Ultra-high brightness fluorescent nanoconstructs as a versatile enhancer

Abstract

This invention provides fluorescent nanoconstructs with high fluorescence intensity, methods for constructing fluorescent nanoconstructs, and methods for detecting analytes. [Solution] A fluorescent nanoconstruct comprising a plasmon 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), wherein the fluorescence intensity is at least 500 times greater than that of the fluorescent agent alone.

Description

[Technical Field]

[0001] Cross-reference of 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 government support, under funding from the National Science Foundation (CBET1254399) and the National Institutes of Health (CA141521). The federal government has certain rights in this invention. [Background technology]

[0003] The field of this disclosure generally relates to plasmonic-fluor (PF) nanoconstructions, which are ultra-bright fluorescent nanoconstructions that can be used to enhance biological assays. Specifically, the present invention relates to the use of novel combinations comprising plasmon nanostructures, spacer layers, and fluorophores, which result in nanoconstructions spectrally similar to the fluorophores but at least 500 times brighter than the individual fluorophores alone. These ultra-bright fluorescent nanoconstructions can be conjugated to at least one biorecognition element and can be used to enhance the detection performance of various biological assays and processes and to improve their detection limits.

[0004] The associated concentrations of biomolecules or biomarkers for diseases such as cancer, heart disease, inflammation, and neurological disorders can range from μg / ml levels to sub-fg / ml, and some of these biomolecules or biomarkers may remain unspecified due to the lack of highly sensitive biological analytical techniques. It is also highly desirable to utilize small sample volumes for multiple detections in valuable biofluids such as inhaled air condensate, ocular fluid, cerebrospinal fluid, or serum from neonatal or small animal models, where further dilution of the sample is necessary to reduce the concentration. Fluorescence-based biological analytical methods are widely used as a foundation for biomedical science and clinical research for the detection, quantification, and imaging of a wide range of biological analytes. Several methods have been explored to improve the sensitivity of fluorescence immunoassays, including increasing antibody affinity, reducing background fluorescence, enhancing mass transfer, and increasing substrate surface area. However, the weak signal-to-noise ratio of weak fluorescence signals and their associated fluorescent labels remains a challenge, limiting the maximum sensitivity of current fluorescence-based assays.

[0005] Most conventional plasmon-enhanced fluorescence assays rely on the design of plasmon-active substrates by either depositing metal islands or adsorbing plasmon nanostructures. These methods, naturally, require the use of special surfaces, and potentially significant modifications to reading devices and bioassay protocols. Therefore, these methods are not readily applicable to a wide variety of systems or bioassays.

[0006] Attempts have been made to use particles in the solution phase in some plasmon-enhanced fluorescence assays, but these particles have problems: they are unstable, they bind very nonspecifically, causing unacceptable background signals, and most importantly, they provide poor fluorescence enhancement. The degree of fluorescence enhancement is typically less than 10-fold.

[0007] Fluorescent probes and fluorescence analysis techniques have been used in biomedical research not only as imaging tools to visualize the location and dynamics of cells and various subcellular species, as well as 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, transcriptome, and proteomics signatures of disease onset, progression, and response to treatment. However, "weak signals" have remained a persistent and recurring problem in a range of fluorescence-dependent detection and imaging techniques. Overcoming this fundamental challenge without using dedicated reagents, instruments, or significant 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 ultra-sensitive fluorescence immunoassays that can be widely adopted by most biolaboratories and clinical laboratories to detect low-abundance target species.

[0008] 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 intracellular, on the cell surface, in tissues, plates, beads, solutions, etc.)], but fluorescence is fundamentally limited by its weak signal. In plate-based assays, improved sensitivity of fluorescence detection is achieved using complex schemes such as poly-HRP, PCR-ELISA, avidin-biotin-complex (ABC) ELISA, and tyramide signal amplification (TSA). These are all more complex, more expensive, and generally have a poorer dynamic range than their alternative assay types. Complex techniques for very high detection sensitivity, such as digital ELISA (Quanterix Simoa System) or electrochemical luminescence (Meso Scale Discovery), each require dedicated substrates, instruments, and workflows.

[0009] Generally, when an assay uses an antibody or streptavidin labeled with HRP that catalyzes a reaction converting a substrate to either a luminescent species (such as in chemiluminescence assays) or a species that absorbs light of a certain wavelength (such as in ELISA), the performance of this assay can be improved by using a plasmon fluorophore conjugated to the antibody or streptavidin. Examples of such assays are membrane-based immunoassays such as ELISA (colorimetric and chemiluminescent), and Western blot (colorimetric and chemiluminescent).

[0010] Improving the signal-to-noise ratio of an assay without completely deviating from existing assay protocols would relax the stringent requirements of high-sensitivity and large-sized photodetectors, reduce implementation costs, eliminate discrepancies between laboratories and platforms, and potentially propel these technologies into point-of-care, in-field, and resource-limited environments. Various techniques, including multiple fluorophore labeling, rolling circle amplification, and enhancement by photonic crystals, have been introduced to improve the signal-to-noise ratio of fluorescence-based imaging and detection techniques. Despite the improvement in sensitivity, these technologies have not been widely adopted in research and clinical settings. Most of these technologies require significant modifications to existing practices, such as additional steps that greatly extend the overall operation time, dedicated expensive reading systems, unconventional data processing and analysis, or temperature-sensitive reagents that require tightly controlled transportation and storage conditions.

[0011] Enhanced emission of fluorophores in the vicinity of plasmonic nanostructures is due to enhanced electromagnetic fields (local excitation regions) at the surface of the plasmonic nanostructures and a reduction in fluorescence lifetime due to coupling between the excited fluorophores and the surface plasmons of the nanostructures. To date, various plasmonic substrates, such as metal nanoislands, have been shown to provide moderate fluorescence enhancement, but these plasmon-active surfaces require the use of glass slides deposited with metal nanostructures in place of prefabricated substrates, usually standard products or sometimes non-replaceable biological analysis platforms and bioimaging platforms. The need for dedicated substrates limits the consistency between platforms and laboratories and seamless integration with widely used biological analysis procedures, which significantly restricts its widespread application in biomedical research and clinical settings. Further challenges are posed to its widespread use due to unconventional bioconjugation procedures and poor stability of biomolecules (e.g., antibodies) immobilized on metal surfaces. The solution of solution-phase plasmon-enhanced fluorescence has generally been limited by poor fluorescence enhancement and unstable particles. Summary of the Invention Problems 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-platform assays such as enzyme-linked immunosorbent assays (ELISA), this technique allows researchers and clinicians to test a large number of biomarkers in parallel, enabling patient stratification and multifactorial disease monitoring using limited sample volume, thereby minimizing assay cost and time and allowing multiple individual biomarker assays. Furthermore, high-throughput biomarker profiling enables personalized medicine using comprehensive molecular fingerprinting of diseases, leading to improved diagnostic resolution between closely related disease phenotypes. Sensitivity and specificity for diagnosing kidney disease have been demonstrated to be significantly increased by combining urinary levels of multiple biomarkers compared to a single individual biomarker. However, despite the availability of various commercially available products, this multiplex method has lower sensitivity and a relatively high limit of detection (LOD) compared to ELISA, which hinders its broad application.

[0013] One technique for addressing the low sensitivity of various fluorescence assays is disclosed in U.S. Provisional Application 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 technique, plasmonic nanostructures are deposited on a polymer film surface, and the polymer film is then placed with the plasmonic structures facing downwards, aligning with the entire top surface of a plate or assay pre-coated with fluorophores. By placing the film in this orientation, the plasmonic nanostructures approach the fluorophores, resulting in superior fluorescence enhancement and a significant increase in sensitivity of the assay. Some assay techniques, while highly useful, are not compatible with this method (for example, this assay is not performed on flat, rigid surfaces such as microplates or glass slides). Therefore, there is a need to develop a method that is compatible with many more assay techniques while addressing each of these drawbacks. [Means for solving the problem]

[0014] In one embodiment, a fluorescent nanoconstruct is disclosed herein. This nanoconstruct generally comprises a plasmon 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). This fluorescent nanoconstruct has a fluorescence intensity at least 500 times greater than that of the fluorescent agent alone.

[0015] In another embodiment, a method for constructing fluorescent nanoconstructs is disclosed herein. This method generally includes coating a plasmon nanostructure with at least one spacer coating, optionally coating at least one spacer coating with a functional layer, conjugating a fluorescent agent to at least one spacer coating or the functional layer, and optionally conjugating a biorecognition element to at least one spacer coating or the functional layer.

[0016] In yet another embodiment, a method for detecting an analyte using an assay is disclosed herein. This method generally includes adding a fluorescent nanoconstruct to an assay to generate a fluorescent signal, and detecting the analyte by analyzing the fluorescent signal. [Brief explanation of the drawing]

[0017] [Figure 1] Figure 1 shows exemplary embodiments of the fluorescence intensities of conventional Cy3 and plasmon-fluer-Cy3 at various molar concentrations as described herein.

[0018] [Figure 2] Figure 2 shows exemplary embodiments of the fluorescence intensities of conventional Flür-800CW and plasmon-Flür-800CW at various molar concentrations as described herein.

[0019] [Figure 3] Figure 3 shows exemplary embodiments of the fluorescence intensities of conventional FITC and plasmon-fluer-FITC at various molar concentrations as described herein.

[0020] [Figure 4] Figure 4 shows an exemplary embodiment of the plasmon-fluer design according to this disclosure.

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

[0022] [Figure 6] Figures 6A and 6B illustrate exemplary embodiments of the importance of absorbance overlap of plasmon particles and the absorbance / excitation spectra of the conjugated dye according to this disclosure.

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

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

[0025] [Figure 9]Figure 9A is an exemplary embodiment of a fluorescence intensity histogram corresponding to the ErbB2 receptor obtained using the Fluor and fluorescent nanoconstructs according to the present disclosure. Figure 9B is an exemplary embodiment of a fluorescence intensity histogram according to the present disclosure.

[0026] [Figure 10] Figure 10 shows an exemplary embodiment of the plasmon-fluer design according to the present disclosure.

[0027] [Figure 11] Figure 11 shows an exemplary embodiment of an alternative plasmon-flur design according to the present disclosure.

[0028] [Figure 12] Figure 12 shows an exemplary embodiment of an additional alternative design for plasmon-fluer according to the present disclosure.

[0029] [Figure 13] Figure 13 shows another exemplary embodiment of the plasmon nanostructure according to this disclosure. The plasmon nanostructure (silver-coated gold nanorods) is embedded in a dielectric material matrix. The dielectric material matrix is ​​coated with a functional layer (blue cloud). A target agent (pink "y" shape, e.g., antibody) is conjugated to the functional layer.

[0030] [Figure 14] Figure 14 shows an exemplary embodiment of the extinction spectrum (excitation maximum = 784 nm) of a plasmon-fluer conjugated to IRDye 800CW according to the present disclosure.

[0031] [Figure 15] Figure 15 is an exemplary embodiment of the discrepancy between the LSPR maximum of plasmon-flur and the excitation maximum of IRDye 800CW as described herein.

[0032] [Figure 16]Figure 16 shows an exemplary embodiment of the extinction spectrum (excitation maximum = 550 nm) of a plasmon-flure (AuNR@Ag cuboid plasmon nanostructure) conjugated to Cy3 according to the present disclosure.

[0033] [Figure 17] Figure 17 is an exemplary embodiment of the discrepancy between the LSPR maxima of a plasmon-flure (AuNR@Ag cuboid plasmon nanostructure) and the excitation maxima of Cy3 as described herein.

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

[0035] [Figure 19] Figure 19 is an exemplary embodiment of a plot showing a standard curve (dose-dependent colorimetric signal) for a human NGAL ELISA that takes 280 minutes to complete, as described herein.

[0036] [Figure 20] Figure 20 is an exemplary embodiment of a plot showing the dose-dependent fluorescence intensity of human NGAL from p-FLISA taken within 20 minutes, as described herein.

[0037] [Figure 21] Figure 21 is an exemplary embodiment of NGAL concentrations in urine samples from renal patients and healthy volunteers, as determined using p-FLISA completed within 20 minutes, as described herein.

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

[0039] [Figure 23] Figure 23 shows an exemplary embodiment of a typical sandwich immunoassay method using biotinylated plasmon-flure according to the present disclosure.

[0040] [Figure 24] Figure 24 shows an exemplary embodiment of the enhancement of a common sandwich immunoassay method using streptavidin-conjugated plasmon-flua according to the present disclosure.

[0041] [Figure 25] Figure 25 shows an exemplary embodiment of a typical sandwich immunoassay method according to this disclosure, using a plasmon-flur conjugated with a secondary antibody, in which case the antibody conjugated with the plasmon-flur recognizes the detection antibody.

[0042] [Figure 26] Figure 26 shows an exemplary embodiment of a typical sandwich immunoassay method according to this disclosure, using a plasmon-flur conjugated with a primary antibody, in which case the antibody conjugated with the plasmon-flur recognizes the analyte.

[0043] [Figure 27] Figure 27A is an exemplary embodiment of a TEM image of a gold nanorod (AuNR) used as a nanostructure in a plasmon-fluer-800CW according to the present disclosure. Figure 27B is an exemplary embodiment of a finite-difference time-domain (FDTD) simulation showing the distribution of electric field intensity around the AuNR according to the present disclosure.

[0044] [Figure 28]Figure 28 is a schematic, exemplary embodiment illustrating the process involved in the formation of polymer spacers on the surface of the plasmon nanostructure AuNR according to this disclosure.

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

[0046] [Figure 30] Figure 30 shows exemplary embodiments of the UV-Vis spectrum of AuNR under various polymerization conditions according to this disclosure.

[0047] [Figure 31] Figure 31 is an exemplary embodiment of a plot showing the increase in the diameter of AuNR (twice the thickness of the polymer layer) under each polymerization condition measured from AFM images according to this disclosure.

[0048] [Figure 32] Figure 32 shows exemplary embodiments of the zeta potentials of AuNR, AuNR / MPTMS, AuNR / MPTMS / polysiloxane (AuNR / polymer) and plasmon-fluer-800CW (AuNR / polymer / BSA-biotin-800CW) according to the present disclosure.

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

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

[0051] [Figure 35]Figure 35 shows exemplary embodiments of various capacities of core AuNR plasmon nanostructures for enhancing 800 CW according to the present disclosure.

[0052] [Figure 36] Figure 36 shows exemplary embodiments of various capacities of core AuNR plasmon nanostructures for enhancing 800 CW according to the present disclosure.

[0053] [Figure 37] Figure 37 shows an exemplary embodiment of the extinction spectrum for an AuNR@Ag cuvoid according to this disclosure.

[0054] [Figure 38] Figure 38 shows exemplary embodiments of plasmon nanostructures 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] Figures 39A and 39B show exemplary embodiments of TEM of PF-532(Cy3) according to the present disclosure.

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

[0057] [Figure 41] Figure 41 shows exemplary embodiments of fluorescence enhancement coefficients obtained using Plasmon-Fleur-800CW with various polymer spacer thicknesses according to the present disclosure.

[0058] [Figure 42]Figure 42 shows exemplary embodiments of the plasmon-enhanced fluorescence and colloidal stability of plasmon-fluer according to this disclosure. Error bars correspond to standard deviation (independent test for n≧3). Statistically significant P-values ​​for the data are 0.0013, **P<0.01 (by two-sided independent t-test with Welch's correction). The left-hand plot of Figure 42 shows the stability of plasmon-fluer suspensions stored at 4°C and restored from lyophilized powder. Error bars correspond to standard deviation (repeat tests for n=6). NS: No significant difference. 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-fluer before and after restoration.

[0059] [Figure 43] Figure 43 is an exemplary schematic embodiment illustrating the concept of plasmon-fluer-800CW enhanced FLISA (p-FLISA) as performed in conventional FLISA (800CW) and the standard 96-well plate according to this disclosure.

[0060] [Figure 44] Figure 44 is an exemplary embodiment of the fluorescence intensity maps of human IL-6 FLISA and p-FLISA at various analyte concentrations as described herein.

[0061] [Figure 45] Figure 45 is an exemplary embodiment of the present disclosure, showing fluorescence intensity maps of human IL-6 FLISA and p-FLISA (with magnified scale bars), and a photograph of the colorimetric signal of the “gold standard” human IL-6 ELISA.

[0062] [Figure 46] Figure 46 is an exemplary embodiment of individual data points, mean, and standard deviation from human IL-6 FLISA, p-FLISA, and ELISA according to this disclosure.

[0063] [Figure 47]Figure 47 is an exemplary embodiment of a plot of human IL-6 dose-dependent fluorescence intensity from conventional FLISA as described herein.

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

[0065] [Figure 49] Figure 49 is an exemplary embodiment of a plot of human IL-6 dose-dependent fluorescence intensity from p-FLISA according to this 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. Figure 50B is an exemplary embodiment of plasmon-flure-800CW nonspecific binding according to the present disclosure.

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

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

[0069] [Figure 53] Figure 53 shows an exemplary embodiment of the IL-6 concentration in a human serum sample (diluted 10-fold) as measured using p-FLISA according to the present disclosure.

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

[0071] [Figure 55] Figures 55A and 55B are exemplary embodiments of TEM images of plasmon-fluer-Cy3 utilizing AuNR@Ag as a plasmon nanostructure according to the present disclosure.

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

[0073] [Figure 57] Figure 57 is an exemplary embodiment of SEM images of microbeads before and after examination using plasmon-fluer-Cy3 according to the present disclosure.

[0074] [Figure 58] Figure 58A is an exemplary embodiment of the present disclosure, showing bright-field and fluorescence images of Luminex microbeads before examination with plasmon-fluer-cy3. Figure 58B is an exemplary embodiment of the present disclosure, showing bright-field and fluorescence images of Luminex microbeads after examination with plasmon-fluer-cy3.

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

[0076] [Figure 60] Figure 60 is an exemplary embodiment of fluorescence images of microbeads before and after examination with plasmon-fluer-Cy3 according to the present disclosure.

[0077] [Figure 61] Figure 61 shows exemplary embodiments of mouse IL-6 standard curves obtained before (left) and after (right) the application of plasmon-fluer-Cy3 according to the present disclosure.

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

[0079] [Figure 63] Figure 63 is an exemplary embodiment of individual data points, mean values, and standard deviations from the Luminex assays of mouse IL-6, plasmon-fluer-cy3 enhanced mouse IL-6, mouse TNF-α, and plasmon-fluer-cy3 enhanced mouse TNF-α 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 fluorescence 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 fluorescence immunoassay (Luminex) for TNF-alpha according to the present disclosure.

[0081] [Figure 65] Figure 65 is an exemplary embodiment illustrating the method of using biotinylated plasmon-fluer to enhance a typical multiplex microarray according to the present disclosure.

[0082] [Figure 66] Figure 66 shows an exemplary embodiment of the method according to this disclosure of using streptavidin-conjugated plasmon-fluer to enhance a typical multiplex microarray.

[0083] [Figure 67] Figures 67(A-B) illustrate an exemplary embodiment of the present disclosure for identifying specific analytes (or controls) for each pair of fluorescent spots in a kidney biomarker array. The fluorescent spots shown in Figure 67A are identified by their coordinates in Figure 67B.

[0084] [Figure 68] Figure 68 is an exemplary SEM image showing a uniform distribution of plasmon-fluer-800CW (several highlighted by yellow circles) on and within the subsurface region of a nitrocellulose film according to the present disclosure.

[0085] [Figure 69] Figure 69 is an exemplary embodiment of a fluorescence intensity map representing a renal disease protein biomarker profile in a patient with renal disease, obtained using a conventional fluorophore (streptavidin-800CW), including a fluorescence intensity scale bar from 0 to 13, as described herein.

[0086] [Figure 70] Figure 70 is an exemplary embodiment of a fluorescence intensity map representing the renal disease protein biomarker profile of Figure 45, including a fluorescence intensity scale bar from 0 to 5000, as described herein.

[0087] [Figure 71] Figure 71 is an exemplary embodiment of a fluorescence intensity map representing the renal disease protein biomarker profile of a patient with renal disease shown in Figures 69 and 70, after the addition of Plasmon-Fleur-800CW, including a fluorescence intensity scale bar from 0 to 5000, as described herein.

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

[0089] [Figure 73] Figure 73 shows an exemplary embodiment of individual data points, mean, and standard deviation using plasmon-flur according to the present disclosure.

[0090] [Figure 74] Figure 74 shows an exemplary embodiment of individual data points, mean, and standard deviation without the use of plasmon-flur according to the present disclosure.

[0091] [Figure 75] Figure 75 is an exemplary embodiment of a photograph taken from a mobile phone, showing the color change of a nitrocellulose membrane after the addition of plasmon-fluer-800CW according to the present disclosure, using a urine sample from a patient with renal disease.

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

[0093] [Figure 77] Figure 77 is an exemplary embodiment of a plot showing the correlation between two reading modes (fluorescence readings versus color readings) of a kidney biomarker array according to the present disclosure.

[0094] [Figure 78] Figure 78 shows exemplary embodiments of confocal laser scanning microscopy (CLSM) images of breast cancer cells (SK-BR-3) examined using conventional Flür (800CW, top row) and the plasmon-Flür-800CW (bottom row) described herein, at various concentrations of ErbB2 primary antibody.

[0095] [Figure 79]Figure 79A is an exemplary embodiment of bright-field microscopy images of SK-BR-3 cells before (top) and after (bottom) labeling with plasmon-fluer-800CW according to the present disclosure. Figure 79B is an exemplary embodiment of SEM images of conventionally fluer-labeled SK-BR-3 cells according to the present disclosure. Figure 79C is an exemplary embodiment of SEM images of SK-BR-3 cells labeled with plasmon-fluer-800CW according to the present disclosure, in which the inset shows plasmon-fluer uniformly distributed on the cell membrane surface.

[0096] [Figure 80] Figure 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 according to the present disclosure.

[0097] [Figure 81] Figure 81A is an exemplary embodiment of 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-fluer (800CW)] at various dilutions of ERbB2 primary antibody according to the present disclosure. Figure 81B is an exemplary embodiment of confocal laser scanning microscopy (CLSM) images of ErbB2-stained breast cancer cells (SK-BR-3) after the addition of plasmon-fluer-800CW at various dilutions of ERbB2 primary antibody according to the present disclosure.

[0098] [Figure 82] Figure 82 shows 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]Figure 83 is an exemplary schematic embodiment of flow cytometry of ErbB2-stained SK-BR-3 cells examined by conventional Fluor (680LT) followed by plasmon-Fluor-680LT, as described herein.

[0100] [Figure 84] Figures 84A and 84B are exemplary embodiments of TEM images and extinction spectra of the 680LT according to this disclosure.

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

[0102] [Figure 86] Figure 86 is an exemplary embodiment of a pseudocolor plot of lateral and forward scattering of SK-BR-3 cells before (left) and after (right) labeling with plasmon-fluer-680LT, as described herein, including an example of a gate strategy for including single cells.

[0103] [Figure 87] Figure 87 is an exemplary embodiment of the flow contour plot (including outliers) of fluorescence-versus-forward scattering (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] Figure 88 is an exemplary embodiment of the 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-fold dilution), as described herein.

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

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

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

[0108] [Figure 92] Figure 92 shows an exemplary embodiment of two schemes for using antibody-labeled plasmon-flure to label target antigens on cells, as described herein.

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

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

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

[0112] [Figure 96] Figure 96 is an exemplary embodiment of a plot showing the mean fluorescence intensity of BMDC (corresponding to the expression level of CD80) 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 levels (corresponding to CD80 expression levels) of BMDCs after stimulation with various amounts of LPS, as examined using conventional immunofluorescence staining, according to the present disclosure. Figure 97B is an exemplary embodiment of a plot showing the mean fluorescence levels (corresponding to CD80 expression levels) of BMDCs after stimulation with various amounts of LPS, as examined using Plasmon-Fleur-680LT, according to the present disclosure.

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

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

[0116] [Figure 100]Figures 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 illustrate experiments performed independently over various days using different batches of Plasmon-Fleur-800CW.

[0117] [Figure 101] Figures 101(A-B) show exemplary embodiments of bead-based mouse TNF-α standard curves obtained after applying plasmon-fluer-Cy3 according to the present disclosure. Figures 101A and 101B illustrate independently conducted experiments for different batches of plasmon-fluer-Cy3.

[0118] [Figure 102] Figures 102(A-B) show exemplary embodiments of bead-based mouse IL-6 standard curves obtained after applying plasmon-fluer-Cy3 according to the present disclosure. Figures 102A and 102B illustrate independently conducted experiments for different batches of plasmon-fluer-Cy3.

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

[0120] [Figure 104]Figure 104A is another exemplary embodiment of 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-fluer (800CW)] at various dilutions of ERbB2 primary antibody according to the present disclosure. Figure 104B is another exemplary embodiment of confocal laser scanning microscopy (CLSM) images of ErbB2-stained breast cancer cells (SK-BR-3) after the addition of plasmon-fluer-800CW at various dilutions of ERbB2 primary antibody according to the present disclosure.

[0121] [Figure 105] Figure 105A is yet another exemplary embodiment of 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-fluer (800CW)] at various dilutions of ERbB2 primary antibody according to the present disclosure. Figure 105B is yet another exemplary embodiment of confocal laser scanning microscopy (CLSM) images of ErbB2-stained breast cancer cells (SK-BR-3) after the addition of plasmon-fluer-800CW at various dilutions of ERbB2 primary antibody according to the present disclosure.

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

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

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

[0125] [Figure 109] Figure 109A is yet another exemplary embodiment of the fluorescence intensity distribution corresponding to naive (control) and LPS-stimulated BMDCs obtained using conventional Fluor (680LT) according to the present disclosure. Figure 109B is yet another exemplary embodiment of the fluorescence intensity distribution corresponding to naive (control) and LPS-stimulated BMDCs obtained using plasmon-Fluor-680LT according to the present disclosure. Figure 109C is yet another exemplary embodiment of a plot showing the mean fluorescence intensity (corresponding to CD80 expression levels) of BMDCs after stimulation with various amounts of LPS according to the present disclosure. [Modes for carrying out the invention]

[0126] This disclosure is at least in part based on the finding that fluorescent plasmon nanostructures can be tuned to match the wavelength of a conjugated fluorophore, resulting in an enhancement of fluorescence intensity of at least 500 times.

[0127] This disclosure relates to ultra-high-brightness fluorescent nanoconstructs specifically designed for use in the biological detection and quantification of targeted analytes. As an example of their extreme potency, plasmon-flur conjugated to a standard target agent (e.g., streptavidin) is at least 500 times brighter than the same standard target agent conjugated to a fluorescent molecule commonly used in microplate-based fluorescence-coupled immunosorbent assays. This results in a significant improvement in assay performance, resulting in both increased sensitivity (an improvement of more than an order of magnitude in the detection limit) and dynamic range.

[0128] Design advantages of this disclosure The advantages of the design disclosed herein over previous types include, but are not limited to, the following: (1) Plasmon-flure is a considerably more useful solution phase than substrates decorated with plasmon species; (2) it is a direct wet chemical synthesis compared to Cu-Ag NP alloys, structures produced by lithography or vapor deposition, or layer-by-layer synthesis; (3) it offers a high degree of control over particle homogeneity / stability / synthesis, which is important for immunoassays: (a) aggregation is generally a major problem with nanoparticles and can lead to serious unnatural results; (b) it is also a problem where high nonspecific background is perceived; (4) the spacer between the fluorophore and the plasmon nanostructure core using MTPMS / APTMS / TMPS enables precise thickness on the nanometer scale. Therefore, the present invention includes: (5) being able to be strictly controlled and applied in solution; (6) the silane-based spacer layer being readily functionalized; (7) the improvement in assay performance being greater than that of previous methods; (8) the increase in fluorescence per dye molecule (average value) being higher than that previously reported for arrangements suitable for immunoassay applications; and (9) the ability of larger particles used in this disclosure to carry more dye molecules than other designs (i.e., more dye and enhanced conjugated dyes means an ultra-high brightness construct).

[0129] According to this disclosure, plasmon enhancement improves the quantum yield of the conjugated dye (which is a key factor in the "brightness" produced by the dye) and reduces its fluorescence lifetime. Therefore, a higher enhancement factor (improvement in relative brightness) can be achieved for dyes with low quantum yield and / or long fluorescence lifetimes than for dyes that already have high quantum yield and short fluorescence lifetimes.

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

[0131] In some embodiments, the nanoconstruct includes a fluorescent agent having a brightness 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] The features of this disclosure include: (1) A plasmon nanostructure that acts as a nanostructure over a range of light wavelengths, wherein the maximum value is defined by the localized surface plasmon resonance (LSPR) wavelength of the particle. The plasmon particle may have one or more LSPR wavelengths. The plasmon particle "attracts" light of wavelengths corresponding to the LSPR wavelength, effectively collecting light and enhancing the electromagnetic field near the particle surface.

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

[0134] (3) A spacer layer having a barrier that prevents metal-induced quenching to maintain an optimal distance from the plasmon particle surface, and on which a fluorophore can be fixed (either directly to the carrier molecule or via bonding). The spacer material ideally contains functional groups that enable covalent conjugation of the fluorophore and / or biorecognition element at the distal surface of the plasmon particle surface.

[0135] (4) Several objectives: stabilization of nanoconstructs from aggregation and nonspecific bonding; bonding sites for biorecognition elements; and functional layers that can also function as carriers for fluorophores.

[0136] (5) A biorecognition element that can use plasmon-flur for specific detection of its target (for example, if the biorecognition element is an antibody and an aptamer, the target antigen; if the biorecognition element is streptavidin, the target antigen; and if the biorecognition element is a complementary oligonucleotide, the target oligonucleotide).

[0137] It is currently believed that particles possessing the composition and performance characteristics of the fluorescent nanoconstructs described herein do not exist. Numerous previous attempts to generate plasmon-enhanced fluorescent nanoconstructs in solution phase have achieved a "brightness enhancement" of approximately 10 times.

[0138] Fluorescent nanoconstructs The fluorescent nanoconstructs disclosed herein overcome the aforementioned challenges and provide a pathway for the broad application of these fluorescent nanoconstructs to immunoassays and other bioassays. As used herein, the term “fluorescent nanoconstruct” also refers to plasmon-flure (PF). As an example, with regard to the detection of biomarkers related to renal function, the results illustrate that fluorescent nanoconstructs significantly enhance the ability to elucidate low levels of renal function parameters (biomarkers) to provide comprehensive renal disease information. It should be noted that the superior performance of multiplex microarrays stems from the very simple addition of nanostructures to the assay before detection using standard techniques. Furthermore, this technique is inexpensive and easily implemented to enhance fluorescence. Such easily deployable techniques can be seamlessly applied to a wide range of platforms in diagnostics, proteomics, and genetics to address the still unmet need for brighter signal intensity.

[0139] In one embodiment, a fluorescent nanoconstruct is disclosed herein, which generally comprises a plasmon nanostructure, a polymer, a biorecognition element, and a fluorescent agent. In some embodiments, the fluorescent nanoconstruct comprises a plasmon 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 biological analytical parameters (sensitivity, LOD, and dynamic range) of fluorescence immunoassays performed in microplate formats, membrane formats, antibody microarray formats, and bead-based formats, in addition to a number of 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 embodiments, the assay or analysis format is a solution phase. In other embodiments, the assay is applied to cells or tissues. In some embodiments, the fluorescent nanoconstructs result in a fluorescence intensity enhancement of 10-fold, 50-fold, 100-fold, 200-fold, 250-fold, 500-fold, 1000-fold, or even more than 10,000-fold compared to biorecognition elements labeled with fluorescent agents without plasmon enhancement.

[0141] Most existing plasmon-enhanced fluorescence techniques require fluorescence-based bioassays performed on pre-fabricated plasmon substrates, glass slides typically coated with metal nanostructures instead of standards, or sometimes on non-replaceable biological analysis platforms (e.g., 96-well plates, nitrocellulose membranes, or microbeads), which significantly limit the broad applicability of the technique. More importantly, the need for dedicated substrates limits platform- and laboratory-to-platform consistency and seamless integration with widely used biological analysis procedures, which is a major drawback of conventional plasmon-enhanced fluorescence techniques. This disclosure develops a plasmon-flur-based "non-invasive" (no modification of existing assay protocols) ultra-high-intensity fluorescence technique in which plasmon-flur is simply added to microtiter wells (or microarrays, microbeads, or cell surfaces) in place of conventional flur.

[0142] Plasmon-fluer (PF) with special specifications to maximize fluorescence enhancement. According to this disclosure, the optical properties of plasmon-flures (e.g., the LSPR wavelength of the metal nanostructure, which plays a crucial role in the final enhancement efficiency) can be easily tuned and optimized for a given fluorescent emitter (organic dye, quantum dot, or upconversion nanoparticle) by a rational selection of the size, shape, and composition of the nanostructure. This is in stark contrast to conventional plasmon substrates (e.g., metal nanoislands), which result in poor control of the LSPR wavelength and are typically limited to suboptimal "universal" approaches.

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

[0144] High stability, performance, and affordability The techniques disclosed herein for enhancing biological assays are cost-effective solutions for improving bioassay performance, including cost estimates for plasmon-flure for a single treatment of a 96-well microtiter plate, comparable to current industrial standards, and substantially less expensive than the dedicated substrates described above (e.g., glass slides coated with metal island films). The high stability of the metal nanostructures further ensures the integrity and functionality of plasmon-flure under typical storage / transport / handling conditions used in bioassays. Generally, plasmon-flure can be stored and handled in a similar manner to handling fluorescently labeled biorecognition elements. In summary, the enhanced signal-to-noise ratio achieved by the techniques described herein significantly improves assay sensitivity, alleviates stringent instrument requirements (such as low background noise and high sensitivity), reduces the required reagent volume, and / or drastically shortens the total assay time, thereby enabling these assays to be performed in a wide range of research and clinical diagnostic situations with minimal effort or cost and significant improvements in assay performance.

[0145] In assays where fluorescence detection is already used as the reading, the improved fluorescence intensity achieved by using plasmon-flure instead of the current gold-standard fluorophores results in an improvement in the limit of detection (LLOD) of the bioassay. In some embodiments, the LLOD is reduced by at least half, one-fifth, one-tenth, one-twentieth, one-fortieth, one-fiftieth, one-hundredth, one-fiftyth, or even one-thousandth. Furthermore, this improves the dynamic range of detection. In some embodiments, the improvement in dynamic range is greater than 2x, 5x, 10x, 20x, 40x, 50x, 100x, 500x, or even one-thousandth. In assays where fluorescence detection is not yet used as the reading method, but the reading method is chemiluminescent or colorimetric, for example in chemiluminescent / colorimetric ELISA or Western blotting, switching to fluorescence detection and using plasmon-flure with appropriate detection equipment will result in at least equivalent performance in LLOD and dynamic range of the bioassay compared to the gold standard reporter method for said assays. Improvements in biological analytical parameters have been found to be consistent across various assay formats, target biomarkers, and fluorophores. Importantly, this method can be performed using existing bioassays with minimal modifications to standard operating procedures and without additional operational training. In some embodiments, the only modification from the existing assay protocol is the addition of a fluorescent nanoconstruct instead of the existing fluorescent reporter molecule.

[0146] As part of the accurate validation of this technology, urine samples were analyzed from patients with renal disease and healthy volunteers. In contrast to unenhanced fluorescence immunoassays and ELISAs, plasmon-enhanced fluorescence immunoassays enabled the detection and quantification of low concentrations of biomarkers, as well as from all patients and healthy volunteers. The additional sensitivity of the plasmon-enhanced assay allows for the easy quantification of low-abundance biomarkers, providing physiological and pathological information that is often missed by conventional immunoassays.

[0147] Plasmon nanostructures The nanostructures described herein include plasmon nanostructure cores. The plasmon nanostructures used herein are selected based on a number of criteria (see, for example, Table 2) that provide plasmon enhancement to the fluorescence signal. The plasmon nanostructures may include any material having surface plasmons that can resonate at a suitable wavelength of light, such as gold (Au), silver (Ag), copper (Cu), or combinations thereof. Preferred examples of plasmon 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 nanostructures are selected from the group consisting of gold-core silver-shell nanocuboids, nanotubes, gold nanorods, silver nanocubes, silver nanospheres, bimetallic nanostructures, gold nanorod cores, silver-shell (AuNR@Ag) canocuboids, nanostructures with sharp tips, nanostars, hollow nanostructures, nanocages, nanorattles, nanobipyramids, nanoplates, self-assembled nanostructures, nanoraspberries, and combinations thereof.

[0148] One criterion for selecting plasmon nanostructures for use in fluorescent nanoconstructs is the LSPR wavelength. Various plasmon nanostructures have various LSPR wavelengths, as illustrated in Figure 5. Even the same plasmon nanostructure can have multiple LSPR wavelengths corresponding to various resonance modes of surface plasmons. The specific LSPR wavelength that is optimal for fluorescence enhancement of a fluorophore is based on the excitation spectrum of that fluorophore. Specifically, it is important that there is an overlap between the fluorophore's LSPR wavelength and excitation spectrum. Generally, a single layer of overlap results in good enhancement. An ideal situation for fluorescence enhancement occurs when the LSPR wavelength, the fluorophore excitation maximum, and the wavelength of the 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 wavelengths are between approximately 200 and 1200 nm, between approximately 250 and 950 nm, between approximately 300 and 850 nm, between approximately 350 and 800 nm, and between approximately 400 and 750 nm. In yet another embodiment, the LSPR wavelengths are approximately (average ±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, 1050, 1100, 1150, or 1200 nm.

[0149] In some embodiments, fluorescent nanoconstructs based on gold nanorods (AuNR) can have LSPR wavelengths between approximately 600 and 1200 nm. As another example, plasmon-flures having a core of either silver-nanocube, AuNR@Ag cuvoid, or -Au@Ag cube can have LSPR wavelengths between approximately 400 nm and 600 nm.

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

[0151] In some embodiments of the fluorescent nanoconstruct, the plasmon 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, antibodies, or any combination thereof.

[0152] [Table 2]

[0153] Plasmon nanostructure size The size of the plasmon nanostructures forming the plasmon-flure core 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 plasmon 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 plasmon nanostructures and their LSPR wavelengths are adjusted so that the size of the nanostructures has an LSPR wavelength that overlaps with the maximum excitation wavelength of the fluorophore.

[0154] Wavelength matching As described herein, the size, shape, and composition of plasmon nanostructures can be adjusted to have an LSPR wavelength that coincides with the maximum excitation maximum wavelength of the fluorophore. Furthermore, the LSPR maximum(singular) / maximum(plural) is shifted after coating with a spacer layer and / or functional layer, and the overlap / coincidence described below must conform to the plasmon nanostructure core coated with the spacer and / or functional layer. Wavelength coincidence may be a large overlap between the LSPR wavelength and the excitation spectrum of the fluorophore; a large coincidence between the LSPR wavelength and the maximum excitation maximum wavelength of the fluorophore showing LSPR / fluorophore excitation maximum coincidence and overlap [see, for example, Figures 5 and 6 (A-B)]; or the quenching spectrum of the nanostructure in solution shows a large overlap with the quenching spectrum and / or absorption maximum excitation maximum wavelength of the fluorophore in solution. The overlap between the LSPR wavelength and the excitation maximum wavelength of the conjugated fluorophore may be 100% overlap, or the maximum LSPR wavelength and the maximum excitation maximum wavelength of the fluorophore may coincide or overlap 100%. In some embodiments, the result of the coincidence between the LSPR wavelength and the excitation maximum wavelength of the conjugated fluorophore is at least 500 times higher fluorescence intensity for a fluorescent nanoconstruct conjugated with 20 to 2000 fluorescent agents compared to a free fluorescent agent in solution, when obtained under similar excitation and detection conditions.

[0155] The maximum LSPR wavelength of gold nanorods (AuNR) can be easily tuned to match the excitation maximum wavelength of fluorophores between 600 nm and >1200 nm. The maximum LSPR wavelengths of silver cubes, AuNR@Ag cuvoids, and Au@Ag cuvoids can be easily tuned to match the excitation maximum wavelength of fluorophores between 400 nm and 600 nm. Therefore, although these were used as exemplary materials, any plasmon nanostructures that can be tuned to have an LSPR wavelength matching 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 plasmon nanostructures are tuned so that the LSPR wavelength matches the maximum excitation maximum wavelength of the fluorophore. As another example, cuboids having at least one dimension between approximately 60 nm and approximately 130 nm have been found to be sufficient for tunably matching wavelengths <600 nm. As yet another example, gold nanorods having a length between approximately 30 nm and approximately 130 nm have been found to be sufficient for tunably matching wavelengths >600 nm. As an example, wavelengths can be considered matched if the excitation maximum wavelength (λEX) of the fluorophore is within approximately 100 nm of the LSPR wavelength (λLSPR) of the plasmon-flure (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 approximately 75 nm. In some embodiments, the absolute value of Δ is 50 nm (i.e., ±50 nm). A smaller absolute value of Δ is preferable (i.e., closer to zero), but the LSPR absorption peak is generally very broad (>50 nm, or even >100 nm in full width at half maximum), which means that there can be sufficient overlap between the quenching spectra of the plasmon fluer and the fluorophore, resulting in significant fluorescence enhancement even when the maxima of both spectra (λLSPR and λEX, respectively) do not coincide. Windows for pairs of plasmon nanostructures and dyes are also disclosed [for example, 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 are used to coat plasmon nanostructures, and quenching is reduced or prevented by maintaining fluorophores at an average sufficient distance (e.g., at least about 0.5 nm to 4 nm away from the plasmon nanostructure) from the surface of the plasmon nanostructure. The spacer coating is any material that can coat plasmon nanostructures and can be controlled to have a thickness of 0.5 to 100 nm. In some embodiments, the spacers may be functionalized with fluorophores.

[0159] In some embodiments, the fluorescent nanoconstruct further includes spacers in the form of a coating on the plasmon nanostructure. In some embodiments, the spacers are dielectric materials. In some embodiments, the coating thickness can be adjusted to achieve varying amounts of fluorescence enhancement of the fluorescence signal. In some embodiments, the coating thickness (d) is about 0.5 nm to about 100 nm. In yet 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. The "about" used here means ±25%. In some embodiments, the coating thickness is controlled during preparation by increasing the monomer concentration. 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. In some embodiments, the variation in the thickness of the spacer coating on a single fluorescent nanoconstruct is about 2 nm, 3 nm, 4 nm, or less than 5 nm.

[0160] In some embodiments, the coating may comprise any polymer or polymer mixture that can be uniformly deposited over the plasmon nanostructure, 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 may be applied in solution. In some embodiments, the spacer coating is covalently bonded to the plasmon 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 covalent bonding to other molecules such as amines, aldehydes, carboxylic acids, sulfhydryls, ketones, and click chemistry-compatible moieties. 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 by a mercapto-containing moiety that forms a reactive layer on a gold / silver surface, and the siloxane network is constructed from this initiation layer using a mixture of functional silanes. The advantages of silanes are that silanes are: 1) suitable for wet chemistry, 2) a variety of functional groups available for further modification and adjustment of particle characteristics [e.g., PEG, amino, epoxy, mercapto, vinyl, click chemistry moieties (TCO, azide), PEG-biotin, aldehydes, fluorophores, and amino acids], and 3) precise control of the 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 acts as a scaffold for the luminescent element (fluorophore) and the biorecognition element (e.g., biotin, streptavidin, antibody, nucleic acid). In some embodiments, the fluorophore is bound to the functional layer, in which case the functional layer also acts as an additional spacer between the fluorophore and the plasmon nanostructure surface, even in the presence of a separate spacer coating layer. In some embodiments, the spacer coating layer or functional layer acts 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 embodiments, 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 embodiments, the protein on the fluorescent nanoconstruct is bovine serum albumin (BSA), human serum albumin (HSA), or a combination thereof. In some embodiments, the protein is BSA.

[0164] functional base layer In some embodiments, the spacer coating is a functional layer or a fluorescent nanoconstruct, and may further include a functional layer coating the spacer layer. In some embodiments, 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 bond to the surface, can be used as the functional layer. In some embodiments, the polymer contains functional groups for covalent bonding to other molecules such as amines, aldehydes, carboxylic acids, sulfhydryls, ketones, and click chemistry-compatible moieties. In some embodiments, the functional layer includes a polypeptide. In some embodiments, the functional layer is an albumin protein or its homolog. 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 bonded to the spacer layer. In some embodiments, the functional layer is the same material used to "block" in immunoassays. 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 biorecognition elements (see, for example, Table 5). The biorecognition elements target specific analytes or species. For example, the biorecognition element may be an antibody if the target is an antigen, or it may be a streptavidin.

[0166] In some embodiments, the biorecognition element is selected from the group consisting of biotin, streptavidin, antibodies (or functional fragments thereof), oligos (DNA, PNA, etc.), aptamers, "click" moieties (e.g., tetrazine), molecular imprinted polymers ("artificial antibodies"), digoxigenin, peptide tags, protein tags, and combinations thereof. In some embodiments, the target is selected from the group consisting of streptavidin, biotin, target antigen, complementary oligos (DNA, RNA), target analytes, complementary "click" moieties that generate pairs, DIG-binding proteins or anti-digoxigenin antibodies, and combinations thereof.

[0167] In some embodiments, biorecognition elements such as antibodies, streptavidin, aptamers, and nucleic acids are added to the spacer layer or functional layer via many of the same chemical properties that are added to the following fluorophores. Furthermore, in some embodiments, biotinylated plasmon-fluer is directly conjugated to streptavidin, which can be further conjugated to a biotinylated antibody. In some embodiments, the biorecognition elements are bound to PF using a flexible linker. In some embodiments, the flexible linker is PEGx, where x is 2 to 36. In some embodiments, the fluorescent nanoconstruct comprises a plasmon nanostructure having at least one localized surface plasmon resonance (LSPR) wavelength (λLSPR) and a spacer containing a first material; at least five fluorescent organic dyes having an excitation maximum wavelength (λEX); and a biological recognition element, wherein the plasmon nanostructure is substantially coated with a spacer having a thickness between 0.5 and 20 nm, the fluorescent species is bound to the distal spacer surface of the plasmon nanostructure, the spacer is substantially coated with a functional layer, the biological recognition element is bound to the functional layer, the difference between the LSPR wavelength and the excitation maximum wavelength of the fluorescent organic dye is less than 75 nm, and each fluorescent organic dye is at least 10 times brighter than a non-conjugate fluorescent species in aqueous solution under typical irradiation and detection conditions.

[0168] [Table 5]

[0169] fluorescent dye Fluorescent agents are 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 with which the fluorescent agent can bond to the spacer coating of the functional group layer in the fluorescent nanoconstruct. In some embodiments, the fluorescent agent is one of the UV, visible, near-infrared (NIR), or infrared (IR) organic fluorophores. 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 dye, flavone, fluorescein, imidazole, indocarbocyanine, indocyanine, indole, iso The following are selected from the group consisting of indole, isoquinoline, naphthacenedione, naphthalene, naphthoquinone, phenanthrene, phenanthridine, phenoselenazine, phenothiazine, phenoxazine, phenylxanthene, polyfluorobenzene, purine, pyrazine, pyrazole, pyridine, pyrimidone, pyrrole, quinoline, quinolone, rhodamine, squaline, tetracene, thiophene, triphenylmethane dye, xanthene, xanthone, and their derivatives.

[0170] Furthermore, the fluorescent nanoconstructs disclosed herein are suitable for enhancing fluorescent signals originating 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 added via standard chemicals: succinimidyl esters, NHS esters, TFP esters, or isothiocyanates to primary amines; maleimides to mercapto groups; click chemistry by directly adding to functionalized silanes (e.g., tetrazine-linked fluorophores to TCO-PEGn-triethoxysilane) or by first functionalizing another reactive group to create a click moiety; or via hydrazides or hydroxylamines to aldehydes or ketones. Furthermore, in some embodiments, the fluorophores are first conjugated to functionalized layer molecules such as proteins and then adhered to a 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 to 2000 fluorophores per plasmon-flue. In some embodiments, the fluorescent species is covalently bonded to a spacer layer. In some embodiments, the fluorescent species is covalently bonded to a functional layer.

[0172] [Table 6]

[0173] Exemplary Embodiments of Plasmon-Fleur (PF) The plasmon-flure (PF) variants described below differ in the arrangement of the PF's constituent components, but all include a plasmon nanostructure coated with a spacer layer having a fluorophore maintained at a specific distance from the plasmon nanostructure surface, a biological recognition element bonded somewhere to the PF, and a coupling region between the fluorophore and the plasmon nanostructure, defined by the overlap of the excitation maxima of the bonded fluorophore with at least one of the PF's LSPR wavelengths. In some examples, a layer referred to as a "functional layer" is present, which can serve several purposes, such as bonding molecules (fluorophore, biological recognition element) and stabilizing the structure from aggregation and nonspecific bonding.

[0174] In some embodiments, the functional base layer is located on the PF. In particular, a plasmon nanostructure is disclosed having at least one localized surface plasmon resonance wavelength (λLSPR), a spacer material of a specific thickness (d) substantially covering the surface of the plasmon nanostructure, a fluorophore conjugated to the spacer material, a functional base layer material substantially covering the spacer material, and a biorecognition element conjugated to the functional base layer material.

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

[0176] In some embodiments, coating a plasmon nanostructure with at least one spacer coating includes applying a first layer onto the plasmon nanostructure core and applying a polysiloxane coating on top of this first layer. In some embodiments, the initiating layer comprises 3-mercaptopropyl)trimethoxysilane (MPTMS). In some embodiments, the polysiloxane coating comprises trimethoxypropylsilane (TMPS) and 3-aminopropyltrimethoxysilane (APTMS).

[0177] In some embodiments, a plasmon nanostructure is disclosed having a localized surface plasmon resonance (LSPR) wavelength (λLSPR), a spacer material of a specific thickness (d) substantially covering the surface of the plasmon nanostructure, a fluorophore conjugated to the spacer material, and a biorecognition element conjugated to the spacer material. In some embodiments, the fluorescent nanostructure comprises a plasmon 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 a spacer having a thickness (d), the spacer is conjugated by a fluorescent species, the biorecognition element is coupled to the spacer, the fluorescent species has an excitation maximum wavelength (λEX), and the difference between the LSPR wavelength and the excitation maximum wavelength is |Δ|.

[0178] In some embodiments, a plasmon nanostructure is disclosed having a localized surface plasmon resonance wavelength (λLSPR), a spacer material of a specific thickness (d) substantially covering the surface of the plasmon nanostructure, a fluorophore conjugated to the spacer material, a biorecognition element conjugated to the spacer, and a functional base layer material substantially covering the spacer. In some embodiments, the fluorescent nanostructure comprises a plasmon nanostructure having a localized surface plasmon resonance (LSPR) wavelength (λLSPR) and a spacer comprising a first material; a fluorescent species; a biorecognition element; a functional base layer comprising a second material, wherein the nanostructure is substantially covered by a spacer having a thickness (d), the spacer is conjugated by a fluorescent species, the biorecognition element is coupled to the spacer, the functional base layer material is coupled to the spacer, 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 plasmon nanostructure is disclosed having a localized surface plasmon resonance wavelength (λLSPR), a spacer material of a specific thickness (d) substantially covering the surface of the plasmon nanostructure, a functional base material substantially covering the spacer material, a fluorophore conjugated to the functional base material, and a biorecognition element conjugated to the functional base material. In some embodiments, the fluorophore is directly bonded to the spacer, or to the functional base material having a biorecognition element. In some embodiments, the fluorescent nanoconstruct comprises a plasmon nanostructure having a localized surface plasmon resonance (LSPR) wavelength (λLSPR) and a spacer comprising a first material; a fluorescent species; a functional base layer comprising a second material; and a biological recognition element, wherein the nanostructure is substantially coated by a spacer having a thickness (d), the spacer is substantially coated by a functional base layer, the fluorescent species is bonded to the functional base layer, the biological recognition element is bonded to the functional base 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 nanoconstructs have a zeta potential greater than approximately 20 mV, or approximately 25 mV, or approximately 30 mV, or approximately 35 mV, or approximately 40 mV, or approximately 45 mV in water at pH 7.

[0181] In some embodiments, the luminance of the plasmon-flure 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 free fluorescent species under typical irradiation and detection conditions. In some embodiments, the luminance of each fluorescent species coupled to the plasmon-flure 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 typical irradiation 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 the 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 (ELISA), microfluidic chips, and membrane-based assays.

[0183] Methods for improving the performance of a biological assay are disclosed herein. The method generally involves using a fluorescent nanoconstruct described anywhere herein as a reporter molecule in a biological assay, wherein the fluorescent nanoconstruct is targeted to a particular 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 particular analyte of interest (e.g., the biorecognition element is a primary antibody to the analyte or a complementary oligonucleotide to a particular 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] Due to the large fluorescence signal generated by the use of fluorescent nanoconstructs compared to standard fluorophores, the lower limit of detection of fluorescence assays is significantly improved compared to the lower limit of detection achievable using current standard fluorescent reporter molecules (i.e., the LOD is lower, enabling the detection of samples with relatively low concentrations). 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 2× (i.e., the LOD is half of the LOD of the same assay using current standard fluorescent reporter molecules, i.e., the detection sensitivity is doubled). In some embodiments, the LOD is at least 2× better, at least 3× better, at least 4× better, at least 5× better, at least 10× better, at least 25× better, at least 50× better, at least 100× better, at least 500× better, at least 1000× better, at least 5000× better, or even at least 10,000× better than the same assay using current standard fluorescent reporter molecules.

[0185]

Table 7

[0186] Manufacturing method In some embodiments, the plasmon-fluoro 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 obtained from (3) with a functional group layer, and (5) conjugating a biorecognition element to the functional group layer. This synthesis also includes several variant forms and other embodiments.

[0187] Modified form 1: Biorecognition element conjugated to a functional layer: (1) Starting with a plasmon nanostructure; (2) Coating the plasmon nanostructure with a spacer layer [The spacer layer may be a mixture of MPTMS, APTMS, and TMPS, or an alternative silane having various functional group moieties (e.g., aldehyde or tetrazine) for conjugating the fluorescent species (e.g., by hydrazine or TCO, respectively)]; (3) Conjugating the fluorescent species to the spacer layer [The fluorescent species may be covalently bonded to an amine (via an NHS or TFP ester), or an alternative silane having various functional group moieties (e.g., aldehyde or tetrazine) for conjugating the fluorescent species (e.g., by hydrazine or TCO, respectively)]; (4) Conjugating the biorecognition element to the functional layer: (a) The biorecognition element is The functional layer is biotin, the functional layer is bovine serum albumin (BSA), the biotin is covalently bound to BSA (or another suitable protein) using an NHS ester, the biotin is present on a PEGx spacer; or (b) using click chemistry, for example, reacting NHS-PEGx-TCO with BSA and NHS-PEGy-tetrazine with streptavidin or an antibody to coat the nanoconstruct with BSA-TCO, then directly binding streptavidin or an antibody to BSA by mixing in a tetrazine-biorecognition element after step (5); (5) in the functional layer step, coating the nanoconstruct from (3) with the functional layer in (4) using a mixture of biotinylated BSA (or another suitable protein) and innate BSA (or another suitable protein).

[0188] Modified form 2: Fluor-biorecognition element conjugated to a functional group layer: (1) starting with a plasmon nanostructure; (2) coating the plasmon nanostructure with a spacer layer; (3) conjugating the biorecognition element and fluorophores to a functional group layer; and (4) coating particles from (2) with the functional group layer from (3).

[0189] In some embodiments, biotinylated PF is used as a building block and added to other biorecognition elements. While biotin can be used as a biorecognition element, it can also be used to conjugate additional biorecognition elements (e.g., streptavidin). Therefore, an additional step exists in which streptavidin is conjugated to the biotinylated PF nanoconstruct. Similarly, it is possible to employ this streptavidin-conjugated PF to bind a biotinylated antibody. In this case, yet another step exists in which the biotinylated antibody is conjugated to the streptavidin-conjugated PF.

[0190] In some embodiments, 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-flures are designed to enhance the performance of fluorescence-based biological assays. Specifically, they are used as reporter molecules, and the fluorescence signal generated by a plasmon-flure when excited at the appropriate wavelength 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 the PF is used to report on the concentration of the detection antibody present, which is proportional to the amount of the target analyte. This can be done by directly conjugating a detection antibody to a PF (the biorecognition element being a detection antibody) and using the resulting construct to conjugate to the target analyte; by conjugating a streptavidin-conjugated PF (the biorecognition element being streptavidin) to a biotinylated detection antibody already bound to the target analyte; by conjugating a biotinylated PF (the biorecognition element being biotin) to streptavidin bound to a biotinylated detection antibody bound to the target analyte; or by conjugating a PF conjugated to a secondary antibody (the biorecognition element being a secondary antibody) that points to a detection antibody bound to the target analyte.

[0192] Furthermore, PF can be used to detect target nucleic acid sequences by either 1) using complementary nucleic acid sequences as biorecognition elements, or 2) using biotinylated nucleic acid sequences that first bind to the target and detecting them with streptavidin-linked PF (where the biorecognition element is streptavidin).

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

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

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

[0196] In some embodiments, the kit contains reagents in individual containers, such as sterile water or saline solution, to be added to individually packaged components. For example, sealed glass ampoules may contain components in individual ampoules, sterile water, sterile saline solution, or sterile material, each packaged under a neutral, non-reactive gas such as nitrogen. The ampoules may be made of any suitable material, such as glass, polycarbonate, organic polymers such as polystyrene, ceramics, metals, or any other material commonly used to hold reagents. Other examples of suitable containers include bottles, which may be made from a substance similar to that of the ampoules, and envelopes, which may have an interior lined with foil, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, etc. Containers may have a sterile access port, such as a bottle with a stopper that can be punctured with a subcutaneous injection needle. Other containers may have two compartments separated by an easily removable membrane, which allows for mixing of the components upon removal. The removable membrane may be glass, plastic, rubber, etc.

[0197] In certain embodiments, the kit is supplied with instructional materials. These instructions may be printed on paper or other substrates and / or supplied as electronically readable media or video. Detailed instructions do not necessarily have to be physically attached to the kit. Instead, the user may be directed to an internet website designated by the kit's manufacturer or distributor.

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

[0199] Exemplary embodiments of fluorescent nanoconstructs and methods for using them are described in detail above. The fluorescent nanoconstructs and methods described herein are not limited to the specific embodiments described; rather, the components of the apparatus, systems, kits, and / or steps of the Method may be used independently and individually from other components and / or steps described herein. For example, the Method may also be used in combination with other polymers, nanostructures, and bioassays, and is not limited to being carried out using only the apparatus, systems, and methods described herein. Rather, exemplary embodiments may be carried out and used in conjunction with numerous other systems.

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

[0201] In the following specification and claims, reference is made to a number of terms which will be defined to have the following meanings. The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "comprising", "including" and "having" are intended to be inclusive and mean that additional elements other than the recited elements may be present. "Optional" or "optionally" means that the subsequent recited event or circumstance may or may not occur, and includes the case where the event occurs and the case where the event does not occur.

[0202] Throughout this specification and the claims, approximate language, if used herein, may be applied to modify any quantitative representation that could vary to some degree without resulting in a change in the basic function associated with it. Accordingly, values modified by terms such as "about", "approximately" and "substantially" (singular or plural) are not limited to the exact values specified. In at least some instances, the approximate language may correspond to the precision of the instrument used to measure the value. Here, as well as throughout this specification and the claims, range limitations may be combined and / or interchanged. Such ranges are specified and, unless the context or language specifically indicates otherwise, include all sub-ranges subsumed therein.

Examples

[0203] The following examples describe compositions, as well as methods of making and using plasmon-fluors (PFs), for maximizing fluorescence enhancement.

Examples

[0204] In the design and synthesis of fluorescent nanoconstructs, the following two factors must be carefully considered: (i) the bound fluorophore must be sufficiently far from the surface of the plasmon nanostructure to avoid metal-induced fluorescence quenching; and (ii) the fluorophore must be sufficiently close to the surface of the plasmon nanostructure to benefit from the enhanced electromagnetic field, which rapidly attenuates as the distance from the surface of the plasmon nanostructure increases. It is known that the transient nature of the enhancement of the electromagnetic field at the surface of the plasmon nanostructure results in a distance-dependent enhancement of fluorescence at the surface of the plasmon nanostructure. When the fluorophore comes into direct contact with (or is closest to) the plasmon nanostructure, fluorescence quenching occurs due to non-radiative energy transfer between the fluorophore and the metal surface. On the other hand, as the distance between the fluorophore and the metal nanostructure increases, the enhancement decreases due to the attenuation of the electromagnetic field from the surface of the nanostructure. In summary, the optimal distance between the metal surface and the fluorophore is one of the important 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 if the fluorophore is directly bonded to the spacer layer, and between 0.5 and 5 nm if the fluorophore is bonded to the functional group layer.

[0205] To achieve the optimal distance between the surface plasmon nanostructure and fluorophores, a polysiloxane copolymer layer was formed on the surface of the plasmon nanostructure as a spacer layer. An initiation layer for the spacer was generated by bonding 3-mercaptopropyl)trimethoxysilane (MPTMS) to the surface of the plasmon nanostructure. Hydrolysis-unstable trimethoxypropylsilane (TMPS) and 3-aminopropyltrimethoxysilane (APTMS) were copolymerized on the plasmon nanostructure via the initiation layer. The formation of the spacer layer resulted in a red shift in the longitudinal LSPR wavelength of the plasmon nanostructure, due to an increase in the refractive index of the surrounding medium.

[0206] Transmission electron microscopy and atomic force microscopy (AFM) imaging confirmed the successful formation of a spacer layer on the plasmon nanostructure. AFM height profiles of AuNR 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 bonding the nanostructures to a substrate coated with streptavidin-800CW. The transient fluorescence enhancement coefficients of nanostructures with and without the polymer spacer layer, at the same density (confirmed by scanning electron microscopy imaging), were found to be approximately 1000 and 200, respectively, highlighting the importance of the spacer layer for significant fluorescence enhancement (Figure 7A). Note that these particles possess fluorophores bound to BSA, providing some degree of space. If the fluorophores were directly bound to AuNR, fluorescence would likely be minimal. The observed bright emission is caused by both excitation enhancement (enhancement of the EM field) and changes in emissivity. For a detailed examination of the enhancement rate, the excited state lifetimes of 800CW-BSA dispersed in solution and nanostructures composed of 800CW-BSA were determined. The fluorescence lifetime of 800CW-BSA adsorbed on AuNR was less than a quarter shorter (0.18 ns) than that of free 800CW-BSA (0.75 ns) (Figure 7B). This significant decrease in fluorescence lifetime directly contributes to an increase in the luminescence of the fluorophore. [Examples]

[0207] Assay validation Following the synthesis of fluorescent nanoconstructs, the application of these novel materials and techniques was validated in several biological analytical methods, enhancing weak fluorescence signals and associated biological analytical parameters. To compare the enhancement of assay parameters in the commercially available assays described below, the assays were performed according to the supplier's specifications, and plasmon-flur was added at a concentration <10 × of the concentration of the gold standard reporter molecule. Further improvements in performance can be achieved by optimizing reagents, incubation time, and concentration.

[0208] Plasmon-Fleur acts as an ultra-high-brightness fluorescent probe in the final step of a bioassay, enhancing weak fluorescence and signal-to-noise ratio (SNR) without requiring any changes or modifications to existing bioassay protocols (i.e., a "non-invasive" method). The ultra-high brightness of plasmon-Fleur is due to the presence of a metal core, which acts as an antenna to strongly enhance the fluorescence emission of surface fluorophores. The enhancement of fluorophore emission near the metal nanostructure is attributed to an enhancement of the electromagnetic field on the surface of the plasmon nanostructure (local excitation region) and a reduction in fluorescence lifetime due to coupling between the excited fluorophores and surface plasmons of the nanostructure. Plasmon-Fleur is highly versatile and universal, and integrates seamlessly with a variety of existing fluorescence-based biological analytical techniques.

[0209] This disclosure tests the application of plasmon-flua as a fluorescence enhancer in fluorophore-conjugated immunosorbent assays (FLISA). A typical sandwich FLISA includes the following main steps: (i) capture of a target antigen with an immobilized antibody; (ii) binding of a biotinylated detection antibody to the captured antigen; and (iii) binding of fluorescently labeled streptavidin. As shown herein, the addition of biotinylated plasmon-flua after the last step (i.e., binding of fluorescently labeled streptavidin) resulted in a significant increase in fluorescence intensity and a substantial improvement in the limit of detection (LOD). The addition of biotinylated plasmon-flua allows for a direct comparison of assay improvements with current fluorescently labeled reporter standards (fluorescently labeled streptavidin). Adding biotinylated PF to a sample already examined with streptavidin still allows the user to examine the sample over a very high dynamic range of target analyte concentrations, even when the reading device cannot sufficiently attenuate the signal (i.e., with PF, high concentrations of analyte produce fluorescence that saturates the detector, but with standard fluorophore-labeled streptavidin, it becomes readable, in which case the streptavidin can be conjugated to one or more fluorophores). To achieve a high dynamic range, the user first adds fluorescently labeled streptavidin, measures the fluorescence obtained from the assay, then adds biotinylated PF and re-reads the fluorescence obtained from the assay. This is particularly attractive for plate-based and bead-based assays. In practice, end users may prefer to directly use an antibody PF or detection-antibody PF conjugated with streptavidin, rather than first adding fluorescently labeled streptavidin, reading, and then examining again with biotinylated PF. This disclosure describes how FLISA was performed in a heterogeneous solid-phase format by using a 96-well microtiter plate as the sampling platform, which is a standard assay format widely used in biomedical research and clinical diagnostics.

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

[0211] As a signal enhancer in the final step, a nanostructure was added following the conventional fluorescent tag, 800CW-streptavidin. To investigate the enhancement of 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 nanostructure revealed a 2,000-fold increase in fluorescence intensity compared to conventional FLISA. Specifically, the fluorescence signal from conventional Flour (800CW) was detectable only at two maximum concentrations (6 and 0.6 ng / mL). On the other hand, the fluorescence signal from the fluorescent nanostructure could be detected even at concentrations as low as 6 fg / mL. The detection limits (LLOD = mean blank + 3σ) for the unenhanced and plasmon-enhanced IL-6 assays were found to be 600 pg / mL and 6 fg / mL, respectively, representing 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 includes 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 biological analytical parameters [LLOD, limit of quantification (LLOQ = mean blank + 10σ), dynamic range] of commercially available immunoassay kits without requiring cumbersome repetitive processes or special or expensive equipment.

[0212] A second application considered was signal enhancement in protein microarrays. For this purpose, a human kidney biomarker microarray was used on a 3D microporous nitrocellulose membrane. By simply adding a fluorescent nanoconstruct, all 38 protein biomarkers in human patient urine samples were visualized in a single, simple test, compared to 15 biomarkers revealed in assays using fluorescently labeled streptavidin.

[0213] In another aspect, the applicability of fluorescent nanoconstructs to enhancing the sensitivity of immunomicroarrays was investigated. Using a microarray of antibodies against biomarkers for human kidney disease (R&D systems, Inc. ARY019)27) as a representative example, the performance of fluorescent nanoconstructs was tested in a spatially multiplexed high-throughput biosensing platform. This microarray consisted of 38 capture antibodies corresponding to human kidney protein biomarkers, printed in doubles on a three-dimensional nitrocellulose membrane. Biotinylated IgG was printed in doubles as a reference (positive control). Double 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. Subsequently, the captured biomarker proteins were exposed to a biotinylated detection antibody cocktail, followed by exposure to 800CW-streptavidin. This step completed the conventional microarray procedure, at which point the biomarker concentration was (semi)quantified by analyzing the fluorescence intensity corresponding to each analyte. In the plasmon-enhanced assay, a solution of biotinylated fluorescent nanoconstructs was added to the microarray, incubated for 30 minutes, and then thoroughly washed to remove weakly bound nanostructures. This allows for a direct comparison between the gold-standard reporter method and the nanoconstructs; however, in practice, users may prefer to use streptavidin-conjugate PF instead of adding fluorescently labeled streptavidin first and then labeling it with biotinylated PF.

[0214] Fluorescence maps obtained using conventional Fluorescent and fluorescent nanoconstructs with human urine samples illustrate the improvement in fluorescence signaling. First, the brightness and SNR of the positive control were found to be 80-fold enhanced after the addition of the nanostructure. Simultaneously, no signal was detected from the negative control, indicating that the nanostructure made minimal nonspecific binding to the nitrocellulose membrane, which is important for ensuring low background. With conventional Fluorescent, only 14 of the 38 targeted protein biomarkers were detectable, and the majority showed weak intensity. After the addition of the nanostructure, the fluorescence signal intensity from each spot in the microarray increased considerably. SEM images of nitrocellulose after the addition of the fluorescent nanoconstructs revealed a uniform distribution of AuNR on the porous membrane without any signs of aggregation. The fluorescent signals corresponding to cystatin C, β2-microglobulin (beta-2M), serpine A3, and neutrophil gelatinase-associated lipocalin (NGAL) were found to be enhanced by up to 500-fold compared to the fluorescent signals obtained using fluorescently labeled streptavidin. Furthermore, the nanostructure 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 biomarker specific to the early detection of acute kidney injury, could only be detected after the addition of the fluorescent nanostructure. [Examples]

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

[0216] In yet another aspect, the ability of fluorescent nanostructures to enhance SNR in flow cytometry-based cell analysis [Figures 8(A-B) and 9(A-B)] was demonstrated. ErbB2 (human epidermal growth factor receptor 2)-positive epithelial breast cancer cells (SKBR3) were used as a model cell line. The cell surface receptor ErbB2 was immunostained using a standard fluorescent probe, followed by the addition of the nanostructure. A conventional two-step staining procedure was performed by sequentially incubating a formaldehyde-fixed SKBR3 single-cell suspension with biotinylated anti-ErbB2 and streptavidin-fluorophore (streptavidin-680LT). The nanostructure was optimized for 680LT by changing the aspect ratio and adjusting the longitudinal LSPR wavelength to 660 nm. After labeling with streptavidin-fluorophore, the cells were further incubated in the nanostructure suspension for 1 hour. Before proceeding to flow cytometry, enhancement of the fluorescence signal was tested and visually confirmed. Confocal laser scanning microscopy (CLSM) images of cells were obtained using conventional Fluor and nanostructures. As noted above, anti-ErbB2 was diluted to various concentrations and incubated with the cell suspension. Compared to conventional staining (i.e., streptavidin-fluorophores), a considerably brighter fluorescence signal was observed after the addition of 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, 5000 cells were analyzed using Guava InCyte, and a fluorescence signal [RED-R channel (excitation laser: 642 nm; filter: 662 / 15 nm)] was obtained by combining forward scattering (FSC) and side scattering (SSC). Because the nanostructure is nanoscale (approximately 75 nm), its binding to the cell surface did not alter the intensity of forward or side scattering (data not shown). The fluorescence signal histogram demonstrated that the intensity was 60 times greater when using the nanostructure compared to cells treated with streptavidin-fluer (Figure 9A). The fluorescence histogram also showed that after adding the nanostructure, ErbB2 expression on the cell surface could be detected even at a 100,000-fold dilution of the primary antibody (Figures 9B and 8b). On the other hand, when streptavidin-fluer was used alone, the fluorescence signal could not be detected at dilutions higher than 1000-fold. Using Flur and nanostructures, the average fluorescence values ​​obtained with primary antibodies at various dilutions demonstrate the potential of novel nanoconstructs for detecting low-abundance targets on the cell surface (Figure 8B). [Examples]

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

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

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

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

[0222] Figure 13 shows an example of a plasmon nanostructure in a dielectric material matrix acting as a spacer layer / coating. The dielectric material matrix is ​​coated with a functional layer (blue cloud). A target agent (pink "y" shape, e.g., antibody) is conjugated to the functional layer.

[0223] Figure 14 shows the extinction spectrum of a plasmon-fluer (AuNR coated with Ag plasmon nanostructure) conjugated to IRDye 800CW (excitation maximum = 784). The inset shows the LSPR maximum.

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

[0225] Figure 16 shows the extinction spectrum (excitation maximum = 550 nm) of a plasmon-flure conjugated to Cy3 (AuNR@Ag cuboid plasmon nanostructure). The inset shows the maximum LSPR wavelength.

[0226] Figure 17 shows that the discrepancy between the LSPR maxima of the plasmon-flure (AuNR@Ag cuboid plasmon nanostructure) and the excitation maxima of Cy3 does not significantly affect the overall brightness of the resulting plasmon-flure. This is because all plasmon-flures with the AuNR@Ag cuboid plasmon nanostructure exhibit large absorption in the region of the Cy3 excitation maxima (i.e., all of these structures show large overlap with the Cy3 excitation spectrum). Furthermore, even when multiple LSPR peaks exist for these nanostructures, and the LSPR peak with the highest amplitude differs significantly from the Cy3 excitation maxima wavelength, some peaks still exist in the region of the Cy3 excitation maxima. Compared to the extinction spectra in Figures 14 and 15, it is clear that for plasmon-flure conjugated to IRDye 800CW (AuNR coated with Ag plasmon nanostructures), the key parameters for significant enhancement are that the plasmon-flure has a large absorption near the excitation maximum of the fluorescent dye, and that the absorption spectrum of the plasmon-flure has a large overlap with the excitation spectrum of the dye.

[0227] Figure 18 shows plasmon nanostructures (silver-coated gold nanorods) coated within a dielectric matrix of a specific thickness (green shell). Fluorophores (red star shapes) are directly bonded to the outer surface of the dielectric matrix. Biorecognition elements (pink "y" shapes, e.g., antibodies) can be directly conjugated to spacers, which can be coated with functionalized substrate materials (blue clouds). [Examples]

[0228] Plasmon-fluer preparation procedure In some embodiments, plasmon-flu is prepared using plasmon-flu conjugated with streptavidin and / or antibody. To obtain only BSA-biotin-flu, the process can be stopped after step 7. These steps are as follows:

[0229] Step 1: Calibration. Based on the quenching of the coreplasmon nanostructure, a 40 mL solution having quenching 2 at the LSPR maximum value is prepared.

[0230] Step 2: Interface layer. In a ventilated hood, 40 μL of MPTMS was added to the solution of plasmon nanostructures, and this was left on an orbital shaker at 125 RPM for 1 hour.

[0231] Step 3: Spacer layer. In a ventilation hood, 160 μL of APTMS was added, the tube was inverted 10 times, 160 μL of TMPS was added, this was inverted 10 times, and the mixture was left 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 monomers / polymers. The solution from Step 3 is centrifuged. The plasmon nanostructures coated with spacers are collected in a pellet. The supernatant is removed and replaced with 1 mM CTAC to remove free silanes.

[0233] Step 5: Dye labeling. Add 250 μL of 10×PBS buffer (pH 7.4) to 4 mL of the above NP solution (quenching at LSPR maximum value of 20). Add 0.1 to 20 μL of dye molecules conjugated with NHS-ester and 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 with altered biotin density) in a 5 mg / mL BSA-biotin solution at pH > 6, mix thoroughly, and incubate overnight in the dark at 4°C. Purify the coated nanoparticles from the free BSA-biotin by centrifugation.

[0236] Step 8: Streptavidin coating. Resuspend the particles from Step 7 in a 10 mg / mL streptavidin solution 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 the free antibody by centrifugation.

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

[0239] Nanostructures and dye combinations for use in plasmon-flures The following classifications were made in accordance with the commonly used laser excitation wavelengths. Those skilled in the art will recognize that any excitation source that can be used to excite a conjugated fluorescent species can also be used to excite a plasmon-fluer containing such species. It is important to note that, since the LSPR shifts to red after coating with spacers and functional layers, the LSPR wavelength of the optimal plasmon nanostructure is generally shifted to blue (i.e., has a lower wavelength) compared to the optimal LSPR wavelength. The resulting plasmon-fluers have an absorption maximum close to the absorption maximum of the dye, and a large overlap between the plasmon-fluer quenching spectrum and the excitation / absorption spectrum of the dye.

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

[0241] In some embodiments, when using laser excitation wavelengths of 532 nm or 543 nm, preferred dyes include Cy3, AlexaFluor532, AlexaFluor543, AlexaFluor555, Atto532, Atto550, rhodamine / tetramethylrhodamine / rhodamine6G / TAMRA / TRITC, and Cy3.5 (Cy3.5). In some embodiments, preferred plasmon nanostructures include AuNR@Ag nanocuboids characterized by length = 86 nm (variable depending on LSPR, but size and LSPR are closely linked in these particular particles, unlike AuNR, in which case LSPR is a function of aspect ratio), width = 73 nm (see above), and LSPR = 500-570 nm.

[0242] In some embodiments, when a laser excitation wavelength of 633 nm is used, suitable dyes include Cy5, Cy5.5, Alexa fluor633, Alexa fluor647, Alexa fluor660, and Atto633. In some embodiments, suitable plasmon nanostructures include AuNR having 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 plasmon nanostructures include AuNR having an LSPR of 720-800 nm. [Examples]

[0244] Using Plasmon-Fleur, the time required to complete a sandwich immunoassay (compared to a 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 of the standard curve (dose-dependent colorimetric signal) for a human NGAL ELISA that takes 280 minutes to complete. Figure 20 is a plot showing the dose-dependent fluorescence intensity of human NGAL from p-FLISA performed within 20 minutes. Compared to conventional ELISA, p-FLISA, which includes ultra-high-intensity fluorescent nanoconstructs (Plasmon-Fleur-800CW), can be completed in 10 times less time while achieving similar detection limits. Figure 21 shows NGAL concentrations in urine samples from renal patients and healthy volunteers determined using p-FLISA completed within 20 minutes. Figure 22 is a plot showing the correlation between human NGAL concentrations determined using ELISA (280-minute assay) and p-FLISA (20-minute assay), demonstrating a good quantitative correlation (R) between the two methods. 2 The result was 0.984). In summary, the human NGAL detection assay can be completed in 20 minutes using plasmon-flur, in contrast to the 280 minutes required for conventional ELISA (as recommended by the supplier and validated by the experiments described herein). The 20-minute assay based on plasmon-flur showed the same detection limits as the 280-minute ELISA. [Examples]

[0245] Ultra-high luminance plasmon-fluids as inter-platform nanolabeling for detecting femtomole concentrations of biological analytes. As stated throughout this disclosure, the detection, imaging, and quantification of biomolecules in small amounts within biological fluids, cells, and tissues are fundamentally important, but remain significant challenges in biomedical research and clinical diagnostics. Utilizing plasmon-enhanced fluorescence, plasmon-flure-800CW exhibited a signal nearly 6700 times brighter than streptavidin labeled with the corresponding near-infrared (NIR) fluorophore (800CW). It should be noted that fluorescence-labeled streptavidin can be labeled with one or more fluorescent dyes.

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

[0247] Gold nanorods (AuNRs) are used as a representative plasmon nanostructure due to their easy tuning of longitudinal localized surface plasmon resonance (LSPR) wavelengths and aspect ratios, and the large electromagnetic field enhancement at both ends [see Figure 27(A-B)]. Figure 27A shows a TEM image of a gold nanorod (AuNR) used as a nanostructure in a plasmon-fluer-800CW. Figure 27B is a finite difference time-domain (FDTD) simulation showing the distribution of electric field intensity around the AuNR (the polarization of the incident beam is along the longitudinal axis of the AuNR). AuNR (length 83.0 ± 8.0 nm; diameter 24.3 ± 1.8 nm) is modified with (3-mercaptopropyl)trimethoxysilane (MPTMS), which acts as an interfacial layer for copolymerization of two organosilane monomers, namely (3-aminopropyl)trimethoxysilane (APTMS) and trimethoxypropylsilane (TMPS) (Figure 28). Figure 28 is a schematic diagram showing the steps involved in the formation of the polymer spacer on the AuNR. In an aqueous medium, APTMS and TMPS undergo rapid hydrolysis and subsequent condensation around the MPTMS-modified AuNR to generate an amorphous copolymer network (Figure 28). The siloxane copolymer acts 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 method allows for easy control of the spacer layer thickness, reduced to 1 nm (Figures 29-31). Figure 29 is an AFM image illustrating the increase in AuNR / polymer diameter under increasing monomer (MPTMS, TMPS, and APTMS) levels. Figure 30 shows the UV-visible spectra of AuNR under various polymerization conditions. Figure 31 is a plot showing the increase in AuNR diameter (twice the polymer layer thickness) under each polymerization condition measured from the AFM images.Modification of AuNR with MPTMS and subsequent polymerization of APTMS / TMPS reduced the zeta potential of cetyltrimethylammonium bromide-capped AuNR from +38.4±2.3mV to +29±2.6mV and +25.8±1.9mV, respectively, due to the partial replacement of the positively charged capping agent (CTAB) with a less charged siloxane copolymer (Figure 32). Figure 32 shows the zeta potentials of AuNR, AuNR / MPTMS, AuNR / MPTMS / polysiloxane (AuNR / polymer), and plasmon-fluer-800CW (AuNR / polymer / BSA-biotin-800CW). Error bars correspond to the standard deviation (n=3 repeated tests). [Examples]

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

[0249] Following the structural characterization of plasmon-fluer-800CW, the luminance of the fluorescent nanoconstruct was determined. The excited-state fluorescence lifetimes of free 800CW (conjugated to BSA) and plasmon-fluer-800CW were measured as 0.74 ± 0.01 ns and 0.179 ± 0.001 ns, respectively, representing a 7-fold improvement in quantum yield (calculated herein, from approximately 11% to approximately 79%). To further understand the luminance of plasmon-fluer-800CW, the number of fluorophores conjugated to a single AuNR was estimated. Plasmon-fluer-800CW at a concentration of 76.2 pM (approximately 0.63 quenching) contains approximately 16 nM of 800CW (calculated herein). Therefore, it is estimated that approximately 210 fluorophores are conjugated to a single AuNR. It should be noted that the fluorescence intensity from 76.2 pM plasmon-fluer-800CW (containing 16 nM 800CW) was found to be equal to the fluorescence intensity from 544 nM 800CW (measured based on Figure 2). The difference in the slopes of the two curves indicates that a single plasmon-fluer-800CW is as bright as a 6700 (±900) fluorophore. Therefore, it can be concluded that each 800CW is enhanced by approximately 30-fold in the presence of the plasmon nanostructure. Error bars correspond to the standard deviation (n=3 repeated tests). This corresponds to approximately 30-fold enhancement per bound fluorophore. This result was obtained for plasmon-fluer where the 800CW is conjugated to a functional layer, BSA. Figure 2 shows the fluorescence intensities of conventional Flour-800CW and plasmon-Flour-800CW at different molar concentrations, where the plasmon-Flour-800CW is directly bound to a spacer layer approximately 2-4 nm thick. The difference in the slope of the plot of plasmon-Flour based on AuNR conjugated by 800CW versus the fluorescence intensity of free, unconjugated 800CW in solution as a function of the fluorescence species concentration indicates that plasmon-Flour-800CW is approximately 20,000 × brighter than free 800CW.These data were collected using an Azure Sapphire scanner under the same excitation and emission conditions as for plasmon fleur and free 800 CW (excitation at 784 nm and detection through a bandpass filter with a width of 37 nm centered at 832 nm). The observed strong emission may be due to enhanced electromagnetic fields on the surface of the plasmon nanostructure [Figure 27 (A-B)] (locally excited regions) and reduced fluorescence lifetime due to coupling between excited fluorophores and surface plasmons of the nanostructure.

[0250] As shown in Figure 34, a schematic diagram (not to scale) illustrating a model system based on the binding events performed in this study, the feasibility of using plasmon-fluer-800CW as an ultra-high-brightness fluorescent reporter was tested by binding the ultra-high-brightness fluorescent reporter to a substrate coated with streptavidin-800CW. The binding of plasmon-fluer-800CW resulted in an average 1200 (±40)-fold improvement in ensemble fluorescence intensity compared to streptavidin-800CW. This indicates that the fluorescence intensity of 800CW-streptavidin was increased by the biotin-streptavidin interaction, followed by the specific binding of plasmon-fluer-800CW, resulting in an average 1200 (±40)-fold improvement in fluorescence intensity. Significant signal enhancement was achieved by using a relatively low concentration of plasmon-fluer (76 pM). It should be noted that in all quarantine assays to further verify the plasmon enhancement of fluorescence, "non-resonant" gold nanoparticles (AuNP) with a similar surface area to "resonant" AuNRs (7850 nm² / AuNP; 8064 nm² / AuNR) were used [see Figure 5 (A-B)]. To illustrate the importance of the overlap between the absorbance of the plasmon nanostructure and the absorbance / excitation spectrum of the conjugated dye, plasmon-flures were prepared using either gold spheres, AuNPs, or gold nanorods, with AuNR as the plasmon nanostructure core and 800 CW as the conjugated fluorescent species bound to BSA, and then adsorbed onto biotin as a spacer layer and biorecognition element. Their individual quenching spectra, as well as the absorption / excitation and emission spectra of 800 CW, are shown in the plot on the left. The fluorescence of the same concentration of material excited at 784 nm obtained is shown in the plot on the right. AuNP exhibits somewhat higher fluorescence compared to Fluor and 800CW alone, but it is only about 1 / 100th as bright as plasmon-Fluor, which has AuNR as the core plasmon nanostructure.Unsurprisingly, the fluorescence intensity of AuNP-plasmon-fluer-800CW was only increased 18-fold, which is about 1 / 70th of that obtained using AuNR-plasmon-fluer-800CW, confirming the fluorescence enhancement by the plasmon [Figure 5 (A~B)].

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

[0252] Figures 35 and 36 show core AuNR particles of various capacities to enhance the generated 800CW by adjusting the amount of added seed. The most commonly used seed amount in the literature for plasmon nanoparticles is 48 μL. Plasmon-flur was generated from various AuNR core particles and 800CW and, after normalizing to the same molar concentration, the fluorescence intensity was measured using an Azure Sapphire scanner by detection with an excitation wavelength of 784 nm and a bandpass filter with a width of 37 nm centered at 832 nm. Larger AuNRs have considerably higher brightness than the most commonly used AuNR for the same LSPR wavelength (indicated by #).

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

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

[0255] Figure 38 shows plasmon nanostructures suitable for enhancing fluorophores, which can be excited at 488 nm (Au@Ag-490), 658 nm (AuNR-670), and 784 nm (AuNR-760). The typical standard fluorophore excitation forms corresponding to the corresponding plasmon particles are highlighted. [Examples]

[0256] Figure 39(A-B) shows TEM images of AuNR@Ag nanocuboid (left) and plasmon-fluer-Cy3 (right), consisting of a coating of AuNR@Ag nanocuboid, polymer shell, and BSA-biotin-Cy3. The coating (functional layer and spacer layer) is approximately 6 nm thick. Figure 40 shows the extinction spectra of AuNR@Ag nanocuboid, AuNR@Ag nanocuboid coated with polymer spacer, and plasmon-fluer-Cy3, clearly showing a continuous red shift after each coating step. [Examples]

[0257] The optimal distance between the metal surface and the fluorophore is crucial for maximizing fluorescence enhancement by balancing two opposing factors: electromagnetic field enhancement and non-radiative energy transfer. Fluorescence enhancement of plasmon-fluer-800CW with dielectric spacers of various thicknesses (MPTMS, APTMS, and TMPS) was investigated by conjugating dielectric spacers to a substrate coated with streptavidin-800CW. The ensemble fluorescence enhancement coefficient (defined as the ratio of fluorescence intensity after addition of plasmon-fluer to fluorescence intensity before addition) of plasmon-fluer without a polymer spacer layer was found to be approximately 146 ± 81. Enhancement efficiency gradually increased 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 conjugated fluer and the metal surface. This figure represents plasmon fluer with fluorescently labeled BSA. While this figure generally appears to indicate that the optimal spacer thickness lies between 0.8 and 2.9 nm, this is not necessarily the case for plasmon fluer where fluorophores are directly bonded to the spacer coating. In other words, when fluorophores are bonded to BSA acting as a functional layer, the BSA itself acts as a spacer between the fluorophores and the plasmon nanostructure; therefore, the polymer thickness is not actually the distance between the bonded fluer and the surface of the plasmon nanostructure. When using BSA with conjugated fluorophores, some fluorophores are located right next to the spacer layer, while others are approximately 4 nm away. 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.From a study 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, it was found that the optimal space between the fluorophore and the plasmon nanostructure surface is between 2 and 5 nm, which is consistent with the results presented here. Notably, the plasmon-flure colloidal solution exhibited a stable fluorescence signal after being stored in the dark at 4°C for one month (Figure 42). For further ease of storage, transport, and handling, the plasmon-flure can be freeze-dried and, if necessary, restored without significant degradation of the fluorescence signal (Figure 42). [Examples]

[0258] Plasmon-flure-enhanced fluorescence-coupled immunosorbent assay (p-FLISA) and bead-based multiplex assays Among the many applications of plasmon-fluer, plasmon-enhanced fluorophore-conjugated immunosorbent assay (p-FLISA) was performed on standard microtiter plates. Human interleukin-6 (IL-6), a pro-inflammatory cytokine, was used as a representative protein biomarker. Conventional FLISA requires a standard sandwich format of capture antibody, analyte (IL-6), biotinylated detection antibody, and then exposure to streptavidin-fluorophore (800CW in this study) (Figure 43). Figure 43 is a schematic diagram illustrating the concepts of conventional FLISA (800CW) and plasmon-fluer-800CW-enhanced FLISA (p-FLISA) performed on a standard 96-well plate. The p-FLISA assay requires no changes to the standard workflow except for the addition of plasmon-fluer as a new final step. In p-FLISA, plasmon-fluer-800CW is introduced after the final step as a signal enhancer (Figure 43). To determine if sensitivity and detection limit (LOD), defined as the mean value of the blank plus 3σ, were improved, serial dilutions of IL-6 at known concentrations [6 ng / ml to 6 fg / ml in 1% BSA buffered with phosphate-buffered saline (PBS)] were used as standards. Fluorescence signals obtained after applying Plasmon-Fleur-800CW revealed that at the highest analyte concentration tested here (6 ng / ml), the ensemble fluorescence intensity was increased by approximately 1440 times compared to conventional FLISA (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. Standard curves were generated using polynomial fitting.Error bars correspond to the standard deviation (n=2 repeated trials). Figure 46 shows individual data points, mean, and standard deviation from human IL-6 FLISA, p-FLISA, and ELISA. On the other hand, the fluorescence signal from p-FLISA could be detected even when the concentration was reduced to 20 fg / ml (approximately 1 fM) [Figures 49 and 46, 4-parameter logistic (4PL) fitting], which corresponds to a 4750-fold improvement in the limit of detection (LOD) compared to conventional FLISA. Figure 49 shows a plot of dose-dependent fluorescence intensity of human IL-6 from p-FLISA. Compared to conventional FLISA, p-FLISA shows a 4750-fold improvement in the limit of detection (LOD) and a large dynamic range of more than three orders of magnitude. It should be noted that plasmon-flure showed very high specificity (against streptavidin) and low nonspecific binding to interfering biomolecules in the bioassay [Figure 50 (A-B)]. Figure 50A shows the IL-6 dose-dependent fluorescence intensity from p-FLISA. Error bars correspond to the standard deviation (n=2 repeated trials). Figure 50B shows the nonspecific binding of Plasmon-Fleur-800CW. C: Capture antibody; D: Detection antibody; S: Streptavidin; PF: Plasmon-Fleur; Blank: No Plasmon-Fleur. Compared to the blank, no signal was observed after applying Plasmon-Fleur-800CW to BSA, the capture antibody, or the capture and detection antibody. ****P<0.0001 (by one-way ANOVA with Tukey's post-hoc test). NS: No significant difference. The nonspecific signal at zero IL-6 concentration was only present when streptavidin was introduced, suggesting the superior specificity of plasmon-fluer. Error bars correspond to the standard deviation (n=3 repeated trials). This "BSA-blocking" strategy of plasmon-fluer is important for increasing the signal-to-background ratio. Scanning electron microscopy (SEM) images revealed an increase in the density of plasmon-fluer-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, clearly showing an increase in the density of plasmon-fluer-800CW with increasing IL-6 concentration. In blank wells incubated with 1% BSA, a fairly low density of plasmon-fluer was observed, indicating low nonspecific binding of plasmon-fluer (Figure 51).

[0259] Surprisingly, the LOD and lower limit of quantification [(LLOQ), defined as the mean of the blank + 10σ, approximately 82 fg / ml] of p-FLISA were found to be 189-fold and 120-fold lower, respectively, than the “gold standard” enzyme-linked immunosorbent assay (ELISA), which includes 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 showed a 1 / 189th lower LOD and a dynamic range more than two orders of magnitude larger. More importantly, p-FLISA showed a dynamic range of five orders of magnitude (ratio between the upper and lower limits of quantification), which is more than two orders of magnitude larger than the dynamic range of ELISA. To validate assay performance, p-FLISA was used to test healthy human serum samples and IL-6 spiked serum. Serum samples were diluted 10-fold, resulting in only 10 μl of the original sample being required for each individual subject. In healthy individuals, IL-6 concentrations typically range from 0.2 to 7.8 pg / ml. Elevated serum IL-6 levels may indicate systemic inflammation, metabolic stimulation, and physiological stimulation. It should be noted that of ELISA, FLISA, and p-FLISA, only the latter technique can determine IL-6 concentrations in healthy individuals, which, after correction for dilution factors, were measured at 8.1 pg / ml, 1.8 pg / ml, and 2.8 pg / ml (Figure 53). Figure 53 shows IL-6 concentrations in human serum samples (diluted 10-fold) measured using p-FLISA. Error bars correspond to the standard deviation (n=3 repeated tests).

[0260] In addition to the microtiter plate format, the application of plasmon-fluer as a hyperluminescent reporter in microbead-based multiplex fluorescence immunoassays utilizing non-flat sampling surfaces was also investigated. Using the Luminex assay as an example, this assay utilizes magnetic microbeads embedded with ratio-defined fluorophores as barcodes for each specific analyte (Figure 54). Figure 54 is a schematic diagram illustrating the concept of using plasmon-fluer-Cy3 to enhance the sensitivity of a bead-based immunoassay (e.g., the Luminex assay). Antibody-conjugated microbeads, in a typical sandwich format, capture the analyte to facilitate its detection, followed by examination with streptavidin conjugated with phycoerythrin (PE), a hyperluminescent fluorescent protein isolated from red algae or cyanobacteria. However, the PE used in the Luminex assay is structurally unstable and prone to photobleaching. Here, highly stable fluorophores similar to PE, exhibiting absorption and emission at 554 nm and 568 nm, respectively, were used as substitutes. As discussed above, it is crucial to select plasmon nanostructures having LSPR wavelengths that match the excitation maximum wavelength of the fluorophores. For this purpose, plasmon-fluer-Cy3 [Figures 55(A-B), 39(A-B), and 40] were fabricated using AuNR@Ag nanocuvoids with an LSPR wavelength of 520 nm. Figures 55(A-B) are TEM images of plasmon-fluer-Cy3 utilizing AuNR@Ag as the plasmon nanostructure, with a spacer coating thickness of approximately 6 nm. Figure 39(A-B) shows TEM images of AuNR@Ag nanocuboids (left) and plasmon-fluer-Cy3 (right), consisting of an AuNR@Ag nanocuboid, a polymer shell, and a BSA-biotin-Cy3 coating. The coating is approximately 6 nm thick.Figure 40 shows the extinction spectra of AuNR@Ag nanocuboids, AuNR@Ag nanocuboids coated with polymer spacers, and plasmon-fluer-Cy3, clearly showing a continuous red shift after each coating step. It should be noted that the synthesized plasmon-fluer-Cy3 exhibits considerably high 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-fluer-Cy3. Figure 56B shows the corresponding SEM image of the individual plasmon-fluer-Cy3 shown in Figure 56A. Figure 56C is a magnified SEM image corresponding to the rectangles shown in Figures 56A and 56B, showing a single plasmon-fluer-Cy3 (single nanocuboid). These fluorescence images were obtained by a non-laser epifluorescence microscope, which is widely available in a typical laboratory.

[0261] A specially modified Luminex assay was used to simultaneously detect mouse IL-6 and mouse tumor necrosis factor-α (TNF-α), key pro-inflammatory cytokines involved in cell signaling and immune modulation. Microbeads were incubated with serially diluted mixtures of TNF-α and IL-6, and then with the detection antibody cocktail of streptavidin-Cy3 and biotinylated plasmon-fluer-Cy3 (Figure 54). Subsequently, the beads were read using a dual-laser flow-based instrument (Luminex200), where a classification laser (635 nm) read the barcode of each bead, and a reporter laser (532 nm) determined the intensity of Cy3 fluorescence, which is directly proportional to the amount of bound analyte (Figure 54). SEM images of the microbeads showed uniform binding of plasmon-fluer-Cy3 with no signs of aggregation (Figure 57). Figure 57 shows SEM images of microbeads before and after examination with plasmon-fluer-cy3. Binding of plasmon-fluer-cy3 did not alter the size and shape of the beads [Figure 58(A-B)] or the optical barcode signal [Figure 59(A-D)]. Figure 58(A-B) shows bright-field and fluorescence images of Luminex microbeads before (Figure 58A) and after (Figure 58B) examination with plasmon-fluer-cy3. Figure 59(A-D) shows fluorescence images of Luminex microbeads after staining with plasmon-fluer-cy3, showing the barcodes of microbeads at various emission intensities (excited by a 633 nm laser) (Figure 59A) and the fluorescence of bound Cy3 (Figure 59B) (excited by a 543 nm laser). Bright-field image of the microbeads (Figure 59C). A combined bright-field and fluorescence image (Figure 59D). The scale bar corresponds to 50 μm. A significant increase in microbead fluorescence intensity was observed after binding of plasmon-fluer-Cy3 (Figure 60). Figure 60 shows fluorescence images of microbeads before and after examination with plasmon-fluer-Cy3.The LODs for 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 curve obtained before (left) and after (right) application of plasmon-fluer-Cy3. Figure 62 shows the mouse TNF-α standard curve obtained before (left) and after (right) application of plasmon-fluer-Cy3. All standard curves were obtained independently at least three times with different batches of plasmon-fluer over different days. Compared to the unenhanced equivalent [Figures 61, 62, 63, and 64 (A-B)], the plasmon-enhanced assays showed lower LODs for mouse IL-6 and mouse TNF-α, at 1 / 143 and 1 / 814, respectively. Figure 63 shows individual data points, mean, and standard deviation from Luminex assays of mouse IL-6, Luminex assays of plasmon-fluer-cy3 enhanced mouse IL-6, Luminex assays of mouse TNF-α, and Luminex assays of plasmon-fluer-cy3 enhanced mouse TNF-α. Figure 64A is a plot showing the LOD of the unenhanced bead-based fluorescence immunoassay (Luminex) for mouse IL-6. Figure 64B is a plot showing the LOD of the unenhanced bead-based fluorescence immunoassay (Luminex) for TNF-α. The curves were generated using polynomial fitting. Error bars correspond to the standard deviation (n=2 repeated trials). It should be noted that the supplier-specified LOD (using PE-streptavidin) for mouse IL-6 (2.3 pg / ml) and mouse TNF-α (1.47 pg / ml) was found to be inferior to that of plasmon-enhanced Luminex assays, at 1 / 41 and 1 / 196, respectively. Essentially, plasmon-flur acts as a powerful platform technology to enhance various existing immunoassays' biological analytical parameters (LOD, LLOQ, dynamic range) without requiring any cumbersome procedures or specialized equipment. [Examples]

[0262] Plasmon-flur-enhanced high-throughput multiplex proteomics arrays: Fluorescence-based biomolecular (micro)arrays are important clinical and research tools, particularly for simple, high-throughput, and rapid proteomics and genetic analysis, enabling the miniaturization of thousands of assays onto a single small piece of analytical substrate. Despite advantages such as high multiplicity, rapid screening, and small sample volumes, this method suffers from low sensitivity (even inferior to ELISA), which hinders its wide range of applications.

[0263] The applicability of plasmon-flur for increasing the sensitivity of immunoarrays was investigated. All error bars correspond to the standard deviation (n=2 repeated trials). A series of antibodies against human kidney disease biomarkers were used as representative examples (Figure 65). Figure 65 is an explanatory diagram illustrating the use of plasmon-flur-800CW to enhance the biological analysis parameters of a multiplex proteome profiler against human kidney disease biomarkers, performed on a nitrocellulose membrane. This example illustrates the use of biotinylated plasmon-flur to enhance a typical multiplex microarray, in which the capture antibody against the specific analyte is printed as spatially distinct spots on either a membrane, glass slide, or polystyrene substrate. In this method, the user can first label the array with standard fluorescence-labeled streptavidin and then label it with biotinylated plasmon-flur. In some embodiments, streptavidin-conjugated plasmon-flur is used to enhance a typical multiplex microarray, in which case capture antibodies against specific analytes are printed as 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 pairs on a microporous nitrocellulose membrane [Figure 67(A-B)]. Figure 67(A-B) shows the identification of specific analytes (or controls) for each pair of fluorescent spots against the kidney biomarker array. The fluorescent spots shown in Figure 67A are identified by their 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 with blocking buffer, mixed with a biotinylated detection antibody cocktail, and added to the nitrocellulose membrane. After incubation, the membrane was exposed to streptavidin-800CW.Finally, the plasmon-fluer-800CW suspension is added to the array, incubated, and thoroughly washed to remove unbound nanoconstructs (Figure 65).

[0264] SEM images derived from the positive control region revealed a uniform distribution of plasmon-fluer on the membrane (including the porous subsurface region) (Figure 68). Figure 68 is an SEM image showing the uniform distribution of plasmon-fluer-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 renal disease protein biomarker profiles of kidney disease patients shown in Figures 69 and 70 after the addition of plasmon-fluer-800CW (note the difference in fluorescence intensity scale bars). Simultaneously, no signal was detected from the negative control (Figure 71: blue square), indicating that plasmon-fluer was not observed in SEM images from these locations, and their nonspecific binding was minimal [Figure 72(A~B)]. Figure 72A shows the kidney biomarker array from Figure 67A. Figure 72B is an SEM image showing a nitrocellulose membrane in the negative control region [the blue rectangle at the lower right edge of Figure 72A (corresponding to the pair at coordinates F23 and F24 shown in Figure 67A); note that no fluorescence signal or plasmon-fluer-800CW is present] after the addition of plasmon-fluer-800CW, indicating low nonspecific binding of plasmon-fluer-800CW. With conventional fluorophores, only 26 of 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 renal disease protein biomarker profiles of patients with renal disease, obtained using a conventional fluorophore (streptavidin-800CW). Figures 73 and 74 show individual data points, mean, and standard deviation with and without plasmon-fluer, respectively. Figure 75 is a digital photograph taken with a mobile phone showing the color change of a nitrocellulose membrane after the addition of plasmon-fluer-800CW, using a urine sample from a patient with renal disease.After the addition of Plasmon-Fleur-800CW, the fluorescence signal intensity from each spot in 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 Fleur. Furthermore, a commercially available 40-plex cytokine microarray was used as another validation for Plasmon-Fleur, in which case a significant improvement in microarray sensitivity was also observed [Figure 76(A-F)]. Figure 76A shows the arrangement of the 40-plex cytokine microarray. Each antibody is printed horizontally in a quadruple row at each spot with a diameter of approximately 140 μm. Fluorescence maps of the cytokine microarray obtained using conventional fluorophores (Streptavidin-800CW) (Figure 76B) and after the addition of Plasmon-Fleur-800CW (Figure 76C). Plots showing the fluorescence intensity corresponding to each cytokine obtained using a conventional fluorophore (streptavidin-800CW) (Figure 76D) and after adding plasmon-fluer-800CW (Figure 76E). Error bars correspond to the standard deviation (n=4 repeated trials). Dark-field scattering of plasmon-fluer-800CW (AuNR) absorbed by the 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-fluer-800CW) on each microspot is clearly shown and can be digitally counted.

[0265] Plasmon nanostructures exhibit a large extinction cross-section at LSPR wavelengths, which can be up to 5–6 orders of magnitude larger than the light absorption of most organic dyes. This unique property of plasmon nanostructures leads to the potential use of plasmon-fluer as a multimodal biolabel. Indeed, binding of plasmon-fluer to the detection domain results in concentration-dependent colored spots of the analyte, which are directly visible to the naked eye (Figure 75). The color intensity of each spot in digital photographs obtained using a mobile phone camera under ambient light conditions was analyzed and compared to the corresponding fluorescence intensity. A good correlation was observed between the two acquisition modes (R2=0.88, Figure 77), which indicates the potential applicability of this nanoconstruct as a “visible label” in resource-limited situations, mitigating reliance on expensive, dedicated reading equipment. Figure 77 is a plot showing the correlation between the two reading modes (fluorescence readings versus color readings) of a kidney biomarker array. [Examples]

[0266] Immunocytochemistry / immunofluorescence (ICC / IF) enhanced by plasmon-flur: Immunocytochemistry based on immunofluorescence is a well-established semi-quantitative method for analyzing the relative abundance, three-dimensional structure, and intracellular localization of target antigens in cells. However, this method still lacks sensitivity to distinguish low-abundance biomolecules from the noise level due to the weak fluorescence signal of conventional fluorophores. Autofluorescence, the spontaneous emission of light from biological structures, further contributes to the overall low signal-to-noise ratio.

[0267] To test the applicability of plasmon-fluer 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 a standard method (biotinylated ErbB2 primary antibody and streptavidin-800CW), and then plasmon-fluer-800CW was added (Figure 78). Figure 78 shows confocal laser scanning microscopy (CLSM) images of breast cancer cells (SK-BR-3) examined with conventional fluer (800CW, top row) and plasmon-fluer-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 incubated with cells. SEM imaging revealed a uniform distribution of plasmon-fluer 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-fluer-800CW. Figure 79B shows SEM images of SK-BR-3 cells labeled with conventional fluer and SK-BR-3 cells labeled with plasmon-fluer-800CW (Figure 79C), and the inset shows the uniform distribution of plasmon-fluer on the cell membrane. Confocal laser scanning microscopy (CLSM) imaging of cells revealed that the fluorescence signal increased up to 100-fold (after subtracting background) after the addition of plasmon-fluer (20 pM) [Figures 78, 80, 81(A-B), and 82], and ErBb2 receptor expression could be imaged even at a 100,000-fold dilution (10 ng / ml) of the primary antibody [Figures 78, 81(A-B)]. Figure 80 is a plot showing the fluorescence intensity of SK-BR-3 cells stained with conventional fluer and plasmon-fluer-800CW. Error bars correspond to the standard deviation (at three different locations). Conventional immunocytochemistry procedures (sequential labeling of cells using biotinylated primary antibody and streptavidin-fluer (800CW)) were performed at various dilutions of the ERbB2 primary antibody.Confocal laser scanning microscopy (CLSM) images of ErbB2-stained breast cancer cells (SK-BR-3) obtained by first using (see Figure 81A) and then adding Plasmon-Fleur-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. Cells are examined using conventional fluorophores (top) and then Plasmon-Fleur-800CW (bottom). The scale bar corresponds to 1 cm. In stark contrast, the fluorescence signal could only be imaged at a dilution of 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-Fleur in significantly reducing the amount of antibody required for ICC / IF (and the resulting cost), but also the ability to image biomarkers with low abundance on the cell surface using Plasmon-Fleur. [Examples]

[0268] Plasmon-flure enhanced flow cytometry measurement Flow cytometry is widely used in cell analysis to measure the expression and relative abundance of specific analytes on or inside cells at a rate of thousands of cells or more per second (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 by plasmon-Fluor-680LT. However, because the target species cross the laser focal point and are very fast, flow cytometry still faces significant challenges regarding the signal-to-noise ratio of fluorescence and has limited time to read the fluorescence. Also, background fluorescence originating from cells (autofluorescence) makes it difficult to accurately depict small changes in the expression levels of intracellular and extracellular targets.

[0269] To test the ability of plasmon-fluer to enhance the signal-to-noise ratio in flow cytometry-based cell analysis (Figure 83), SK-BR-3 cell suspension was incubated with ErbB2 primary antibody and streptavidin-680LT, followed by the addition of plasmon-fluer-680LT. Labeled cells were then harvested by mild centrifugation (1000 rpm) with removal of unbound plasmon-fluer. To match the excitation laser with fluorophore emission, plasmon-fluer-680LT [Figure 84(A-B)] was generated using an AuNR with an LSPR wavelength of approximately 647 nm as a nanostructure. Figure 84(A-B) shows TEM images and extinction spectra of 680LT. Specific binding of plasmon-fluer-680LT caused a significant color change in the cell pellet [Figure 85(A-B)]. Figure 85A illustrates photographs showing the color changes of SK-BR-3 cells (top: pellet; bottom: suspension) after labeling with plasmon-fluer-680LT. Figure 85B shows the visible-NIR extinction spectra of SK-BR-3 cell suspensions labeled with plasmon-fluer-680LT under various dilutions of the ErbB2 primary antibody. The presence of plasmon-fluer-680LT on the cell surface did not alter the intensity of forward or side scattering (Figure 86), indicating that cell size and particle size / complexity remained virtually unchanged after binding of plasmon-fluer-680LT. Figure 86 shows pseudocolor plots of side and forward scattering of SK-BR-3 cells before (left) and after (right) labeling with plasmon-fluer-680LT (including an example of a gated strategy to include single cells), showing no apparent change in their size profile. Flow cytograms of SK-BR-3 cells with fluorescence paired forward scattering (canceled vertically for clarity) revealed a more pronounced separation of cell populations stained with plasmon-fluer-680LT compared to those obtained using conventional fluorophores (Figure 87).Figure 87 shows a flow contour plot (including outliers) of fluorescence vs. forward scatter of SK-BR-3 cells examined using ErbB2 primary antibodies at various concentrations (subtracted vertically for clarity). (Red: control group without addition of primary antibody, blue: cells treated with primary antibody at various dilutions). The cells were stained by adding conventional fluorophore (680LT, left plot), and then plasmon-fluorophore-680LT (right plot). A histogram of the cell fluorescence signal revealed that, compared to its conventional equivalent, using plasmon-fluorophore-680LT resulted in up to 60-fold higher intensity (subtracting background) (Figure 88). Figure 88 shows fluorescence histograms of SK-BR-3 cells examined using addition of conventional fluorophore (680LT), and then plasmon-fluorophore-680LT (at a 10 3 -fold dilution). Error bars correspond to standard deviation (independent tests with n = 3). **** p < 0.0001 (by two-sided independent t-test with Welch's correction). The fluorescence histogram revealed that the expression of ErbB2 on the cell surface can be detected using plasmon-fluorophore-680LT labeling even with a primary antibody at a dilution of 200,000-fold (5 ng / ml) (Figures 89, 90). Figure 89 is a histogram showing the fluorescence amounts of SK-BR-3 cells before (top) and after (bottom) addition of plasmon-fluorophore-680LT. Red: without primary antibody; blue: 2×10 5 -fold dilution; orange: 10 5 -fold dilution; light green: 10 4 -fold dilution; green: 10 3 -fold dilution; pink: 10 2 -fold dilution of the stock solution provided by the supplier. Figure 90 is a plot showing the mean fluorescence intensity obtained from flow cytometry at various primary antibody concentrations. On the other hand, for conventional labeling, it was necessary to dilute the antibody to less than 1000-fold (i.e., concentration > 0.5 μg / ml) to ensure a detectable increase in the fluorescence signal compared to the background (blank) (Figures 89, 90).

[0270] To further validate the performance of plasmon-fluer in depicting cell populations with small differences in surface receptor expression levels, bone marrow-derived dendritic cells (BMDCs) were used as a model system in which receptor surface expression can be modulated using immunogenic stimulation. After exposure to immunogenic stimulation, dendritic cells undergo activation and maturation, resulting in cytokine secretion and upregulation of maturation markers such as CD40, CD80, CD86, MHC I, and MHC II. Here, BMDCs were isolated from 6-8 week old C57BL / 6 mice and treated with lipopolysaccharide (LPS) as an immunogenic stimulus to induce dose-dependent upregulation of CD80 and cytokine release. The cells were then immobilized and treated with a biotinylated CD80 antibody. Finally, BMDCs were examined with conventional fluorophores (680LT) followed by plasmon-fluer-680LT, and fluorescence levels were compared using a flow cytometer (Figure 91). Figure 91 is a schematic diagram showing bone marrow-derived dendritic cells (BMDCs) treated with an immunostimulator [lipopolysaccharide (LPS)]. Small changes in post-stimulation maturation marker (CD80) expression were detected by immunofluorescence staining followed by the addition of plasmon-flur-680LT. Figure 92 shows two schemes for using antibody-labeled plasmon-flur to label target antigens on cells.

[0271] Histograms of fluorescence intensity distributions for naive (control) and LPS (0.05 μg / ml) stimulated BMDCs, obtained using conventional Fluor (680LT) and plasmon-Fluor-680LT, are shown in Figures 93 and 94, respectively. Clearly, BMDCs stained with plasmon-Fluor showed a significant difference in fluorescence between the activated cell population (blue) and the naive (red) cell population [Figures 93, 94, and 95(A-B)]. Figure 95(A-B) shows a pseudocolor plot of lateral scatter versus CD80 fluorescence of BMDC populations treated with 0.05 μg / ml LPS (right), without LPS stimulation (left: naive), and using conventional immunofluorescence staining (Figure 95A) and plasmon-Fluor-680LT (Figure 95B). Further investigation of dose-dependent (0-0.05 μg / ml) stimulation of BMDCs with LPS revealed that using Plasmon-Fleur-680LT resulted in a rapid increase in mean fluorescence intensity followed by a stable phase at higher LPS doses [Figures 96, 97(A-B)], indicating increased CD80 expression. Figure 96 is a plot showing the mean fluorescence intensity of BMDCs (corresponding to CD80 expression levels) after stimulation with various amounts of LPS. However, BMDCs stained with conventional fluorophores showed shallow fluorescence with increasing LPS dose, which was obscured by a high fluorescence background [Figures 96 and 97(A-B)]. Figures 97(A-B) are plots showing the mean fluorescence intensity of BMDCs (corresponding to CD80 expression levels) after stimulation with various amounts of LPS. BMDCs were examined using conventional immunofluorescence staining (Figure 97A) followed by Plasmon-Fleur-680LT (Figure 97B). Furthermore, the secretion levels of pro-inflammatory cytokines (TNF-α and IL-12) tended to increase with increasing LPS concentration (Figures 98 and 99). Figure 98 shows the secretion levels of pro-inflammatory cytokines (TNF-α and IL-12), confirming 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 precision of plasmon-flure in identifying dose-dependent activation and maturation of BMDCs, as well as subtle changes in cell surface maturation markers. [Examples]

[0272] Synthesis of AuNRs to enhance 800CW and 680LT: AuNR-760 (LSPR wavelength approximately 760 nm), suitable for enhancing 800 CW, was prepared by seed-mediated synthesis. 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 of HAuCl4 (10 mM) (Sigma-Aldrich, Inc., 520918) and 9.75 ml of 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, a growth solution was prepared by sequentially adding aqueous HAuCl4 (0.01M, 2 ml), CTAB (0.1M, 38 ml), AgNO3 (0.01M, 0.5 ml, Sigma-Aldrich, Inc., 204390), HCl (1M, 0.8 ml, Sigma-Aldrich, Inc., H9892), and ascorbic acid (0.1M, 0.22 ml, Sigma-Aldrich, Inc., A92902), and then gently inverting the solution to homogenize it. By changing the volume ratio of AgNO3 and HCl, the correct wavelength can be obtained. 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 quenching 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, this value may vary) and ascorbic acid (0.1 M, 0.32 ml). [Examples]

[0273] Synthesis of AuNR@Ag for Cy3: AuNR with an LSPR wavelength of approximately 711 nm was used as the core for synthesizing AuNR@Ag nanostructures. Specifically, 3 ml of 711 nm AuNR (peak quenching of approximately 4) was 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 added sequentially, and this 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. [Examples]

[0274] Conjugation procedure for fluorescently labeled functional group plasmon fleurs: Biotin and 800CW were sequentially conjugated to BSA using EDC / NHS chemistry. In buffer solutions of pH 7-9, the NHS ester reacted well with the primary amino group (-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 number: 21329) was added to 2.2 ml of BSA (Sigma-Aldrich, Inc., A7030) solution (5 mg / ml in 1× PBS). This 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 number: 21329) pre-equilibriumated with 1× 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 purified BSA-biotin solution to raise the pH. Then, 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. Next, free NHS-800CW was separated from the conjugate using a Zeba desalting column and pre-equilibrium with nanopure water. BSA-biotin-680LT and BSA-biotin-Cy3 were prepared using a similar method, except for a change in the fluorophore. [Examples]

[0275] Synthesis of plasmon-fluer based on fluorescently labeled functional layers To produce highly efficient fluorescence-enhancing plasmon-flures, it is crucial to select a plasmon nanostructure that is "on-resonant" to a given fluorophore. For 800CW, AuNR-760 (with lengths and diameters of 83 and 24 nm, respectively) was used as the nanostructure. 1 μl of MPTMS (Sigma Aldrich, Inc., 175617) was added to 1 ml of AuNR with a quenching of approximately 2.0, and the mixture was shaken for 1 hour to form an interface 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 at 6000 rpm for 10 minutes each time to remove free monomers. After the second centrifugation, the AuNR / polymer was concentrated to a final volume of 10 μl.

[0276] Next, the BSA-biotin-800CW conjugate was coated around the AuNR / polymer. Specifically, 1 μl of 20 mg / ml citric acid (Alfa Aesar, 36664) was added to 100 μl of BSA-biotin-800CW (approximately 4 mg / ml) to lower the pH. Subsequently, the concentrated AuNR / polymer solution was added to this mixture and sonicated in the dark for 20 minutes. Next, the nanostructures were collected using mild centrifugation (5000 rpm, 3 minutes). Subsequently, the AuNRs were 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 redispersed in 1% BSA (buffered with 1×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, and the grid was made 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 Dimension3000 in light-trapping mode using a silicon cantilever with a nominal spring constant of 40 N / m. Extinction spectra of plasmon nanostructures were obtained using a Shimadzu UV-1800 spectrophotometer. Fluorescence lifetimes 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° relative to a PMT R928P detector (Hamamatsu Photonics KK, Japan). Unless otherwise specified, most fluorescence mapping was recorded using the LI-COR Odyssey Clx imaging system. Fluorescence signals from microbeads were read using the Luminex200 system. Cell imaging was performed under a 40× water immersion objective using Olympus FV1000 LSM confocal laser scanning microscopy (785nm excitation laser). Flow cytometry data was acquired using Guava easyCyte.

[0278] Calculation of Protein / Biotin Ratio: The BSA / biotin ratio was calculated by the 4-hydroxyazobenzene-2-carboxylic acid (HABA) assay. Specifically, biotinylated BSA (0.4 mg / ml × 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 replaced HABA from avidin, and the absorbance at 500 nm decreased proportionally. The change in absorbance is given by the following formula: ΔA 500=(0.9 × A 500 )-A 500 B (1) (In the formula, A 500 and A 500 B was calculated using the absorbance of HABA / avidin before and after the addition of biotinylated BSA. 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 determined by Baer's Law: A λ =ε λ bC (2) [In the formula, A λ The wavelength is λnm (ΔA 500 This is the absorbance of the sample at ε. λ This is the extinction coefficient at a wavelength λnm (34,000M -1 cm -1 The formula can be used to calculate the biotin concentration using the following equation: b is the cell path length (1 cm, using a quartz cuvette), and c is the sample concentration. Using this formula, the biotin concentration is:

number

number

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

number

[0280] [Table 8]

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

number

number

[0282] Estimation of the amount of 800CW absorbed by a sample plasmon-fluer-800CW: To estimate the amount of fluorophore on AuNR, the amount of BSA (-conjugate) was first estimated using a bicinchoninic acid assay (BCA assay). Since the pigment-to-protein ratio was found to be 1.2 (as above), the concentration of 800CW could be calculated from the amount of BSA. A microBCA protein assay kit (Thermo Scientific Inc., product number 23235, lot number QG218473A) was used for this test. 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-Fleur-800CW (extension approximately 4.6) was mixed with 150 μl of working reagent, and the mixture was incubated at 60°C for 1 hour. Absorbance at 562 nm was measured by a plate reader to obtain a BSA standard curve. Based on the standard curve, the concentration of BSA absorbed around Plasmon-Fleur-800CW was calculated to be 6.2 μg / ml. Therefore, Plasmon-Fleur-800CW with an extion 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 approximately 83 nm and diameter approximately 24 nm) is ε ≈ 8.27 × 10⁻⁶ 9 (LM -1 cm -1 ) was calculated. The molar concentration of AuNR corresponding to 0.63 optical quenching can be derived from Beer's law, which is calculated to be 76.2 pM. Therefore, the number of 800 CW(n) on a single AuNR is as follows:

number

[0284] Exemplary conditions for fluorescence enhancement of 800CW-streptavidin using AuNR-plasmon-fluer-800CW and AuNP-plasmon-fluer-800CW The experimental procedure used in this study (the results of which are disclosed elsewhere in this specification) is shown in Figure 34, and the data are shown in Figure 5 (A-B). Specifically, BSA-biotin was first immobilized at the bottom of the wells by incubating a 96-well plastic plate containing 50 ng / ml of BSA-biotin (in 1× PBS) at room temperature for 15 minutes. This plate was washed three times with PBST (0.05% Tween20 in 1× PBS) and then blocked with Odyssey® Blocking Buffer (PBS) (LI-COR, P / N 927-40100). Next, the BSA-biotin-coated wells were incubated with 1 μg / ml of streptavidin-800CW [in Odyssey® Blocking Buffer] for 10 minutes to specifically bind streptavidin to biotin. Next, the plate was washed three times with PBST and then incubated with approximately 76 pM Plasmon-Fleur-800CW (in 1% BSA). The plate was washed three more times with PBST to remove free Plasmon-Fleur. Finally, 200 μl of PBST was added to each well, and the fluorescence signals before and after the addition of Plasmon-Fleur were recorded using a LI-COR CLx fluorescence imaging system with the following scanning parameters: laser power approximately L2; resolution approximately 169 μm; channel: 800; height: 4 mm. This experiment was independently repeated four times, and the fluorescence intensity before and after the addition of Plasmon-Fleur-800CW was compared. The data were statistically significant, and the P-value was 0.0044. ** The result was calculated as P<0.01 (using a two-sided independent t-test with Welch's correction). [Examples]

[0285] Exemplary conditions for human IL-6 ELISA: The results were obtained using the Human IL-6 DuoSet ELISA Kit (R&D Systems, Inc., catalog number DY206, lot number P173353), which are disclosed somewhere in this specification. Specifically, a 96-well plate was first coated with capture antibody (2 μg / ml in PBS) by incubation overnight at room temperature, and then blocked with 300 μl of reagent diluent (1× PBS containing 3% BSA, filtered to 0.2 μm). 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. Next, the plate was washed and incubated with biotinylation detection antibody (product number 840114, 50 ng / ml in reagent diluent) for 2 hours. It was then washed again with PBST and incubated with HRP-labeled streptavidin (product number 893975, diluted 200-fold using reagent diluent) for 20 minutes. 100 μl 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 to 450 nm. [Examples]

[0286] Human IL-6 FLISA and p-FLISA: Human IL-6 FLISA was performed using a method similar to the ELISA described above, except that HRP-labeled streptavidin was replaced with 800CW-labeled streptavidin (LI-COR P / N 926-32230, 50 ng / ml, 20 minutes). The plate was washed three times with PBST, and then with nanopure water. For p-FLISA, plasmon-fluer-800CW was added (quenching approximately 1), incubated for 1 hour, and the plate was washed three times each with reagent diluent and then with PBST. The plate was imaged using a LI-COR CLx fluorescence imaging system with the following scanning parameters: laser power approximately L2; resolution approximately 169 μm; channels: 800; height: 4 mm. Results from independent experiments are shown in Figures 47 and 49, and Figure 100 (A-C). Figures 100(A-C) show plots of IL-6 dose-dependent fluorescence intensity from p-FLISA. In Figures 100A, 100B, and 100C, the data represent experiments conducted independently over various number of days using different batches of Plasmon-Fleur-800CW. Error bars correspond to the standard deviation (for repeated trials with n≧2). [Examples]

[0287] Assay based on plasmon-fluer-enhanced Luminex beads: A mouse magnetic Luminex assay was purchased from R&D Systems, Inc. (catalog number: LXSAMSM-03, lot number: L126064) and specially modified to simultaneously detect mouse TNF-α and mouse IL-6. To begin, 50 μl of standards containing various concentrations of TNF-α and IL-6 (product number 984658) were 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 to 800 rpm. Subsequently, the microbeads were collected using a magnetic device (Millipore Sigma 40-285) designed to accommodate microplates, washed by removing the liquid, and filled with washing buffer (product number 895003). The washing process was repeated three times. Next, 50 μl of diluted biotin-antibody cocktail (product code 894666) was introduced into each well and incubated on an 800 rpm shaker for 1 hour. The microbeads were washed again three times and then incubated with 100 ng / ml Cy3-streptavidin (in 3% BSA buffered with 1× PBS) at 800 rpm for 30 minutes. After three washes, the microbeads were incubated with 50 μl of plasmon-flure-Cy3 (quenching was approximately 5 at the LSPR maximum) at 800 rpm for 1 hour and washed three times each with 3% BSA and washing buffer. Finally, the microbeads were resuspended in 100 μl of washing buffer and incubated at 800 rpm for 2 minutes before reading. A Luminex200 instrument was used for fluorescence reading. Dual-mode fluorescence of microbeads was observed using a 40× water immersion objective lens in a Conconfor II LSM system (Carl Zeiss-Evotec, Jena, Germany). 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 the mouse TNF-α standard curve based on the beads, obtained after applying plasmon-fluer-Cy3.Figures 101A and 101B show data from three independent experiments conducted over varying lengths of time using different batches of plasmon-fluer-Cy3. Error bars correspond to the standard deviation (n=2 repeated trials). Figures 102(A-B) show the bead-based mouse IL-6 standard curve obtained after applying plasmon-fluer-Cy3. Figures 102A and 102B show data from three independent experiments conducted over varying lengths of time using different batches of plasmon-fluer-Cy3. Error bars correspond to the standard deviation (n=2 repeated trials). [Examples]

[0288] Plasmon-flure-enhanced human kidney biomarker array The human kidney biomarker array kit was purchased from R&D systems, Inc. (catalog number ARY019, lot number 1311110). Urine samples from a patient with renal disease (ID number 25, age 61, male) were 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 the participant. Nitrocellulose membranes (product number 893967) were blocked by incubation in a 4-well multi-dish with 2 ml of blocking buffer (product number 893573) for 1.5 hours under gentle shaking. During the blocking process, the urine sample (150 μl) from the patient with renal disease (ID number 25) was diluted with 500 μl of blocking buffer and 850 μl of array buffer (product number 895876) to a total dilution of 10-fold. Diluted urine samples were mixed with 15 μl of reconstituted detection antibody cocktail (product code 893966), and this mixture was incubated at room temperature for 1 hour. The nitrocellulose membrane was removed from the blocking solution and incubated overnight at 4°C with the urine sample and biotinylated detection antibody mixture. Subsequently, the membrane was washed with 20 ml of 1× washing buffer (product code 895003) for 10 minutes with gentle shaking, and this washing process was repeated two more times. Next, the membrane was incubated with 800CW-streptavidin (50 ng / ml in 1% BSA) with gentle shaking for 30 minutes, washed three times, and incubated for another hour with plasmon-fluer-800CW (quenching approximately 0.5). Finally, the membrane was scanned. Next, the membrane was imaged using a LI-COR CLx imaging system at a focal height of 0.5 mm and a resolution of 169 μm at laser power L2. Protein array images were acquired using an iPhone 6 camera, and these images were analyzed using Image Studio Lite software to measure the median intensity of each spot (after subtracting background). Results from independent experiments are shown in Figure 103 (A-B).Figure 103(A-B) shows the second independent experiment of the kidney biomarker array. Fluorescence intensities corresponding to the concentrations of various urinary biomarkers (typical assays using conventional fluorophores) before (Figure 103A) and after (Figure 103B) the addition of Plasmon-Fleur-800CW. Error bars correspond to the standard deviation (n=2 repeated trials). [Examples]

[0289] Exemplary conditions for plasmon-flure-enhanced human cytokine microarrays: The efficacy of plasmon-flur was further tested using a 40-plex human cytokine microarray (RayBiotech, Inc., catalog number: QAH-CYT-4), and the results are disclosed somewhere in this specification. To begin, the glass substrate of the microarray was blocked with 100 μL of sample diluent (catalog number: QA-SDB) and then incubated with the sample standard (catalog number: QAH-CYT-4-STD) at room temperature for 2 hours with gentle shaking. The microarray was washed five times with 1× Wash Buffer I (catalog number: AA-WB1-30ML) and then twice with 1× Wash Buffer II (catalog number: AA-WB2-30ML). Next, 80 μL of the restored detection antibody cocktail was added to each well and incubated for a further 2 hours with gentle shaking. After incubation, the washing process was repeated as described above. Next, 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-Fleur-800CW (quenching approximately 1) for 1 hour. This 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. [Examples]

[0290] Immunocytochemistry / immunofluorescence (ICC / IF) enhanced by plasmon-flur: Human epithelial breast cancer cells SK-BR-3 [SKBR3] [ATCC® HTB30®] were purchased from ATCC (Manassas, VA) and secondary cultured 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). The cells were grown in T-25 tissue culture flasks in an incubator with a water jacket at 37°C in a 5% CO2 humidified atmosphere. Once the cells reached 90% confluence, they were washed with PBS and detached from the bottom of the flask using a scraper. After centrifugation, the cells were redispersed in culture medium and seeded overnight into 6-well plates, ligated to the bottom of the plates. Next, the cells were immobilized using 3.7% formaldehyde (in 1× PBS) for 30 minutes, washed three times with 1× PBS, and blocked with 3% BSA for 1 hour. Then, ErbB2 primary antibody (anti-human HER-2 / biotin, eBioscience, clone 2G11, REF number BMS120BT, lot number 186281000) was diluted with 1% BSA and incubated with SK-BR-3 cells for 1.5 hours. Subsequently, these cells were washed three times, incubated with 800CW-streptavidin (in 1% BSA, 1 μg / ml) for 30 minutes, washed three more times, and examined with Plasmon-Fleur-800CW (quenching approximately 0.3). Finally, the cells were imaged under a 40× water immersion objective lens using Olympus FV1000 LSM confocal laser scanning microscopy (785 nm excitation laser). Results from independent experiments are shown in Figure 78, and in Figures 104(A-B) and 105(A-B). Figure 104(A-B) shows the second independent immunocytochemistry experiment. Conventional immunocytochemistry procedure at various dilutions of ERbB2 primary antibody [cells are sequentially labeled with biotinylated primary antibody and streptavidin-fluer (800CW)].Confocal laser scanning microscopy (CLSM) images of ErbB2-stained breast cancer cells (SK-BR-3) obtained using the conventional immunocytochemistry procedure [see Figure 104A] followed by the addition of Plasmon-Fleur-800CW (see Figure 104B). 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 the conventional immunocytochemistry procedure [cells are sequentially labeled with biotinylated primary antibody and streptavidin-Fleur (800CW). See Figure 105A] followed by the addition of Plasmon-Fleur-800CW (see Figure 105B). The scale bar corresponds to 15 μm. [Examples]

[0291] SK-BR-3 flow cytometry measurement: 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 was removed, and then the cells were fixed with 3.7% formaldehyde in 1× PBS for 30 minutes. The cell suspension was centrifuged again to remove free formaldehyde, and then the cells were blocked overnight with 3% BSA. Next, varying amounts of ErbB2 primary antibody were added to the cell suspension, and this mixture was incubated for 1 hour with gentle shaking. These cells were centrifuged at 1000 rpm, washed once with 1× 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-fluer-680LT (quenching approximately 2.0) for 1 hour. Finally, 5000 cells were analyzed using Guava easyCyte to obtain a combined fluorescence signal [RED-R channel (excitation laser: 642 nm; filter: 662 / 15 nm)] with forward scattering (FSC) and side scattering (SSC). Results from independent experiments are shown in Figures 89 and 90, as well as Figures 106(A-B) and 107(A-B). Figure 106(A-B) shows the second independent SK-BR-3 flow cytometry experiment. Figure 106A is a histogram showing the fluorescence levels of SK-BR-3 cells before (top) and after (bottom) addition of plasmon-fluer-680LT. Red: No primary antibody; Blue: 2 × 10⁻⁶ 5 Dilute twice; orange color: 10 5 Dilute twice; light green: 10 4 Dilute twice; green: 10 3 Dilution twice; rose color: 10 times the amount of the preservative solution provided by the supplier. 2Dilution 1-2 times (1 mg / ml). Figure 106B is a plot showing the average fluorescence intensity obtained from flow cytometry at various primary antibody concentrations. Figure 107(A-B) shows the third independent SK-BR-3 flow cytometry experiment. Figure 107A is a histogram showing the fluorescence levels of SK-BR-3 cells before (top) and after (bottom) addition of Plasmon-Fleur-680LT. Red: No primary antibody; Blue: 2 × 10⁻⁶ 5 Dilute twice; orange color: 10 5 Dilute twice; light green: 10 4 Dilute twice; green: 10 3 Dilution twice; rose color: 10 times the amount of the preservative solution provided by the supplier. 2 Dilution 1:1 (1 mg / ml). Figure 107B is a plot showing the average fluorescence intensity obtained from flow cytometry at various primary antibody concentrations. [Examples]

[0292] BMDC isolation and flow cytometry measurement: Female C57BL / 6(H-2b) mice aged 5-6 weeks were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). The mice were maintained under pathogen-free conditions. All experiments using mice were performed in accordance with laboratory animal protocols approved by the Animal Review Board of Washington University (School of Medicine, St. Louis). Mice were euthanized using CO2 asphyxia and cervical vertebral dislocation. Euthanized mice were kept in 70% (v / v) ethanol for 1 minute. Both the femur and tibia were separated, and the muscle junctions were carefully removed using a gauge pad. Both ends of the bone were cut with scissors, and the bone marrow was centrifuged in a suitable centrifuge tube (a 0.6 ml perforated tube inserted into a 1.5 ml tube) at 1000 rpm for 10 seconds. The pellet was resuspended in RPMI1640 medium by vigorous pipetting. Cells were passed through a 70 μm cell strainer to prepare a single-cell suspension. After one wash (1200 rpm, 5 minutes), red blood cells were depleted using RBC lysis buffer (Sigma-Aldrich, Inc.). Bone marrow cells were collected and refractory to 10% thermally inactivated FBS, 50 IU mL. -1 Penicillin, 50 μg mL -1 The cells were cultured in 100 mm petri dishes containing 10 mL of RPMI medium supplemented with streptomycin and 20 ng mL-1 mouse recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF, R&D Systems, Inc., MN, USA). 1 × 10 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 collected using a cell scraper for further staining and flow cytometry analysis.

[0293] CD80 overexpressed on the cell surface was examined using conventional fluorophores, followed by plasmon-fluer-680LT. Specifically, stimulated BMDCs were washed once with 1× PBS to remove the culture medium (centrifugation at 2000 rpm for 5 minutes) and fixed with 10% neutral buffered formalin for 20 minutes. Next, the cells were washed (at 2000 rpm for 5 minutes) and blocked overnight at 4°C with 3% BSA. Then, biotinylated CD80 primary antibody [anti-Mo CD80 / biotin (Invitrogen, reference no. 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. BMDC cells were washed once (at 2000 rpm for 5 minutes) and then incubated with 1 μg / ml streptavidin-680LT (in 1% BSA) for 40 minutes. Finally, the cells were washed twice more and incubated with plasmon-fluer-680LT (quenching approximately 2) for 1 hour, followed by one more wash to remove unbound plasmon-fluer-680LT. 10,000 cells were analyzed using Guava easyCyte to obtain fluorescence signals [RED-R channel (excitation laser: 642 nm; filter: 662 / 15 nm)] combined with forward scattering (FSC) and side scattering (SSC). Results from independent experiments are shown in Figures 108(A-C) and 109(A-C). Figure 108(A-C) shows the second independent flow cytometry measurement of BMDC maturation markers (maker) investigated by plasmon-fluer-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) Plots showing the mean fluorescence intensity of BMDCs (corresponding to CD80 expression levels) after stimulation with various amounts of LPS. Figures 109(A-C) show third independent flow cytometry measurements 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 of BMDCs (corresponding to CD80 expression levels) after stimulation with various amounts of LPS.

[0294] Statistics: A two-sided independent t-test with Welch's correction was used to analyze the statistical difference between two groups. For statistical differences between more than two groups, a one-way ANOVA with Tukey's post-hoc honest significance test was used. Statistical significance of the data was calculated with a 95% CI (p<0.05). All values ​​are expressed as mean ± standard deviation. GraphPad Prism6 (San Diego, CA, USA) was used for all statistical analyses. The detection limit was calculated on the standard curve of the bioassay using either a 4-parameter logistic (4PL) fit or a polynomial fit. The detection limit was defined as the analyte concentration corresponding to the mean fluorescence intensity of the blank plus three times its standard deviation (mean + 3σ). Origin2016 (Northampton, MA, USA) was used to calculate the detection limit.

Claims

1. Plasmon nanostructures 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) A fluorescent nanoconstruct comprising, The fluorescent nanoconstruct has a fluorescence intensity at least 500 times greater than that of at least one fluorescent agent alone. Fluorescent nanoconstructs.

2. The fluorescent nanoconstruct according to claim 1, wherein the difference between at least one λLSPR and λEX is less than 75 nm.

3. A fluorescent nanoconstruct according to any one of the claims, wherein at least one fluorescent agent comprises at least about 5, at least about 10, at least about 20, at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, or at least about 1000 of the fluorescent agent.

4. A fluorescent nanoconstruction according to any one of the claims, further comprising at least one biorecognition element.

5. The fluorescent nanoconstruction according to any one of the claims, wherein the spacer coating further comprises a functional layer, or the fluorescent nanoconstruction further comprises a functional layer.

6. The fluorescent nanoconstruction according to any one of claims 2 to 6, wherein the plasmon nanostructure comprises a plasmon-active material selected from the group consisting of gold (Au), silver (Ag), copper (Cu), or a combination thereof.

7. A fluorescent nanoconstruct according to any one of claims 2 to 6, wherein the plasmon nanostructure is selected from the group consisting of nanorods, nanocubes, nanospheres, bimetallic nanostructures, gold-core silver-shell nanocubes, nanotubes, gold nanorods, silver nanocubes, silver nanospheres, gold nanorod-core-silver-shell (AuNR@Ag) nanocubes, nanostructures having sharp tips, nanostars, hollow nanostructures, nanocages, nanorattles, nanobipyramids, nanoplates, self-assembled nanostructures, nanoraspberries, and combinations thereof.

8. A fluorescent nanoconstruct according to any one of the claims, wherein the λLSPR is between approximately 200 nm and approximately 1000 nm, between approximately 250 nm and approximately 950 nm, between approximately 300 nm and approximately 850 nm, between approximately 350 nm and approximately 800 nm, or between approximately 400 nm and approximately 750 nm.

9. A fluorescent nanoconstruct according to any one of the claims, wherein the λLSPR is approximately (average ±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.

10. The fluorescent nanoconstruction according to any of the claims, wherein the spacer coating is thick enough to reduce or prevent the quenching of the fluorescent agent.

11. A fluorescent nanoconstruction according to any one of the claims, wherein the spacer coating on the nanostructure has a thickness of about 0.5 nm to about 100 nm or about 1 nm to about 20 nm.

12. The fluorescent nanoconstruct according to any one of the claims, wherein the spacer coating has a thickness of at least 1 nm, at least 2 nm, or at least 3 nm.

13. A fluorescent nanoconstruction according to any one of the claims, wherein the variation in the thickness of the spacer coating is less than 2 nm.

14. The fluorescent nanoconstruction according to any one of the claims, wherein the spacer coating is a reactive polymer coating containing a dielectric rigid polymer that can be functionalized.

15. The fluorescent nanoconstruction according to any one of the claims, wherein the spacer coating comprises at least one polymer coating containing a dielectric matrix.

16. A fluorescent nanoconstruction according to any one of the claims, wherein the spacer coating is a rigid polymer network.

17. The fluorescent nanoconstruction according to any one of the claims, wherein the spacer coating comprises a siloxane network.

18. The fluorescent nanoconstruction according to any one of the claims, wherein the spacer coating comprises at least one polymer coating selected from the group consisting of mercaptosilane, aminopropylsilane, trimethoxypropylsilane, and combinations thereof.

19. The fluorescent nanoconstruction according to any of the claims, wherein the spacer coating substantially covers the plasmon nanostructure.

20. A fluorescent nanoconstruct according to any one of the claims, having an absolute value of zeta potential greater than approximately 20 mV, or approximately 25 mV, or approximately 30 mV, or approximately 35 mV in water at pH 7.

21. A fluorescent nanoconstruction according to any one of the claims, wherein the spacer coating encapsulates a plasmon nanostructure.

22. A fluorescent nanoconstruction according to any one of the claims, wherein the spacer coating comprises a material selected from the group consisting of APTMS / APTES, TMPS, MPTMS, titanium dioxide, polydopamine, polyoctopamine, silane and silane mixtures, PEG, metal oxides, polyelectrolyte bilayers, layer-by-layer assembled multilayer films, zinc oxide, alumina, polysaccharides, silica, proteins, polypeptides, peptides, polyproline, DNA / RNA, PSS / PAH, chitosan, alginates and combinations thereof.

23. The fluorescent nanoconstruction according to any one of the claims, wherein at least one fluorescent agent is selected from the group consisting of fluorescent proteins, organic dyes, quantum dots, upconversion nanoparticles, nanodiamonds, carbon dots, metal nanoclusters (e.g., Au and Ag nanoclusters), fluorophore-doped nanoparticles, Eu-doped nanoparticles, transition metal complexes, lanthanide complexes, and derivatives thereof.

24. At least one fluorescent agent is fluorescein, Cy3, Cy5, 680LT, 800CW, acridine, acridone, anthracene, anthracycline, anthraquinone, azaazulene, azoazulene, benzene, benzimidazole, benzofuran, benzoindocarbocyanine, benzoindole, benzothiophene, carbazole, coumarin, cyanine, dibenzofuran, dibenzothiophene, dipyrrolo dye, flavone, fluorescein, imidazole, indocarbocyanine, indocyanine, indole, isoindole A fluorescent nanoconstruct according to any one of the claims, selected from the group consisting of isoquinoline, naphthacenedione, naphthalene, naphthoquinone, phenanthrene, phenanthtridine, phenanthtridine, phenoselenazine, phenothiazine, phenoxazine, phenylxanthene, polyfluorobenzene, purine, pyrazine, pyrazole, pyridine, pyrimidone, pyrrole, quinoline, quinolone, rhodamine, squaline, tetracene, thiophene, triphenylmethane dye, xanthene, xanthone, and derivatives thereof.

25. The fluorescent nanoconstruction according to claim 4, wherein at least one biorecognition element is selected from the group consisting of streptavidin, biotin, antibodies, nucleic acids, and combinations thereof.

26. The fluorescent nanoconstruction according to any one of the claims, wherein the spacer coating includes a flexible linker, and further, at least one biorecognition element is coupled to the flexible linker.

27. The fluorescent nanoconstruction according to any one of the claims, wherein the functional group layer comprises a flexible linker, and further, at least one biorecognition element is bonded to the flexible linker.

28. The fluorescent nanoconstruction according to any one of the claims, wherein the flexible linker is PEGx, and x is equal to 2 to 36.

29. The fluorescent nanoconstruction according to any one of the claims, wherein at least one biorecognition element is streptavidin conjugated to a spacer coating with biotin.

30. The fluorescent nanoconstruct according to any one of the claims, wherein at least one biorecognition element is a biotinylated antibody conjugated to streptavidin, and the streptavidin is conjugated to a spacer coating via biotin.

31. A fluorescent nanoconstruct according to any one of the claims, further comprising a functional group layer.

32. The fluorescent nanoconstruct according to claim 31, wherein the functional group layer is a polymer.

33. The fluorescent nanoconstruct according to claim 31, wherein the functional layer comprises a material selected from the group consisting of bovine serum albumin, human serum albumin, hemoglobin, ovalbumin, lysozyme or its homolog, albumin or its homolog, polymers (homo, diblock, triblock, random, alternating, and statistical copolymers), amphoteric polymers, zwitterionic polymers, carboxybetaine, sulfobetaine, carboxybetaine polymer, sulfobetaine polymer, PEG / betaine polymer, polynucleotides, polysaccharides, polypeptides, and combinations thereof.

34. The fluorescent nanoconstruct according to claim 31, 32, or 33, wherein the functional layer is selected from polypeptides, albumin proteins, or albumin protein homologs.

35. A fluorescent nanoconstruct according to any one of claims 31 to 34, wherein the functional layer substantially covers at least one spacer coating, or the functional layer encapsulates at least one spacer coating.

36. The fluorescent nanoconstruct according to any one of claims 31 to 35, wherein the functional layer is adsorbed onto at least one spacer coating by hydrophobic interactions, electrostatic interactions, or a combination thereof.

37. A fluorescent nanoconstruct according to any one of claims 31 to 36, wherein the functional layer stabilizes the nanoconstruct from aggregation and nonspecific bonding.

38. A fluorescent nanoconstruct according to any one of claims 31 to 37, wherein at least one fluorescent agent and at least one biorecognition element are each conjugated to a functional group layer.

39. A fluorescent nanoconstruct according to any one of claims 31 to 37, wherein at least one fluorescent agent is conjugated to at least one spacer coating, and at least one biorecognition element is conjugated to a functional base layer.

40. A fluorescent nanoconstruct according to any one of claims 31 to 37, wherein at least one fluorescent agent is conjugated to a functional group layer, and at least one biorecognition element is conjugated to at least one spacer coating.

41. The fluorescent nanoconstruct according to claim 38 or 39, wherein the functional layer contains reactive groups, and at least one biorecognition element is covalently bonded to the reactive groups on the functional layer.

42. A fluorescent nanoconstruction according to any one of claims 1 to 37, wherein at least one fluorescent agent and at least one biorecognition element are each conjugated to a spacer coating.

43. A fluorescent nanoconstruction according to any one of claims 1 to 37, wherein at least one biorecognition element is covalently bonded to a reactive group on a spacer coating.

44. A fluorescent nanoconstruction according to any one of the claims, wherein the fluorescent agent is maintained within approximately 0.5 nm to approximately 10 nm of the surface of the plasmon nanostructure.

45. A fluorescent nanoconstruction according to any one of the claims, wherein the fluorescence intensity of the fluorescent nanoconstruction is 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, at least about 7,000 times, or at least about 10,000 times higher than the fluorescence intensity of at least one fluorescent agent alone.

46. The plasmon nanostructure includes gold nanorods or silver-coated gold nanorods. The spacer coating comprises a stable silane network and reactive groups that can be functionalized. The biorecognition element contains biotin. The fluorescent nanoconstruction according to claim 4.

47. The fluorescent nanoconstruct according to claim 46, wherein the stable silane network comprises a polysiloxane.

48. The fluorescent nanoconstruct according to claim 47, wherein the polysiloxane comprises 3-mercaptopropyl)trimethoxysilane (MPTMS), trimethoxypropylsilane (TMPS), (3-aminopropyl)trimethoxysilane (APTMS), or a combination thereof.

49. The fluorescent nanoconstruction according to any one of claims 46 to 48, wherein a biorecognition element is covalently bonded to a reactive group on a spacer coating.

50. The fluorescent nanoconstruct according to any one of claims 46 to 49, wherein the spacer coating further comprises a functional layer containing BSA, and the biorecognition element is covalently bonded to a reactive group on the functional layer or covalently bonded to the spacer coating.

51. The spacer coating has a thickness of approximately 1 nm to 10 nm. The plasmon nanostructure has at least one dimension longer than 60 nm. A fluorescent nanoconstruction according to any one of claims 46 to 50.

52. A method for constructing a fluorescent nanoconstruction according to any of the above claims, Coating the plasmon nanostructure with at least one spacer coating, The functional base layer appropriately coats at least one spacer coating. Conjugate a fluorescent agent to at least one of the spacer coatings or functional base layers, and A biorecognition element is appropriately conjugated to at least one of the spacer coatings or functional substrate layers. A method that includes this.

53. The method according to claim 52, wherein the coating of the plasmon nanostructure with at least one spacer coating comprises applying a first layer to the surface of the plasmon nanostructure and applying a polysiloxane coating on the first layer.

54. The method according to claim 53, wherein the starting layer comprises 3-mercaptopropyl)trimethoxysilane (MPTMS).

55. The method according to claim 53, wherein the polysiloxane coating comprises trimethoxypropylsilane (TMPS) and 3-aminopropyltrimethoxysilane (APTMS).

56. A method for detecting an analyte using an assay, Adding the fluorescent nanoconstruct described in any of the above claims to the assay to generate a fluorescent signal, and Detecting the target of analysis by analyzing the fluorescence signal. A method that includes this.

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