability of Plasmon-Fluor to enhance the sensitivity of immunoarrays was investigated. All error bars represent standard deviations (n ​​= 2 replicates). A series of antibodies to human renal disease biomarkers was used as a representative example (Figure 65). Figure 65 is an illustration showing the application of Plasmon-Fluor-800CW to enhance the biological analysis parameters of a multiplexed proteome profiler for human renal disease biomarkers implemented on a nitrocellulose membrane. This example illustrates the use of biotinylated Plasmon-Fluor to enhance a typical multiplexed microarray, where capture antibodies for specific analytes are printed in spatially distinct spots on either a membrane, glass slide, or polystyrene substrate. In this method, users can first label the array with standard fluorescently labeled streptavidin and then label it with biotinylated Plasmon-Fluor. In some embodiments, streptavidin-conjugated plasmon fluors are used to enhance a typical multiplex microarray, in which capture antibodies for specific analytes are printed in spatially distinct spots on either a membrane, glass slide, or polystyrene substrate (Figure 66). This array consists of 38 capture antibodies corresponding to human kidney disease protein biomarkers printed in duplicate on a microporous nitrocellulose membrane [Figure 67(A-B)]. Figures 67(A-B) show the identification of the specific analyte (or control) of each pair of fluorescent spots for the kidney biomarker array. The fluorescent spots shown in Figure 67A are identified by the coordinates in Figure 67B. Biotinylated IgG and PBS were printed as reference positive and negative controls, respectively [Figure 67(A-B)]. Human urine samples from patients with kidney disease were diluted 10-fold using blocking buffer, mixed with a biotinylated detection antibody cocktail, and applied to the nitrocellulose membrane. After incubation, the membrane was exposed to streptavidin-800CW.Finally, the Plasmon-Fluor-800CW suspension is added onto the array, incubated, and washed extensively to remove unbound nanoconstructs (Figure 65).

[0264] SEM images from the positive control area revealed a uniform distribution of Plasmon-Fluor on the membrane (including the porous subsurface region) (Figure 68). Figure 68 is an SEM image showing the uniform distribution of Plasmon-Fluor-800CW (several highlighted by yellow circles) on and within the subsurface region of the nitrocellulose membrane. Figure 71 shows a fluorescence intensity map representing the kidney disease protein biomarker profile of the kidney disease patient shown in Figures 69 and 70 after the addition of Plasmon-Fluor-800CW (note the difference in the fluorescence intensity scale bars). At the same time, no signal was detected from the negative control (Figure 71: blue square), and Plasmon-Fluor was not observed in the SEM images from these locations, indicating minimal nonspecific binding [Figure 72(A-B)]. Figure 72A shows the kidney biomarker array of Figure 67A. Figure 72B is an SEM image showing the nitrocellulose membrane in the negative control region (the blue rectangle at the bottom right corner of Figure 72A (corresponding to the pair at coordinates F23 and F24 shown in Figure 67A); note the absence of fluorescent signal and Plasmon-Fluor-800CW) after the addition of Plasmon-Fluor-800CW, demonstrating low nonspecific binding of Plasmon-Fluor-800CW. Using conventional fluorophores, only 26 of the 38 target protein biomarkers were detectable, and most of them showed weak intensity (Figures 69, 70, 73, and 74). Figures 69 and 70 show fluorescence intensity maps representing the kidney disease protein biomarker profile of a kidney disease patient obtained using a conventional fluorophore (streptavidin-800CW). Figures 73 and 74 show individual data points, average values, and standard deviations with and without Plasmon-Fluor, respectively. FIG. 75 is a digital photograph taken with a mobile phone showing the color change of a nitrocellulose membrane with a urine sample from a patient with kidney disease after addition of Plasmon-Fluor-800CW.After the addition of Plasmon-Fluor-800CW, the fluorescence signal intensity from each spot on the protein array significantly increased (Figures 71, 73, and 74), enabling the detection and relative quantification of all other targets that could not be detected by conventional Fluor. Furthermore, a commercially available 40-plex cytokine microarray was used as another validation for Plasmon-Fluor, and in this case, a significant improvement in microarray sensitivity was also observed [Figure 76(A-F)]. Figure 76A shows the layout of the 40-plex cytokine microarray. Each antibody is printed horizontally in quadruplicate in each spot with a diameter of approximately 140 μm. Fluorescence maps of the cytokine microarray obtained using a conventional fluorophore (streptavidin-800CW) (Figure 76B) and after the addition of Plasmon-Fluor-800CW (Figure 76C). Plot showing the fluorescence intensity corresponding to each cytokine obtained using a conventional fluorophore (streptavidin-800CW) (Figure 76D) and after adding Plasmon-Fluor-800CW (Figure 76E). Error bars correspond to standard deviation (n=4 replicates). Dark-field scattering of Plasmon-Fluor-800CW (AuNR) absorbed on a cytokine microarray (Figure 76F). Each circle corresponds to one microspot area for each analyte. The scale bar corresponds to 50 μm. The distribution of AuNR (Plasmon-Fluor-800CW) on each microspot is clearly visible and can be digitally counted.

[0265] Plasmonic nanostructures at the LSPR wavelength exhibit large extinction cross-sections, which can be up to 5–6 orders of magnitude larger than the optical absorption of most organic dyes. This unique property of plasmonic nanostructures offers the possibility of utilizing plasmonic-fluorescent nanoparticles as multimodal biolabels. Indeed, binding of plasmonic-fluorescent nanoparticles to the sensing domain results in analyte concentration-dependent colored spots that are directly visible to the naked eye (Figure 75). The color intensity of each spot in digital photographs taken using a mobile phone camera under ambient light conditions was analyzed and compared with the corresponding fluorescence intensity. A good correlation was observed between the two acquisition modes (R2 = 0.88, Figure 77), demonstrating the potential applicability of this nanoconstruct as a "visible label" in resource-limited settings, mitigating the dependency on dedicated, expensive reading equipment. Figure 77 shows a plot showing the correlation between two reading modes (fluorescence readout vs. color readout) of a kidney biomarker array. [Example]

[0266] Plasmon-Fluor-Enhanced Immunocytochemistry / Immunofluorescence (ICC / IF): Immunocytochemistry, based on immunofluorescence, is a well-established semi-quantitative method for analyzing the relative abundance, conformation, and subcellular localization of target antigens in cells. Again, this method lacks the sensitivity to distinguish low-abundance biomolecules from the noise level due to the weak fluorescent signals of conventional fluorophores. Autofluorescence, the spontaneous emission of light by biological structures, further contributes to the overall low signal-to-noise ratio.

[0267] To test the applicability of Plasmon-Fluor in ICC / IF, ErbB2 (human epidermal growth factor receptor 2)-positive epithelial breast cancer cells (SK-BR-3) were used as a model cell line. Surface receptor ErbB2 was immunostained using standard techniques (biotinylated ErbB2 primary antibody and streptavidin-800CW), followed by the addition of Plasmon-Fluor-800CW (Figure 78). Figure 78 shows confocal laser scanning microscopy (CLSM) images of breast cancer cells (SK-BR-3) examined with conventional Fluor (800CW, top row) and Plasmon-Fluor-800CW (bottom row) at various concentrations of ErbB2 primary antibody. The scale bar corresponds to 10 μm. ErbB2 primary antibody (1 mg / ml) was diluted to various concentrations and then incubated with the cells. SEM images revealed a uniform distribution of Plasmon-Fluor on the cell membrane [Figure 79(A-C)]. Figure 79A shows bright-field microscopy images of SK-BR-3 cells before (top) and after (bottom) labeling with Plasmon-Fluor-800CW. SEM images of conventional Fluor-labeled SK-BR-3 cells (Figure 79B) and Plasmon-Fluor-800CW-labeled SK-BR-3 cells (Figure 79C), with the inset showing the uniform distribution of Plasmon-Fluor on the cell membrane. Confocal laser scanning microscopy (CLSM) images of the cells revealed that the addition of Plasmon-Fluor (20 pM) resulted in up to a 100-fold higher fluorescence signal (after background subtraction) [Figures 78, 80, 81(A-B), and 82], and ErBb2 receptor expression could be imaged even at a 100,000-fold dilution of the primary antibody (10 ng / ml) [Figures 78, 81(A-B)]. Figure 80 shows a plot of the fluorescence intensity of SK-BR-3 cells stained with conventional Fluor and Plasmon-Fluor-800CW. Error bars represent the standard deviation (from three different locations). Cells are sequentially labeled using conventional immunocytochemistry procedures (biotinylated primary antibody and streptavidin-Fluor (800CW) at various dilutions of the ERbB2 primary antibody.Confocal laser scanning microscopy (CLSM) images of ErbB2-stained breast cancer cells (SK-BR-3) obtained using Plasmon-Fluor-800CW (see Figure 81A) followed by the addition of Plasmon-Fluor-800CW (Figure 81B). The scale bar corresponds to 15 μm. Figure 82 shows fluorescence mapping of SK-BR-3 cells cultured in a 6-well plate. The cells are probed with a conventional fluorophore (top) and then with Plasmon-Fluor-800CW (bottom). The scale bar corresponds to 1 cm. In stark contrast, fluorescent signals could only be imaged at a dilution 100-fold (typical dilution; 10 μg / ml) of the primary antibody using conventional fluorophores (Figure 78). These results demonstrate not only the applicability of Plasmon-Fluor in significantly reducing the amount of antibody required (and the resulting cost) for ICC / IF, but also the ability to image low-abundance biomarkers on the cell surface using Plasmon-Fluor. [Example]

[0268] Plasmon-Fluor enhanced flow cytometry measurements Flow cytometry is widely used in cell analysis to measure the expression and relative abundance of specific analytes on or within cells at rates of thousands of cells per second or more (Figure 83). Figure 83 is a schematic diagram showing flow cytometry of ErbB2-stained SK-BR-3 cells examined by conventional Fluor (680LT) and then Plasmon-Fluor-680LT. However, flow cytometry still faces significant challenges in terms of the signal-to-noise ratio of fluorescence due to the high velocity of the target species as they cross the laser focal spot, limiting the time available for reading fluorescence. Again, background fluorescence (autofluorescence) from cells poses difficulties in accurately depicting small changes in the expression levels of intracellular and extracellular targets.

[0269] To test the ability of Plasmon-Fluor to enhance the signal-to-noise ratio in flow cytometry-based cell analysis (Figure 83), SK-BR-3 cell suspensions were incubated with the ErbB2 primary antibody, streptavidin-680LT, and then Plasmon-Fluor-680LT was added. Subsequently, labeled cells were harvested by mild centrifugation (1000 rpm), with concomitant removal of unbound Plasmon-Fluor. To match the excitation laser with the fluorophore emission, AuNRs with an LSPR wavelength of approximately 647 nm were used as nanostructures to generate Plasmon-Fluor-680LT [Figure 84(A-B)]. Figure 84(A-B) shows the TEM image and extinction spectrum of 680LT. Specific binding of Plasmon-Fluor-680LT caused a significant color change in the cell pellet [Figure 85(A-B)]. Figure 85A illustrates photographs showing the color change of SK-BR-3 cells (top: pellet; bottom: suspension) after labeling with Plasmon-Fluor-680LT. Figure 85B shows the visible-NIR extinction spectra of Plasmon-Fluor-680LT-labeled SK-BR-3 cell suspensions at various dilutions of ErbB2 primary antibody. The presence of Plasmon-Fluor-680LT on the cell surface did not alter the forward or side scattering intensity (Figure 86), indicating that cell size and granularity / complexity remained virtually unchanged after Plasmon-Fluor-680LT binding. Figure 86 shows pseudocolor plots of side and forward scattering (including an example of a gating strategy to include single cells) of SK-BR-3 cells before (left) and after (right) labeling with Plasmon-Fluor-680LT, demonstrating no obvious change in their size profile. Flow cytograms of fluorescence versus forward scatter (vertically offset for clarity) of SK-BR-3 cells revealed a clearer separation of cell populations stained with Plasmon-Fluor-680LT compared to that obtained with conventional fluorophores (Figure 87).Figure 87 shows a flow contour plot (including outliers) of fluorescence versus forward scatter (vertically offset for clarity) of SK-BR-3 cells probed with various concentrations of ErbB2 primary antibody (red: control without primary antibody; blue: cells treated with various dilutions of primary antibody). Cells were stained by adding conventional Fluor (680LT, left plot) followed by Plasmon-Fluor-680LT (right plot). Histograms of the cellular fluorescence signal revealed up to 60-fold higher intensity (subtracting background) using Plasmon-Fluor-680LT compared to its conventional counterpart (Figure 88). Figure 88 shows the fluorescence versus forward scatter plots ... 3 Fluorescence histograms of SK-BR-3 cells probed with the addition of 100 μg ... **** p<0.0001 (two-tailed unpaired t-test with Welch's correction). Fluorescence histograms revealed that cell surface ErbB2 expression could be detected even with a 200,000-fold dilution of primary antibody (5 ng / ml) using Plasmon-Fluor-680LT labeling (Figure 89, Figure 90). Figure 89 shows histograms showing the fluorescence intensity of SK-BR-3 cells before (top) and after (bottom) the addition of Plasmon-Fluor-680LT. Red: no primary antibody; Blue: 2x10 5 10x dilution; Orange: 10 5 10x dilution; pale green: 10 4 fold dilution; green: 10 3 10-fold dilution of the stock solution provided by the supplier; 2 Figure 90 is a plot showing the mean fluorescence intensity obtained from flow cytometry at various primary antibody concentrations. In contrast, conventional labeling requires antibody dilutions less than 1000-fold (i.e., concentrations >0.5 μg / ml) to ensure a detectable increase in fluorescent signal compared to background (blank) (Figures 89 and 90).

[0270] To further validate the performance of Plasmon-Fluor in delineating cell populations with small differences in surface receptor expression levels, we used bone marrow-derived dendritic cells (BMDCs) as a model system in which receptor surface expression can be modulated using immunogenic stimuli. After exposure to immunogenic stimuli, dendritic cells undergo activation and maturation, which leads to cytokine secretion and upregulation of maturation markers such as CD40, CD80, CD86, MHC I, and MHC II. Here, BMDCs were isolated from 6- to 8-week-old C57BL / 6 mice, and lipopolysaccharide (LPS) was used as an immunogenic stimulus, causing CD80 upregulation and cytokine release in a dose-dependent manner. Subsequently, the cells were fixed and treated with a biotinylated CD80 antibody. Finally, BMDCs were examined with a conventional fluorophore (680LT) followed by Plasmon-Fluor-680LT, and fluorescence levels were compared using a flow cytometer (Figure 9). Figure 91 is a schematic diagram showing bone marrow-derived dendritic cells (BMDCs) treated with an immunostimulatory agent [lipopolysaccharide (LPS)]. Small changes in maturation marker (CD80) expression after stimulation were detected by immunofluorescence staining followed by addition of Plasmon-Fluor-680LT. Figure 92 shows two schemes for using Plasmon-Fluor-labeled antibodies to label target antigens on cells.

[0271] Fluorescence intensity distribution histograms corresponding to naive (control) and LPS (0.05 μg / ml)-stimulated BMDCs obtained using conventional Fluor (680LT) and Plasmon-Fluor-680LT are shown in Figure 93 and Figure 94, respectively. Notably, BMDCs stained with Plasmon-Fluor showed significant differences in fluorescence between activated (blue) and naive (red) cell populations [Figure 93, Figure 94, and Figure 95(A-B)]. Figure 95(A-B) shows pseudocolor plots showing side scatter versus CD80 fluorescence of BMDC populations without LPS stimulation (left: naive) and after treatment with 0.05 μg / ml LPS (right) using conventional immunofluorescence staining (Figure 95A) and Plasmon-Fluor-680LT (Figure 95B). Further investigation of dose-dependent stimulation of BMDCs with LPS (0–0.05 μg / ml) revealed that, using Plasmon-Fluor-680LT, a rapid increase in mean fluorescence intensity was observed, followed by a plateau at higher LPS doses [Figure 96, Figure 97(A–B)], indicating increased CD80 expression. Figure 96 shows a plot of the mean fluorescence intensity (corresponding to the CD80 expression level) of BMDCs after stimulation with various amounts of LPS. However, BMDCs stained with conventional fluorophores showed shallower fluorescence with increasing LPS dose, which was obscured by the high fluorescence background [Figure 96 and Figure 97(A–B)]. Figure 97(A–B) shows a plot of the mean fluorescence (corresponding to the CD80 expression level) of BMDCs after stimulation with various amounts of LPS. BMDCs were examined using conventional immunofluorescence staining (Figure 97A) followed by Plasmon-Fluor-680LT (Figure 97B). Furthermore, the secretion levels of pro-inflammatory cytokines (TNF-α and IL-12) tended to increase with increasing LPS concentrations (Figures 98 and 99). Figure 98 shows the secretion levels of pro-inflammatory cytokines (TNF-α and IL-12), confirming the dose-dependent activation and maturation of BMDCs. Figure 99 shows the individual ELISA data points (absorbance and concentration), mean concentration, and standard deviation corresponding to the inflammatory cytokines secreted after LPS stimulation.This further confirmed the specificity and accuracy of Plasmon-Fluor in discerning dose-dependent activation and maturation of BMDCs and subtle changes in cell surface maturation markers. [Example]

[0272] Synthesis of AuNR for enhancing 800CW and 680LT: AuNR-760 (LSPR wavelength approximately 760 nm), suitable for enhancing 800 CW, was prepared by a seed-mediated method. Au seeds were synthesized by adding 0.6 ml of ice-cold NaBH4 solution (10 mM) (Sigma-Aldrich, Inc., 71321) to a solution containing 0.25 ml HAuCl4 (10 mM) (Sigma-Aldrich, Inc., 520918) and 9.75 ml CTAB (0.1 M) (Sigma-Aldrich, Inc., H5882) under vigorous stirring at room temperature for 10 minutes. The color of the solution changed from yellow to brown, indicating the formation of Au seeds. For the synthesis of AuNR, the growth solution was prepared by sequentially adding aqueous HAuCl4 (0.01 M, 2 ml), CTAB (0.1 M, 38 ml), AgNO3 (0.01 M, 0.5 ml, Sigma-Aldrich, Inc., 204390), HCl (1 M, 0.8 ml, Sigma-Aldrich, Inc., H9892), and ascorbic acid (0.1 M, 0.22 ml, Sigma-Aldrich, Inc., A92902), followed by gentle inversion to homogenize the solution. The AgNO3 and HCl volume ratios can be varied to obtain the correct wavelength. Subsequently, 5 μl of seed solution was added to the above growth solution and allowed to stand in the dark for 24 hours. The AuNR solution was centrifuged at 7000 rpm for 40 minutes, the supernatant was removed, and the AuNR was redispersed in nanopure water to obtain a final peak extinction of approximately 2.0. For AuNR-647 (LSPR wavelength approximately 647 nm), which is suitable for enhancing 680LT, the growth solution contained HAuCl4 (0.01 M, 2 ml), CTAB (0.1 M, 38 ml), AgNO3 (0.01 M, 0.2 ml, although this value may vary), and ascorbic acid (0.1 M, 0.32 ml). [Example]

[0273] Synthesis of AuNR@Ag for Cy3: AuNRs with an LSPR wavelength of approximately 711 nm were used as the core for synthesizing AuNR@Ag nanostructures. Specifically, 3 ml of 711 nm AuNRs (peak extinction approximately 4) were incubated with 8 ml of CTAC (20 mM) at 60 °C for 20 minutes under stirring. Next, 8 ml of AgNO3 (4 mM), 4 ml of CTAC (20 mM), and 0.8 ml of ascorbic acid (0.1 M) were sequentially added, and the mixture was incubated at 60 °C for 4 hours under magnetic stirring to form AuNR@Ag nanocuboids. Finally, the AuNR@Ag nanocuboid solution was centrifuged at 6000 rpm, and the nanocuboids were redispersed in nanopure water. [Example]

[0274] Conjugation procedure of fluorescently labeled functionalized layer Plasmon Fluor: Biotin and 800CW were sequentially conjugated to BSA using EDC / NHS chemistry. In pH 7-9 buffers, the NHS ester reacts well with primary amino groups (-NH2) via nucleophilic attack, forming an amide bond and releasing NHS. Specifically, 2 mg of NHS-activated biotin (NHS-PEG4-biotin, Thermo Scientific Inc., Product No. 21329) was added to 2.2 ml of BSA (Sigma-Aldrich, Inc., A7030) solution (5 mg / ml in 1x PBS). The mixture was incubated at room temperature (approximately 22°C) for 1 hour to complete the reaction. Excess NHS-PEG4-biotin was removed from the solution using a desalting column (5 mL, 7000 MWCO, Thermo Scientific Inc., Product No. 21329) pre-equilibrated with 1x PBS. Next, 800CW was conjugated to BSA-biotin. 0.1 ml of 1 M potassium phosphate buffer (K2HPO4, pH = 9) was added to 1 ml of the purified BSA-biotin solution to raise the pH. Next, 25 μl of 4 mg / ml NHS-800CW (LI-COR, 929-70020) was added to this mixture, and the solution was incubated at 23 °C for 2.5 hours. Free NHS-800CW was then separated from the conjugate using a Zeba desalting column pre-equilibrated with nanopure water. BSA-biotin-680LT and BSA-biotin-Cy3 were prepared using a similar method, except for the fluorophore change. [Example]

[0275] Synthesis of plasmonic fluorescein-based fluorescently labeled functional layers To create a plasmon-fluor with high fluorescence enhancement efficiency, it is crucial to select plasmon nanostructures that are "on-resonant" with a given fluorophore. For 800 CW, AuNR-760 (length and diameter: 83 and 24 nm, respectively) were used as nanostructures. 1 μl of MPTMS (Sigma Aldrich, Inc., 175617) was added to 1 ml of AuNR with an extinction ratio of approximately 2.0, and the mixture was shaken for 1 hour to form an interfacial layer on the AuNR. The MPTMS-modified AuNR was further mixed with various volumes of APTMS (Sigma Aldrich, Inc., 281778) and TMPS (Sigma Aldrich, Inc., 662275) (0.5 μl to 2 μl) to form a polymer spacer layer on the AuNR. Finally, the AuNR / polymer solution was centrifuged twice, for 10 minutes at 6000 rpm each time, to remove free monomers. After a second centrifugation, the AuNR / polymer was concentrated to a final volume of 10 μl.

[0276] Next, BSA-biotin-800CW conjugate was coated around the AuNR / polymer. Specifically, 100 μl of BSA-biotin-800CW (approximately 4 mg / ml) was mixed with 1 μl of 20 mg / ml citric acid (Alfa Aesar, 36664) to lower the pH. The concentrated AuNR / polymer solution was then added to this mixture and sonicated for 20 minutes in the dark. The nanostructures were then harvested using mild centrifugation (5000 rpm, 3 minutes). The AuNRs were then incubated with 0.5 ml of BSA-biotin-800CW (approximately 0.4 mg / ml, pH = 10) for 3 days at 4°C in the dark. Finally, the nanostructures were washed four times by centrifugation at 6000 rpm using nanopure water (pH = 10). After the final washing step, the particles were resuspended in 1% BSA (buffered with 1x PBS).

[0277] Material Characterization: Transmission electron microscope (TEM) images were obtained using a JEOL JEM-2100F field emission (FE) instrument. A drop of aqueous solution was dried on a carbon-coated grid, which was then rendered hydrophilic by glow discharge. SEM images were obtained using an FEI Nova 2300 field emission scanning electron microscope at an accelerating voltage of 10 kV. AFM imaging was performed on a Dimension 3000 using a silicon cantilever with a nominal spring constant of 40 N / m in optical trapping mode. Extinction spectra of the plasmonic nanostructures were obtained using a Shimadzu UV-1800 spectrophotometer. Fluorescence lifetimes were measured using time-correlated single-photon counting (TCSPC performed on a Fluorolog-3, Horiba Jobin Yvon GmbH) with a 740 nm excitation source NanoLed® (1 MHz impulse repetition rate) at 90° to a PMT R928P detector (Hamamatsu Photonics KK, Japan). Unless otherwise stated, most fluorescence mapping was recorded using a LI-COR Odyssey Clx imaging system. Fluorescence signals from microbeads were read using a Luminex200 system. Cells were imaged using an Olympus FV1000 LSM confocal laser scanning microscope (785 nm excitation laser) under a 40x water immersion objective. Flow cytometry data were acquired using a Guava easyCyte.

[0278] Calculation of protein / biotin ratio: The BSA / biotin ratio was calculated by 4-hydroxyazobenzene-2-carboxylic acid (HABA) assay. Specifically, biotinylated BSA (0.4 mg / ml x 100 μl) was added to a mixture of HABA (Thermo Scientific Inc., 1854180) and avidin solution (900 μl, Thermo Scientific Inc., 21121). Due to its higher affinity for avidin, biotin displaced HABA from avidin, and the absorbance at 500 nm decreased proportionally. The change in absorbance was calculated using the following formula: ΔA 500=(0.9×A 500 )-A 500 B (1) (In the formula, A 500 and A 500 B represents the absorbance of HABA / avidin before and after the addition of biotinylated BSA, respectively. A correction factor (0.9) was used to adjust for the dilution of the HABA / avidin solution due to the addition of biotinylated BSA. The concentration of the sample was calculated using Beer's law: A λ =ε λ bC (2) [In the formula, A λ is the wavelength λ nm (ΔA 500 ) is the absorbance of the sample at λ is the extinction coefficient at wavelength λ nm (34,000 M -1 cm -1 where a represents the cell path length (1 cm, using a quartz cuvette), b is the cell path length (1 cm, using a quartz cuvette), and C is the sample concentration. Using this formula, the biotin concentration can be calculated as:

number

number

[0279] Fluorescence lifetime measurements: Fluorescence lifetimes (FLTs) were measured using time-correlated single-photon counting (TCSPC performed on Fluorolog-3, Horiba Jobin Yvon GmbH) with a 740 nm excitation source NanoLed® (1 MHz impulse repetition rate) at 90° to a PMT R928P detector (Hamamatsu Photonics KK, Japan). A 20 nm bandpass was used, and the detector was set to 800 nm. Data were collected until the peak signal reached 2,000 counts. Details of this system have been published in a previous study. The instrument response function was obtained using Rayleigh scattering of Ludox-40 (0.05% in MQ water; Sigma-Aldrich, Inc.) in an acrylic clear cuvette with emission at 740 nm. Lifetime values ​​were calculated as follows:

number

[0280] [Table 8]

[0281] Quantum Yield Calculation: The radiative and non-radiative decay rates can be calculated from the measured lifetime and quantum yield of 800CW (conjugated to BSA):

number

number

[0282] Estimation of the amount of 800CW absorbed by an exemplary Plasmon-Fluor-800CW: To estimate the amount of fluorophore on the AuNR, the amount of BSA (-conjugate) was first estimated using a bicinchoninic acid assay (BCA assay). Since the dye-to-protein ratio was determined to be 1.2 (see above), the concentration of 800CW can be calculated from the amount of BSA. A micro BCA protein assay kit (Thermo Scientific Inc., product number 23235, lot number QG218473A) was used for this study. Specifically, the BCA working reagent was prepared by mixing 2.5 ml of reagent MA, 2.4 ml of reagent MB, and 0.1 ml of reagent MC. 150 μl of BSA standard (0-40 μg / ml) or Plasmon-Fluor-800CW (extinction approximately 4.6) was mixed with 150 μl of working reagent, and the mixture was incubated at 60°C for 1 hour. The absorbance at 562 nm was measured using a plate reader to obtain a BSA standard curve. The concentration of BSA absorbed around Plasmon-Fluor-800CW was calculated to be 6.2 μg / ml based on the standard curve. Therefore, Plasmon-Fluor-800CW, with an extinction of approximately 0.63, contains approximately 0.9 μg / ml of BSA (approximately 13.5 nM) and approximately 16.2 nM of 800CW.

[0283] The extinction coefficient of AuNR (length ∼83 nm and diameter ∼24 nm) is ε ≈ 8.27 × 10 9 (LM -1 cm -1 ) was calculated. The molar concentration of AuNR corresponding to an optical extinction of 0.63 can be derived from Beer's law, which is calculated to be 76.2 pM. Therefore, the number of 800CW(n) on a single AuNR is as follows:

number

[0284] Exemplary conditions for fluorescence enhancement of 800CW-streptavidin using AuNR-Plasmon-Fluor-800CW and AuNP-Plasmon-Fluor-800CW The experimental procedure used in this study (the results of which are disclosed elsewhere herein) is shown in Figure 34, and the data are shown in Figure 5 (A-B). Specifically, BSA-biotin was first immobilized to the bottom of the wells by incubating a plastic 96-well plate with 50 ng / ml BSA-biotin (in 1x PBS) at room temperature for 15 minutes. The plate was washed three times with PBST (0.05% Tween 20 in 1x PBS) and then blocked with Odyssey® Blocking Buffer (PBS) (LI-COR, P / N 927-40100). The BSA-biotin-coated wells were then incubated with 1 μg / ml streptavidin-800CW (in Odyssey® Blocking Buffer) for 10 minutes to allow specific binding of streptavidin to biotin. The plate was then washed three times with PBST and then incubated with approximately 76 pM Plasmon-Fluor-800CW (in 1% BSA). The plate was washed three more times with PBST to remove free Plasmon-Fluor. Finally, 200 μl of PBST was added to each well, and the fluorescence signal before and after the addition of Plasmon-Fluor was recorded using a LI-COR CLx fluorescence imager with the following scanning parameters: laser power approximately L2; resolution approximately 169 μm; channels: 800; height: 4 mm. This experiment was repeated four times independently, and the fluorescence intensity before and after the addition of Plasmon-Fluor-800CW was compared. The data were statistically significant, with a P value of 0.0044 ( ** P<0.01, calculated by two-tailed unpaired t-test with Welch's correction). [Example]

[0285] Exemplary conditions for human IL-6 ELISA: A human IL-6 DuoSet ELISA kit (R&D Systems, Inc., catalog number DY206, lot number P173353) was used, and the results are disclosed elsewhere herein. Specifically, a 96-well plate was first coated with a capture antibody (2 μg / ml in PBS) by overnight incubation at room temperature, and then blocked with 300 μl of reagent diluent (1× PBS containing 3% BSA, 0.2 μm filtered). After washing three times with PBST, 100 μl of serially diluted standard samples and patient serum samples (diluted 10-fold using reagent diluent) were added to various wells, and the plate was incubated at room temperature for 2 hours. The plates were then washed and incubated with biotinylated detection antibody (product number 840114, 50 ng / ml in reagent diluent) for 2 hours, washed again with PBST, and incubated with HRP-conjugated streptavidin (product number 893975, diluted 200-fold using reagent diluent) for 20 minutes. One hundred microliters of substrate solution (a 1:1 mixture of color reagent A (H2O2) and color reagent B (tetramethylbenzidine) (R&D Systems, Inc., catalog number DY999)) was added to each well, and after 20 minutes, the reaction was stopped by adding 50 μl of H2SO4 (2N) (R&D Systems, Inc., catalog number DY994). The optical density of each well was immediately determined using a microplate reader set at 450 nm. [Example]

[0286] Human IL-6 FLISA and p-FLISA: FLISA for human IL-6 was performed using a similar procedure to the above ELISA, except that HRP-labeled streptavidin was replaced with 800CW-labeled streptavidin (LI-COR P / N 926-32230, 50 ng / ml, 20 min). The plate was washed three times with PBST and then with nanopure water. For p-FLISA, Plasmon-Fluor-800CW was then added (extinction approximately 1), incubated for 1 hour, and the plate was washed three times with reagent diluent and then PBST. The plate was imaged using a LI-COR CLx fluorescence imager with the following scanning parameters: laser power approximately L2; resolution approximately 169 μm; channels: 800; height: 4 mm. Results from an independent experiment are shown in Figures 47 and 49, and Figure 100 (A–C). Figure 100 (A-C) shows plots of IL-6 dose-dependent fluorescence intensity from p-FLISA. In Figures 100A, 100B, and 100C, the data represent independent experiments performed on various days using different batches of Plasmon-Fluor-800CW. Error bars represent standard deviation (n≧2 replicates). [Example]

[0287] Plasmon-Fluor-enhanced Luminex bead-based assay: A mouse magnetic Luminex assay was purchased from R&D systems, Inc. (catalog number: LXSAMSM-03, lot number L126064) and customized to simultaneously detect mouse TNF-α and mouse IL-6. To begin, 50 μl of standard containing various concentrations of TNF-α and IL-6 (product number 984658) was mixed with 50 μl of diluted microbead cocktail (product number 894724) in a 96-well plate. This mixture was homogenized by horizontal shaking for 2 hours using a microplate orbital shaker (0.12-inch circumference) set at 800 rpm. The microbeads were then collected using a magnetic device (Millipore Sigma 40-285) designed to accommodate microplates, washed by removing the liquid, and filled with wash buffer (product number 895003). The washing process was repeated three times. Next, 50 μl of diluted biotin-antibody cocktail (product number 894666) was added to each well and incubated for 1 hour on a shaker at 800 rpm. The microbeads were washed three times again and then incubated with 100 ng / ml Cy3-streptavidin (in 3% BSA buffered with 1× PBS) at 800 rpm for 30 minutes. After washing three times, the microbeads were incubated with 50 μl of Plasmon-Fluor-Cy3 (quenching ∼5 at the LSPR maximum) at 800 rpm for 1 hour and washed three times each with 3% BSA and wash buffer. Finally, the microbeads were resuspended in 100 μl of wash buffer and incubated for 2 minutes at 800 rpm before reading. A Luminex200 instrument was used for fluorescence reading. Dual-mode fluorescence of the microbeads was observed with a Confocor II LSM system (Carl Zeiss-Evotec, Jena, Germany) using a 40x water-immersion objective. Results from independent experiments are shown in Figures 61 and 62, and Figures 101(A-B) and 102(A-B). Figure 101(A-B) shows a bead-based mouse TNF-α standard curve obtained after application of Plasmon-Fluor-Cy3.In Figures 101A and 101B, the data represent three independent experiments performed on various days using different batches of Plasmon-Fluor-Cy3. Error bars represent standard deviation (n=2 replicates). Figures 102 (A-B) show the bead-based mouse IL-6 standard curve obtained after applying Plasmon-Fluor-Cy3. In Figures 102A and 102B, the data represent three independent experiments performed on various days using different batches of Plasmon-Fluor-Cy3. Error bars represent standard deviation (n=2 replicates). [Example]

[0288] Plasmon-Fluor-Enhanced Human Kidney Biomarker Array The human kidney biomarker array kit was purchased from R&D systems, Inc. (catalog number ARY019, lot number 1311110). A urine sample from a patient with kidney disease (ID number 25, age 61, male) was used in this study. This study was approved by Washington University IRB201601082, "Nanotech Biomarkers for Renal Cancer Intervention: Clinical Validation and Utility." Informed consent was obtained from participants. A nitrocellulose membrane (product number 893967) was blocked by incubating with 2 ml of blocking buffer (product number 893573) in a 4-well multi-dish with gentle rocking for 1.5 hours. During the blocking process, a urine sample (150 μl) from patient with kidney disease (ID number 25) was diluted with 500 μl of blocking buffer and 850 μl of array buffer (product number 895876) for a total of 10-fold dilution. The diluted urine sample was mixed with 15 μl of reconstituted detection antibody cocktail (product number 893966), and the mixture was incubated at room temperature for 1 hour. The nitrocellulose membrane was removed from the blocking solution and incubated with the urine sample and biotinylated detection antibody mixture overnight at 4°C. The membrane was then washed with 20 ml of 1× Wash Buffer (product number 895003) for 10 minutes with gentle rocking, and this washing process was repeated two more times. The membrane was then incubated with 800CW-streptavidin (50 ng / ml in 1% BSA) for 30 minutes with gentle rocking, washed three times, and incubated with Plasmon-Fluor-800CW (extinction approximately 0.5) for an additional hour. Finally, the membrane was scanned. The membrane was then imaged using a LI-COR CLx imager at laser power L2 with a focal height of 0.5 mm and a resolution of 169 μm. Photographs of the protein arrays were taken using an iPhone 6 camera, and the images were analyzed using Image Studio Lite software to measure the median intensity of each spot (background subtracted). Results from an independent experiment are shown in Figure 103 (A-B).Figure 103 (A-B) shows a second independent experiment of a kidney biomarker array. Fluorescence intensity corresponding to the concentration of various urinary biomarkers (typical assay using conventional fluorophores) before (Figure 103A) and after (Figure 103B) the addition of Plasmon-Fluor-800CW. Error bars represent standard deviation (n=2 replicates). [Example]

[0289] Exemplary conditions for plasmon-fluor-enhanced human cytokine microarray: The effectiveness of Plasmon-Fluor was further tested using a 40-plex human cytokine microarray (RayBiotech, Inc., Catalog No. QAH-CYT-4), the results of which are disclosed elsewhere herein. To begin, the glass substrate of the microarray was blocked with 100 μL of sample diluent (Cat. No. QA-SDB) and then incubated with sample standard (Cat. No. QAH-CYT-4-STD) at room temperature with gentle rocking for 2 hours. The microarray was washed five times with 1× Wash Buffer I (Cat. No. AA-WB1-30ML) and then twice with 1× Wash Buffer II (Cat. No. AA-WB2-30ML). Next, 80 μL of reconstituted detection antibody cocktail was added to each well and incubated for an additional 2 hours with gentle rocking. After incubation, the washing step was repeated again as described above. Subsequently, 80 μl of 800CW-streptavidin (50 ng / ml in 1% BSA) was added to the array slide, incubated for 20 minutes, washed, and immersed in Plasmon-Fluor-800CW (extinction approximately 1) for 1 hour. The slide was scanned using a LI-COR CLx scanner with the following parameters: laser power approximately 3.5; resolution approximately 21 μm; channels: 800; height: 1.8 mm. [Example]

[0290] Plasmon-Fluor-Enhanced Immunocytochemistry / Immunofluorescence (ICC / IF): Human epithelial breast cancer cells SK-BR-3 [SKBR3][ATCC® HTB30™] were purchased from ATCC (Manassas, VA) and subcultured in McCoy's 5A medium containing 10% fetal bovine serum (FBS) and antibiotics (100 μg / ml penicillin and 100 μg / ml streptomycin) (Sigma-Aldrich, Inc., St. Louis, MO). Cells were grown in T-25 tissue culture flasks in a water-jacketed incubator at 37°C in a 5% CO2 humidified atmosphere. Once the cells reached 90% confluence, they were washed with PBS and detached from the flask bottom using a scraper. After centrifugation, the cells were resuspended in culture medium and seeded into 6-well plates overnight to allow attachment to the plate bottom. Cells were then fixed with 3.7% formaldehyde (in 1x PBS) for 30 minutes, washed three times with 1x PBS, and blocked with 3% BSA for 1 hour. ErbB2 primary antibody (anti-human HER-2 / biotin, eBioscience, clone 2G11, REF# BMS120BT, lot# 186281000) was diluted with 1% BSA and incubated with SK-BR-3 cells for 1.5 hours. The cells were then washed three times, incubated with 800CW-streptavidin (1 μg / ml in 1% BSA) for 30 minutes, washed three more times, and examined with Plasmon-Fluor-800CW (extinction approximately 0.3). Finally, cells were imaged using an Olympus FV1000 LSM confocal laser scanning microscope (785 nm excitation laser) under a 40x water-immersion objective. The results from an independent experiment are shown in Figure 78, as well as Figures 104(A-B) and 105(A-B). Figure 104(A-B) shows a second independent immunocytochemistry experiment. Cells were sequentially labeled with biotinylated primary antibody and streptavidin-fluor (800CW) at various dilutions of ERbB2 primary antibody.Confocal laser scanning microscopy (CLSM) images of ErbB2-stained breast cancer cells (SK-BR-3) obtained using a conventional immunocytochemistry procedure [cells are sequentially labeled with biotinylated primary antibody and streptavidin-Fluor (800CW) (see Figure 105A)] at various dilutions of the ERbB2 primary antibody, followed by the addition of Plasmon-Fluor-800CW (Figure 105B). The scale bar corresponds to 15 μm. Figures 105 (A-B) show a third independent immunocytochemistry experiment. Confocal laser scanning microscopy (CLSM) images of ErbB2-stained breast cancer cells (SK-BR-3) obtained using a conventional immunocytochemistry procedure [cells are sequentially labeled with biotinylated primary antibody and streptavidin-Fluor (800CW) (see Figure 105A)] at various dilutions of the ERbB2 primary antibody, followed by the addition of Plasmon-Fluor-800CW (Figure 105B). The scale bar corresponds to 15 μm. [Example]

[0291] SK-BR-3 flow cytometry assay: SK-BR-3 cells were grown and harvested using the method described above. The cells were centrifuged at 1000 rpm for 10 minutes, the culture medium removed, and then fixed using 3.7% formaldehyde in 1x PBS for 30 minutes. The cell suspension was centrifuged again to remove free formaldehyde, and the cells were then blocked overnight with 3% BSA. Various amounts of ErbB2 primary antibody were then added to the cell suspension, and the mixture was incubated for 1 hour with gentle shaking. The cells were centrifuged at 1000 rpm, washed once with 1x PBS to remove free antibody, incubated with streptavidin-680LT (LI-COR:P / N926-6803; 1 μg / ml in 1% BSA) for 1 hour, washed twice more, and incubated with Plasmon-Fluor-680LT (extinction approximately 2.0) for 1 hour. Finally, 5,000 cells were analyzed by Guava easyCyte to obtain a fluorescence signal [RED-R channel (excitation laser: 642 nm; filter: 662 / 15 nm)] combined with forward scatter (FSC) and side scatter (SSC). Results from an independent experiment are shown in Figures 89 and 90, as well as Figures 106(A-B) and 107(A-B). Figure 106(A-B) shows a second independent SK-BR-3 flow cytometry experiment. Figure 106A is a histogram showing the fluorescence of SK-BR-3 cells before (top) and after (bottom) the addition of Plasmon-Fluor-680LT. Red: no primary antibody; blue: 2x10 5 10x dilution; Orange: 10 5 10x dilution; pale green: 10 4 fold dilution; green: 10 3 10-fold dilution of the stock solution provided by the supplier; 2106B is a plot showing the mean fluorescence intensity obtained from flow cytometry at various primary antibody concentrations. Figure 107 (A-B) shows three independent SK-BR-3 flow cytometry experiments. Figure 107A is a histogram showing the fluorescence intensity of SK-BR-3 cells before (top) and after (bottom) the addition of Plasmon-Fluor-680LT. Red: no primary antibody; Blue: 2x10 5 10x dilution; Orange: 10 5 10x dilution; pale green: 10 4 fold dilution; green: 10 3 10-fold dilution of the stock solution provided by the supplier; 2 107B is a plot showing the mean fluorescence intensity obtained from flow cytometry at various primary antibody concentrations. [Example]

[0292] BMDC isolation and flow cytometry measurement: Five- to six-week-old female C57BL / 6 (H-2b) mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Mice were maintained under pathogen-free conditions. All experiments using mice were performed in accordance with laboratory animal protocols approved by the Institutional Animal Review Committee of Washington University (School of Medicine, St. Louis). Mice were euthanized using CO2 asphyxiation and cervical dislocation. Euthanized mice were placed in 70% (v / v) ethanol for 1 minute. Both femurs and tibias were separated, and muscle attachments were carefully removed using a gauge pad. Both ends of the bones were cut with scissors, and the bone marrow was centrifuged at 1000 rpm for 10 seconds in a compatible centrifuge tube (a 0.6 ml tube with a slot inserted into a 1.5 ml tube). The pellet was resuspended in RPMI 1640 medium by vigorous pipetting. The cells were passed through a 70 μm cell strainer to prepare a single cell suspension. After one wash (1200 rpm, 5 min), red blood cells were depleted using RBC lysis buffer (Sigma-Aldrich, Inc.). Bone marrow cells were harvested and lysed in 10% heat-inactivated FBS, 50 IU mL -1 Penicillin, 50 μg mL -1 The cells were cultured in a 100 mm Petri dish containing 10 mL of RPMI medium supplemented with streptomycin and 20 ng mL of mouse recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF, R&D Systems, Inc., MN, USA). 6 BMDCs were cultured in 6-well plates and stimulated for 24 hours by adding 1 ml of various concentrations of LPS (0.5 μg / ml, 0.2 μg / ml, 0.1 μg / ml, 0.05 μg / ml, 0.01 μg / ml, and 0 μg / ml). Cells were harvested using a cell scraper for further staining and flow cytometry analysis.

[0293] CD80 overexpression on the cell surface was examined using conventional fluorophores followed by Plasmon-Fluor-680LT. Specifically, stimulated BMDCs were washed once with 1x PBS to remove the culture medium (centrifugation at 2000 rpm for 5 minutes) and fixed with 10% neutral-buffered formalin for 20 minutes. The cells were then washed (2000 rpm for 5 minutes) and blocked with 3% BSA overnight at 4°C. Next, biotinylated CD80 primary antibody [anti-Mo CD80 / biotin (Invitrogen, ref. 13-0801-82, clone 16-10A1, lot no. 1934784)] was added to the BMDC suspension to a final antibody concentration of 100 ng / ml, and the mixture was incubated for 1 hour. BMDCs were washed once (2000 rpm for 5 min) and then incubated with 1 μg / ml streptavidin-680LT (in 1% BSA) for 40 min. Finally, cells were washed twice more and incubated with Plasmon-Fluor-680LT (extinction ∼2) for 1 h, followed by one more wash to remove unbound Plasmon-Fluor-680LT. 10,000 cells were analyzed using a Guava easyCyte to obtain a fluorescence signal [RED-R channel (excitation laser: 642 nm; filter: 662 / 15 nm)] combined with forward scatter (FSC) and side scatter (SSC). Results from an independent experiment are shown in Figures 108(A–C) and 109(A–C). Figures 108(A–C) show a second independent flow cytometry measurement of BMDC maturation markers probed by Plasmon-Fluor-680LT. Fluorescence intensity distributions corresponding to naive BMDCs (control) and LPS-stimulated BMDCs obtained using conventional Fluor (680LT) (Figure 108A) and Plasmon-Fluor-680LT (Figure 108B). (Figure 108C) Plot showing the mean fluorescence intensity (corresponding to the expression level of CD80) of BMDCs after stimulation with various amounts of LPS. Figure 109 (A-C) shows a third independent flow cytometry measurement of BMDC maturation markers examined by Plasmon-Fluor-680LT.Fluorescence intensity distributions corresponding to naive BMDCs (control) and LPS-stimulated BMDCs obtained using conventional Fluor (680LT) (Figure 109A) and Plasmon-Fluor-680LT (Figure 109B). Figure 109C is a plot showing the mean fluorescence intensity (corresponding to the expression level of CD80) of BMDCs after stimulation with various amounts of LPS.

[0294] Statistics: A two-tailed unpaired t-test with Welch's correction was used to analyze statistical differences between two groups. A one-way ANOVA with post-hoc Tukey's honest significance test was used to analyze statistical differences between more than two groups. Statistical significance of the data was calculated with a CI of 95% (p<0.05). All values ​​are expressed as mean ± standard deviation. GraphPad Prism 6 (San Diego, CA, USA) was used for all statistical analyses. A four-parameter logistic (4PL) fit or polynomial fit was used to calculate the detection limit for the bioassay standard curve. The detection limit is defined as the analyte concentration corresponding to the mean fluorescence intensity of the blank plus three times its standard deviation (mean + 3σ). Origin 2016 (Northampton, MA, USA) was used to calculate the detection limit.

Claims

1. 1. A method of detecting an analyte using an assay, comprising: adding a fluorescent nanoconstruct to the assay to generate a fluorescent signal; and Detecting the analyte by analyzing the fluorescent signal Including; The fluorescent nanoconstructs: a plasmonic nanostructure having at least one localized surface plasmon resonance wavelength (λLSPR); at least one spacer coating on the plasmonic nanostructure; at least one fluorescent agent having a maximum excitation wavelength (λ) conjugated onto the at least one spacer coating; at least one functional layer coated on the at least one spacer coating; and At least one biorecognition element conjugated to at least one spacer coating or at least one functional group layer. wherein at least one biorecognition element is targeted to an analyte; a spacer coating on the nanostructures, (1) when the fluorescent agent is directly conjugated to the spacer coating, it has a thickness between 1 and 10 nm; (2) when the fluorescent agent is conjugated to the functional group layer, it has a thickness between 0.5 and 5 nm; the fluorescent nanoconstruct has a fluorescence intensity at least 500 times greater than the fluorescence intensity of the at least one fluorescent agent alone; The difference between at least one λLSPR and λEX is less than 75 nm; method.

2. Analyte detection: Exciting the fluorescent nanoconstruct with an appropriate excitation wavelength; and Detecting the emitted light wherein the amount of light detected is proportional to the concentration of the analyte; The method of claim 1.

3. The assay: (i) capture of target antigen by immobilized antibody; (ii) binding of a biotinylated detection antibody to the target antigen, yielding a biotinylated detection antibody-target antigen; and (iii) Binding of fluorescently labeled streptavidin to the target antigen, yielding a biotinylated detection antibody-target antigen. The method of claim 1 , comprising:

4. 4. The method of claim 3, further comprising adding a fluorescent nanoconstruct to the assay after step (iii) to generate a fluorescent signal.

5. The method of claim 1, wherein the assay is performed in a plate.

6. The method of claim 1 , wherein the assay is a protein microarray.

7. The method of claim 1 , wherein the assay is an immunomicroarray.

8. 2. The method of claim 1, wherein the assay comprises an immune targeting-based assay selected from the group consisting of FLISA, FACS, flow cytometry, Western blot, protein microarray, bead-based multiplex immunoassay, immunohistochemistry, lateral flow assay, microfluidics, ELISPOT, fluorescence microscopy, FLIM, dot blot, single-cell Western, in-cell Western, competitive immunoassay, digital immunoassay, ImmunoCAP assay, simple ELLA assay of proteins, and combinations thereof.

9. 10. The method of claim 1, wherein the assay comprises a nucleic acid-based assay selected from the group consisting of Northern blot, microarray, next generation sequencing, RNA-seq, FISH, EMSA, and combinations thereof.

10. The method of claim 1 , wherein the at least one fluorescent agent comprises at least about five fluorescent agents.

11. 10. The method of claim 1, wherein the plasmonic nanostructure is selected from the group consisting of nanorods, nanocubes, nanospheres, bimetallic nanostructures, gold core silver shell nanocuboids, nanotubes, gold nanorods, silver nanocubes, silver nanospheres, gold nanorod core-silver shell (AuNR@Ag) nanocuboids, nanostructures with sharp tips, nanostars, hollow nanostructures, nanocages, nanorattle, nanobipyramids, nanoplates, self-assembled nanostructures, nanoraspberries, and combinations thereof.

12. 10. The method of claim 1, wherein the spacer coating comprises a material selected from the group consisting of APTMS / APTES, TMPS, MPTMS, titanium oxide, polydopamine, polyoctopamine, silane and mixtures of silanes, PEG, metal oxides, polyelectrolyte bilayers, layer-by-layer assembled multilayers, zinc oxide, alumina, polysaccharides, silica, proteins, polypeptides, peptides, polyproline, DNA / RNA, PSS / PAH, chitosan, alginate, and combinations thereof.

13. The method of claim 1 , wherein the spacer coating is a rigid polymer network.

14. The method of claim 1 , wherein the spacer coating comprises a siloxane network.

15. 10. The method of claim 1, wherein the spacer coating comprises at least one polymer coating selected from the group consisting of mercaptosilane, aminopropylsilane, trimethoxypropylsilane, and combinations thereof.

16. 10. The method of claim 1, wherein the spacer coating comprises an initiating layer of mercaptosilane bound to the plasmonic nanostructures and further comprises a siloxane network added to the initiating layer selected from the group consisting of aminopropylsilane, trimethoxypropylsilane, and combinations thereof.

17. 10. The method of claim 1, wherein the functional layer comprises a material selected from the group consisting of bovine serum albumin, human serum albumin, hemoglobin, ovalbumin, lysozyme or a homolog thereof, albumin or a homolog thereof, polymers (homo-, diblock-, triblock-, random-, alternating-, and statistical-copolymers), amphoteric polymers, zwitterionic polymers, carboxybetaine, sulfobetaine, carboxybetaine polymers, sulfobetaine polymers, PEG / betaine polymers, polynucleotides, polysaccharides, polypeptides, and combinations thereof.

18. 10. The method of claim 1, wherein the at least one biorecognition element is selected from the group consisting of streptavidin, biotin, an antibody, a nucleic acid, and combinations thereof.

19. The method of claim 1 , wherein the functional layer comprises at least one reactive group to which at least one biorecognition element is covalently attached.

20. 2. The method of claim 1, wherein the functional layer comprises a mixture of proteins bound to at least one biotin (biotinylated proteins) and proteins not bound to biotin (native proteins), and the percentage of biotinylated proteins to native proteins can be between 0% and 100%.

21. 10. The method of claim 1, wherein the fluorescent agent is maintained within about 0.5 nm to about 10 nm of the surface of the plasmonic nanostructure.

22. 2. The method of claim 1, wherein λLSPR is about (mean value ±25 nm) 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, or 1000 nm.

23. The method of claim 1 , wherein at least one biorecognition element is attached to a flexible linker.

24. The method of claim 1 , wherein the functional layer substantially covers or encapsulates the at least one spacer coating.

25. 10. The method of claim 1, wherein the spacer coating substantially covers the plasmonic nanostructures or the spacer coating encapsulates the plasmonic nanostructures.

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