Multiplexed surface-enhanced raman spectroscopy (SERS) detection of serum cardiac markers using plasmonic metasurfaces

Abstract

A biosensing system and a biosensing method are disclosed. The biosensing system includes a functionalized plasmonic metasurface, a biosensing platform that is integrated with the functionalized plasmonic metasurface, and a detection system for detecting one or more chemical and / or biological species introduced to the functionalized plasmonic metasurface.

Description

Attorney Docket No. 0184.0334-PCTClient Reference No. Pl 8519-02MULTIPLEXED SURFACE-ENHANCED RAMAN SPECTROSCOPY (SERS) DETECTION OF SERUM CARDIAC MARKERS USING PLASMONICMETASURFACESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefitto U.S. Provisional Application Serial No. 63 / 733,135 filed on December 12, 2024, the contents of which are hereby incorporated in its entirety.FIELD OF THE DISCLOSURE

[0002] The present disclosure is directed to Multiplexed SERS Detection of Serum Cardiac Markers Using Plasmonic Metasurfaces.GOVERNMENT SUPPORT

[0003] This invention was made with government support under grant R35GM149272 awarded by the National Institute of General Medical Sciences. The government has certain rights in the invention.BACKGROUND

[0004] Surface-enhanced Raman spectroscopy (SERS) possesses exquisite molecular- specific properties with single-molecule sensitivity. Yet, translation of SERS into a quantitative analysis technique remains elusive owing to considerable fluctuation of the SERS intensity, which can be ascribed to the SERS uncertainty principle, a tradeoff between “reproducibility” and “enhancement”. As a non-invasive and non-contact analytical technique with distinct molecular fingerprinting capability and single-molecule sensitivity, SERS has recently dramatically advanced biomedical diagnostics and therapeutics, accelerated the development of biopharmaceuticals, facilitated detection of antimicrobial resistance, and found promising applications in areas including forensic science and homeland security. Despite its great promise, the translation of SERS into a quantitative analysis technique in clinical settings is hindered by two major challenges. First, despite the electromagnetic (EM) field enhancement mechanism, the prevailing SERS techniques primarily rely on the electric field (E-field) component to amplify the intrinsically inefficient Raman scattering by accelerating the vibrational transition dynamics, while the magnetic field (H-field) component remains largely unexplored. The absence of the H-field component in SERS enhancement can be attributed to the weak permeability of most metals at optical frequencies, and this asymmetric contributionAttorney Docket No. 0184.0334-PCTClient Reference No. Pl 8519-02 of the EM field to SERS highlights the incompleteness of the widely accepted EM enhancement mechanism, and underscores the untapped potential of the largely ignored H-field in producing enhancement mechanism. Second, despite single-molecule sensitivity, quantitative SERS analysis is constrained by the SERS intensity uncertainty principle, which is manifested as a considerable fluctuation of SERS signals that are amplified by intense hotspots. There are two primary mechanisms underlying the SERS intensity fluctuations. One is the spatial heterogeneity of SERS hotspots, which are mostly located at sparsely distributed sharp vertices and edges and narrow gaps with sub-nanometer dimensions. Such intense SERS hotspots cover far less area than do the bonded Raman molecules on plasmonic substrates.

[0005] Consequently, only a small fraction of Raman molecules present contributes to most of the detected SERS signals. The other mechanism relates to complex dynamic processes involving the interplay between Raman molecules and the plasmonic substrate, which include but are not limited to the molecular adsorption, detachment, diffusion, and reorientation, as well as the possible atomic reconstruction of the SERS hotspots. As the distribution of the H- field is spatially complementary to that of the E-field, given the origin of the E-field from surface polarization charges and the H-field origin from fictitious current loops enclosed by those surface polarization charges, simultaneously enhanced E- and H-fields could potentially alleviate the SERS intensity uncertainty possibly by providing denser SERS hotspots. One way to approach this goal is the development of plasmonic substrates containing sites that are both E- and H-fields active.

[0006] Recently, photonic metamaterials have emerged as a promising platform for manipulating EM fields over a wide frequency range. Photonic metamaterials are artificially engineered media consisting of nanoscale constituent meta-atoms patterned as sub wavelength- structured arrays and can exhibit novel optical effects not commonly observed in nature. Particularly, as opposed to most naturally occurring materials which only support significant magnetism up to a certain frequency in the gigahertz range, photonic metamaterials can be made to exhibit considerable optical magnetism by rationally structuring the meta-atoms, and therefore they hold great promise to provide a highly desirable H-field complementary to the E-field capable of alleviating the spatial heterogeneity of SERS hotspots. Historically, split ring resonators have underscored the initial realization of such effects and catalyzed the development of artificial magnetism from the microwave to optical frequencies. Recently, layered hybrid metal-dielectric nano-constructs were established as favorable meta-atoms for creating more SERS-active plasmonic metasurfaces. They simultaneously support significant and spatially complementary E- and H-field, afford fine tuning of the EM properties, and allowAttorney Docket No. 0184.0334-PCT Client Reference No. Pl 8519-02 manufacturing with ease and at scale. Importantly, the simultaneous excitation of spatially complementary E- and H-fields yields SERS hotspots of denser population on the plasmonic metasurface, thus mitigating the spatial heterogeneity of SERS hotspots, and improving the consistency of the SERS intensity-based signals. Nevertheless, intensity-based SERS signals are intrinsically limited by molecular blinking and dynamic behaviors of molecule-metal binding, highlighting the long-standing challenge in translating SERS into a quantitative spectroscopic tool.

[0007] While SERS is subject to intensity variation, its frequencies have been recently recognized as robust signal outputs that are less susceptible to fluctuation. This is because the frequencies of the SERS peaks correspond to the energy of a particular vibrational transition, and are not directly related to the magnitude of the SERS intensity or the number of molecules in hotspots. Previous research has established that nanomechanical perturbations induced by antibody-antigen interactions can lead to structural deformation of the antibody-conjugated Raman molecules, which results in a characteristic frequency shift in SERS. Notably, the frequency -based SERS immunoassays only require the capture antibody for specific detection of antigen analytes. As compared to the prevailing sandwich immunoassays that require both capture and detection antibodies, the use of a single type of antibody significantly simplifies the immunoassay design. This can further eliminate the repeated incubation and washing steps that are often time-consuming and demand additional hardware, and can thereby potentially lower the cost to shrink the gap of health inequity. Such a unique SERS frequency shift-based method that requires only a single type of antibody has been utilized to create novel bioanalytical tools for detecting a wide range of biological analytes, such as small and macromolecules, proteins, and even circulating tumor DNA and serum microRNA where the detection antibodies were replaced with single-stranded DNA sequences that are complementary to those nucleic acid analytes.

[0008] Therefore, the monitoring of SERS frequency shifts provides an alternative that holds great promise for achieving quantitative SERS analysis, unaffected by the uncertainty of SERS intensity measurements.SUMMARY

[0009] According to examples of the present disclosure, SERS-active plasmonic metasurfaces are combined with the monitoring of SERS frequency shifts to provide a singleantibody SERS biosensing platform for multiplexed detection of a panel of cardiac biomarkers in serum. Creatine kinase-myocardial band (CK-MB), myoglobin (Mb), and cardiac troponin-Attorney Docket No. 0184.0334-PCT Client Reference No. Pl 8519-02I (cTnl) are three biomarkers of acute myocardial infarction (AMI). Prompt and accurate diagnosis of AMI can inform immediate medical intervention, particularly reperfusion therapy, which can be crucial to improving patient outcomes. While intensity-based SERS approaches were previously explored for detecting cardiac biomarkers, the potential of the integrated strategy combining frequency shifts in SERS with both the E- and H-fields-active plasmonic metasurfaces for multiplexed detection of serum cardiac biomarkers has not been reported. To fill this gap, a pyramidal plasmonic metasurface is described and was fabricated by the inventors that comprises alternately stacked gold-silica meta-atoms. Finite-difference timedomain (FDTD) simulations were used to unambiguously demonstrate the spatially extended and weakly wavelength-dependent SERS enhancement, which is contributed by both the E- and H-fields.

[0010] According to examples of the present disclosure, a compartmentalized biosensing platform is described and was created by the inventors on the plasmonic metasurface using 3D printing devices and methods. This provides an alternative approach to significantly increase the number of spots available for analysis on the same substrate, whereas regeneration strategies were traditionally used to address the infamous SERS memory effect. The SERS memory effect is an interference involving irreversible biochemical processes for measurements performed on the same substrate. This allows the present method to provide simultaneous, high-throughput, and cross interference-free detection of multiple serum samples containing different cardiac biomarkers of varied concentrations.

[0011] According to examples of the present disclosure, the presently disclosed integrated multiplexed SERS biosensing system, device, and method include at least two distinct advantages. First, a subwavelength-structured plasmonic metasurface, which includes alternately stacked metal-dielectric pyramidal meta-atoms, is fabricated and can provide simultaneously enhanced electric and magnetic fields to enable spatially extended and weakly wavelength-dependent SERS. Second, nanomechanical perturbations are harnessed to transduce signals in the form of SERS frequency shifts, which are not directly affected by the SERS uncertainty principle. By also employing 3D printing methods, multiplexed detection of a panel of serum cardiac biomarkers for acute myocardial infarction can be performed. The development of both the electric and magnetic fields-active plasmonic metasurfaces could transform future designs of SERS substrates with newly endowed functionalities, and that frequency shift-based SERS multiplexing can provide new opportunities to develop innovative quantitative optical techniques for applications in chemistry, biology, and medicine.Attorney Docket No. 0184.0334-PCTClient Reference No. Pl 8519-02

[0012] According to examples of the present disclosure, a biosensing device is disclosed that comprises a functionalized plasmonic metasurface; a biosensors that is integrated with the functionalized plasmonic metasurface; and an optical detector for detecting one or more chemical and / or biological species introduced to the functionalized plasmonic metasurface. The functionalized plasmonic metasurface comprises a top surface with a plurality of hexagonally arranged periodic structures formed using nanosphere lithography. Each of the plurality of hexagonally arranged periodic structures comprise a nanopyramidal meta-atom comprising alternately layered gold-silica thin films. The one or more biological species comprise one or more serum cardiac biomarkers.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 A, FIG. IB, FIG. 1C, FIG. ID, FIG. IE, FIG. IF, FIG. 1G, FIG. 1H, and FIG. II show fabrication and characterization of the gold-silica-gold alternately layered pyramidal plasmonic metasurfaces according to examples of the present disclosure, where FIG. 1 A, FIG. IB, FIG. 1C, and FIG. ID show experimental protocol for fabricating the plasmonic metasurface on quartz substrates, FIG. IE, FIG. IF, and FIG. 1G show tilted-view and FIG. 1H and FIG. II show top-view SEM images of the fabricated plasmonic metasurfaces. The gold-silica layered structures can be seen in the FIG. IF, FIG. 1G, and FIG. II micrographs.

[0014] FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E, and FIG. 2F show FDTD calculations of the E- and H-fields for SERS enhancement on the plasmonic metasurface and Au nanostructure according to examples of the present disclosure.

[0015] FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, and FIG. 3F show wavelengthdependent SERS enhancement according to examples of the present disclosure.

[0016] FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, and FIG. 4E show separate detection of each type of cardiac biomarkers in serum, according to examples of the present disclosure, where FIG. 4A shows Raman molecules and corresponding monoclonal antibodies, FIG. 4B shows experimental protocol for separate detection of each type of cardiac biomarkers in serum. SERS spectra depicting the frequency shift of the peak of interest (top panel) and the regression analysis for the relative frequency shift with respect to the concentration of the detected cardiac biomarker (lower panel) for detection of CK-MB in FIG. 4C, Mb in FIG. 4D, and cTnl in FIG. 4E.

[0017] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, and FIG. 5E show multiplexed detection of cardiac biomarkers in serum, according to examples of the present disclosure.Attorney Docket No. 0184.0334-PCT Client Reference No. Pl 8519-02

[0018] FIG. 6A, FIG. 6B, and FIG. 6C show extended wavenumber range for SERS spectra collected for separate detection of each type of serum cardiac biomarkers according to examples of the present disclosure, where FIG. 6A shows SERS spectra of CK-MB monoclonal antibody-conjugated MBA after capturing CK-MB antigens with various concentrations, FIG. 6B shows SERS spectra of myoglobin monoclonal antibody-conjugated DTNB after capturing myoglobin antigens with various concentrations, and FIG. 6C shows SERS spectra of troponin- I monoclonal antibody-conjugated MP after capturing troponin-I antigens with various concentrations.

[0019] FIG. 7A, FIG. 7B, and FIG. 7C shows extended wavenumber range for SERS spectra collected for multiplexed detection of serum cardiac biomarkers according to examples of the present disclosure, where FIG. 7A shows SERS spectra of CK-MB monoclonal antibody- conjugated MBA after capturing CK-MB antigens with various concentrations, FIG. 7B shows SERS spectra of myoglobin monoclonal antibody-conjugated DTNB after capturing myoglobin antigens with various concentrations, and FIG. 7C shows SERS spectra of troponin- I monoclonal antibody-conjugated MP after capturing troponin-I antigens with various concentrations.

[0020] FIG. 8A, FIG. 8B, and FIG. 8C show specificity test for detecting according to examples of the present disclosure, where FIG. 8A shows CK-MB, FIG. 8B shows Mb, and FIG. 8C shows cTnl. The upper panel showed the averaged SERS spectra; the lower panel showed the corresponding frequency shifts. The relative frequency shift Av was defined as Av = v0is the frequency measured for the sample(i = 1, 2, 3, 4) and v0is that for a sample Mo. The definition of Ml to M4 are discussed herein.DETAILED DESCRIPTION

[0021] Reference will now be made in detail to example implementations, illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. In the following description, reference is made to the accompanying drawings that form a part thereof, and in which is shown by way of illustration specific exemplary embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention and it is to be understood that other embodiments may be utilized and that changes may be made without departing from the scope of the invention. The following description is, therefore, merely exemplary.Attorney Docket No. 0184.0334-PCT Client Reference No. Pl 8519-02

[0022] FIG. 1A, FIG. IB, FIG. 1C, FIG. ID, FIG. IE, FIG. IF, FIG. 1G, FIG. 1H, and FIG. II show fabrication and characterization of the gold-silica-gold alternately layered pyramidal plasmonic metasurfaces according to examples of the present disclosure where FIG. 1 A, FIG. IB, FIG. 1C, and FIG. ID show an example experimental protocol for fabricating the plasmonic metasurface on quartz substrates, FIG. IE, FIG. IF, and FIG. 1G shows a tilted- view and FIG. 1H and FIG. II show a top-view SEM images of the fabricated plasmonic metasurfaces. The gold-silica layered structures can be seen in the FIG. IF, FIG. 1G, and FIG. II micrographs.

[0023] Fabrication of plasmonic metasurfaces are now discussed. The plasmonic metasurfaces can be fabricated based on nanosphere lithography, as schematically shown in FIG. 1A, FIG. IB, FIG. 1C, FIG. ID, FIG. IE, FIG. IF, FIG. 1G, FIG. 1H, and FIG. II. Following cleaning a substrate 102, such as a quartz substrate, as shown in FIG. 1A, the substate 102 is then patterned by applying a monolayer of polystyrene beads 104 (1 pm in diameter) to a top surface of the substrate 102, as shown in FIG. IB. Gold 106 and silica thin layers 108 (20 nm and 10 nm in thickness, respectively) are alternately deposited by e-beam evaporator to fill the gaps defined by the hexagonally patterned polystyrene beads 104, as shown in FIG. 1C. In some examples, a total of five thin-film layers of gold and four layers of silica can be alternately deposited. Because of the poor adhesion between the metal and dielectric, a thin layer of chromium with a nominal thickness of 5 nm can be deposited as an initial step, and then an ultrathin layer of chromium with a nominal thickness of 2 nm can be deposited between each subsequent gold and silica deposition as shown in FIG. 4C. Given such a small thickness, these chromium layers are unlikely to be continuous. Instead, they are more likely to be discrete nanoparticles and perhaps could be fused with part of the gold and silica layers. The polystyrene beads 104 are then removed, as shown in FIG. ID. Based on SEM characterizations, the fabricated plasmonic metasurfaces display a hexagonally periodic pattern as shown in FIG. IE, FIG. IF, FIG. 1H, and FIG. II, with each constituent meta-atom having a pyramidal shape with sharp vertices and edges as shown in FIG. 1G, FIG. 1H, and FIG. II. The well-defined metal-dielectric layers, as shown in FIG. IF, FIG. 1G, and FIG. II, are characteristic of the plasmonic metasurfaces. Consequently, the capacitive plasmonic coupling at the metal-dielectric-metal interlayer can be expected to dramatically alter the spatial EM field profiles for amplifying signals in SERS.

[0024] FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E, and FIG. 2F show FDTD calculations of the E- and H-fields for SERS enhancement on the plasmonic metasurface and Au nanostructure according to examples of the present disclosure. From left to right: FIG. 2AAttorney Docket No. 0184.0334-PCTClient Reference No. Pl 8519-02 shows a schematic of the plasmonic metasurface, cross-sectional, 3D, and top view of the spatial profiles of SERS enhancement from the E-field (upper panel) and H-field (lower panel) components with an incident wavelength of 785 nm. FIG. 2B shows the same as FIG. 2 A except the metasurface is replaced by a solid Au pyramid with the same dimension as a control. FIG. 2C, FIG. 2D, FIG. 2E, and FIG. 2F shows a comparison of the line profiles of SERS enhancements between the plasmonic metasurface and the control for both the E- and H-fields extracted from the green and pink dashed lines in the top view. The SERS enhancement iscalculated as — for the E-field component and ; — - for the H-field component at the incident l^ol l^ol wavelength with the Stokes shift ignored and represented by log10for easyspatial visualization (as shown in the colorbars).

[0025] Spatially extended simultaneously enhanced E- and H-fields for SERS enhancement are now discussed. To investigate how the EM field profiles were modified by the plasmonic metasurfaces as compared to previously studied solid gold nanopyramid arrays, FDTD numerical simulations were implemented to extract both the E- and H-fields at an incident wavelength of 785 nm. In FDTD simulations, to be consistent with the nominal dimension of the fabricated plasmonic metasurfaces, an alternating gold-silica layered nanopyramid with a base length of about 312 nm was studied while a solid gold nanopyramid with the same dimension was used as a control; these structures are shown schematically in FIG. 2A and FIG. 2D. All the EM field enhancement factors were converted to the SERS enhancement factors at the incident wavelength without considering the Stokes shift and represented by the commonlogarithm for easy visualization, which is log10— - for the E-field component and log10— - mol mol for the H-field component. It was observed that, for both the E- and H-fields, the plasmonic metasurface displayed spatially extended SERS enhancements, featuring a series of intense hotspots at the dielectric layers along both the edges and facets, which could be observed in the cross-sectional, 3D, and top views in FIG. 2A and FIG. 2B. In comparison, SERS enhancements were mostly concentrated at the vertex for the solid gold nanopyramid. To quantify how the SERS enhancements differed spatially, the line profiles of the SERS enhancements extracted along the dashed green and pink lines from the top views are compared, as shown in FIG. 2C, FIG. 2D, FIG. 2E, and FIG. 2F. For the E-field contribution to SERS, the plasmonic metasurface was found to display a series of SERS enhancement peaks originating from the capacitive plasmonic coupling at the gold-silica-gold interlayers, which were not seen on the solid gold nanopyramid (FIG. 2C and FIG. 2E). For the H-fieldAttorney Docket No. 0184.0334-PCT Client Reference No. Pl 8519-02 contribution to SERS, the plasmonic metasurface exhibited similarly spatially extended and larger SERS enhancements as compared to the control. These observations highlight the unique spatially extended SERS enhancements supported by the plasmonic metasurface, not seen in conventional plasmonic nanostructures, such as the solid gold nanopyramid.

[0026] FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, and FIG. 3F show wavelengthdependent SERS enhancement according to examples of the present disclosure, where FIG. 3 A shows a schematic of the plasmonic metasurface, cross-sectional view of the spatial profiles of SERS enhancement from the E-field (upper panel) and H-field (lower panel) components with varying incident wavelength, FIG. 3B show similar representation as shown in FIG. 3 A except now for a solid Au pyramid with the same dimension, as a control, and FIG. 3C, FIG. 3D, FIG. 3E, and FIG. 3F show a comparison of the wavelength-dependent SERS enhancement at different points (marked Pl to P5 as shown in the schematic in between the plasmonic metasurface in FIG. 3 A, and in the schematic of FIG. 3B, for the control in FIG. 3E and FIG.3F for both the E- and H-fields. The SERS enhancement is calculated as ■ — - for the E-field l^ol4component and for the H-field component at the incident wavelength with the Stokes shiftignored and represented by log10for easy spatial visualization.

[0027] The wavelength-dependent SERS enhancement was also studied to understand how the plasmonic metasurface compares with the solid gold nanopyramid over the wavelength range from 700 nm to 1000 nm. In particular, we focused on the variation of the SERS enhancement at the plasmonic hotspots marked as Pl to P5, as schematically shown in FIG. 3 A, FIG. 3B. While the SERS enhancement factors from the E-field component were found to be weakly wavelength dependent for both the plasmonic metasurface and the solid gold nanopyramid (FIG. 3C, FIG. 3E), the SERS enhancement factors from the H-component were found to have a considerable fluctuation over the studied wavelength range (FIG. 3D, FIG. 3F). But as different hotspots were found to have a different dependence on the wavelength, they could compensate each other and enable an overall high signal level and weak wavelength dependence of the SERS enhancement factors for the plasmonic metasurface. Such an ensemble contribution to SERS from all these hotspots combined is very different than the singular hotspot at the vertex of the solid gold nanopyramid. Collectively, the above studies demonstrated the unique capability of the plasmonic metasurface in enabling spatially extended and weakly wavelength-dependent SERS enhancement.Attorney Docket No. 0184.0334-PCTClient Reference No. Pl 8519-02

[0028] FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, and FIG. 4E show separate detection of each type of cardiac biomarkers in serum, according to examples of the present disclosure, where FIG. 4A shows Raman molecules and corresponding monoclonal antibodies, FIG. 4B shows experimental protocol for separate detection of each type of cardiac biomarkers in serum. SERS spectra depicting the frequency shift of the peak of interest (top panel) and the regression analysis for the relative frequency shift with respect to the concentration of the detected cardiac biomarker (lower panel) for detection of CK-MB in FIG. 4C, Mb in FIG. 4D, and cTnl in FIG. 4E. In FIG. 4B, thermoplastic polyurethane was used as the 3D printing material. Error bars indicate the standard deviation of the mean frequency shifts for the studied concentration. The relative frequency shift Av was defined as Av = v0— vbwhere vtis the frequency measured for a sample with a biomarker concentration of i and v0is that for the sample without any biomarkers.

[0029] Separate detection of each type of cardiac biomarkers in serum is now discussed. After elucidating the SERS enhancement mechanism of the plasmonic metasurface, the performance as a biosensing platform for detecting a panel of serum cardiac biomarkers was examined. Conventional SERS intensity-based immunoassays are vulnerable to signal fluctuations A frequency shift-based spectro-immunoassays design is used, which relies on a detection antibody to capture the antigen of interest and transduce a signal in the form of frequency shift. Three metasurface quartz substrates were used in our separate SERS detection studies. The three Raman molecules used for these sensing studies were 4-mercaptobenzoic acid (MBA) 402, 5,5 ’-dithiobis-(2 -nitrobenzoic acid) (DTNB) 404, and 6-thioguanine (TG) 406, with each devoted to detecting a specific cardiac biomarker (CK-MB, Mb, or cTnl, respectively) by including a conjugated antibody for target capture. The Raman molecules and associated capture antibodies are summarized in FIG. 4A, and the experimental protocol which we recently established is indicated in FIG. 4B. Raman molecules 410 were first functionalized on each of the three plasmonic metasurface substrates, such a plasmonic metasurface 408 and Ramen molecule-functionalized metasurface 412, through Au-S covalent bonding. The corresponding monoclonal antibodies 414 were then immobilized onto the Raman molecule- conjugated antibody functionalized metasurface 416 through the functionalized Raman molecule using carbodiimide crosslinker chemistry. Subsequently, 3D printing 418 was implemented to compartmentalize each of the three plasmonic metasurfaces to produce the 3D printing-compartmentalized metasurface 420. Thermoplastic polyurethane (TPU) filament was used and heated to 228 °C at the extrusion nozzle for 3D printing. The printed compartments have a dimension of about 3 mm by 3 mm and are separated by a TPU wall with a thickness ofAttorney Docket No. 0184.0334-PCT Client Reference No. Pl 8519-02 about 1 mm or less. Each formed compartment could then be used to study a particular serum sample without cross interference. Serum samples containing different concentrations of one type of cardiac biomarker 422 were pipetted into each compartment on the three differently functionalized plasmonic metasurfaces. After incubation 424 at 37 °C for 20 minutes, excessive reagents were washed away using PBS buffer and the substrates were dried with compressed air. Biomarkers are then captured, at 426, and Ramen measurements are made, at 428. A total of 5 x 5 spectra were collected with an acquisition time of 10 seconds and three accumulations across areas of 20 pm x 20 pm in each compartment under an excitation wavelength of 785 nm.

[0030] The SERS spectra acquired for varying concentrations of CK-MB, Mb, and cTnl were averaged and presented in the upper panel of FIG. 4C, FIG. 4D, and FIG. 4E, respectively. SERS spectra for FIG. 4C, FIG. 4D, and FIG. 4E with an extended wavenumber range are presented in FIG. 6A, FIG. 6B, and FIG. 6C. The mean SERS spectra were vertically offset for better visualization. Herein, for detecting CK-MB, we focused on the SERS peak of MBA at about 1590 cm'1as shown in FIG. 4C, which originates from the C-C breathing mode. For detecting Mb, we focused on the SERS peak of DTNB at about 1340 cm'1as shown in FIG. 4D, which can be assigned to the N-0 stretching mode. For detecting cTnl, we focused on the SERS peak of TG at about 1300 cm'1(FIG. 4E), which is from the ring C-N stretching mode. These characteristic SERS peaks, rather than the entire spectra, from the three Raman molecules were found to undergo biomarker-induced frequency shifts owing to the nanomechanical perturbations, and therefore, each of them was utilized to detect a particular type of serum cardiac biomarker. The relative frequency shift Av was defined as Av = v0— Vj, is the frequency measured for a sample with a biomarker concentration of i and v0is that for the sample without any biomarkers. Although the specific mechanism underlying frequency shifts in SERS has yet to be fully understood, we observed unequivocal frequency shifts correlated to the concentration variations for the antigen analytes, as shown in FIG. 4C and FIG. 4D. The MBA molecules displayed a red shift at the characteristic SERS peak at about 1590 cm'1with an increasing concentration of CK-MB, which was consistent with previous studies of influenza-Hl antigen and human p53 protein, protein carbonylation, and serum microRNA. Both the DTNB and TG Raman molecules exhibited a blue shift at their respective characteristic SERS peaks at about 1340 cm'1and 1300 cm'1with increasing concentrations of Mb and cTnl, consistent with recent studies of circulating tumor DNA and our studies of thyroid-stimulating hormone. The correlation between frequency shifts in SERS and analytes’ concentrations was confirmed by the regression analysis, which returnedAttorney Docket No. 0184.0334-PCT Client Reference No. Pl 8519-02 coefficients of determination R2of 0.97, 0.99, and 0.94 for the detection of CK-MB, Mb, and cTnl, respectively. These coefficients of determination are overall better than those obtained on the solid gold nanopyramid for detecting thyroid-stimulating hormone we studied previously, implying the advantage of the plasmonic metasurface-based biosensing platform. The linear detection ranges for CK-MB, Mb, and cTnl were found to be from 0.1 to 300 ng / mL, 6 to 4000 ng / mL, and 8 to 567 pg / mL, respectively. Based on the definition of the limit of detecting (LOD) being 3o / S, where c is the standard deviation of blank samples, and S is the slope of the calibration curve, the LOD for separate detection of CK-MB, Mb, and cTnl is about 0.04 ng / mL, 3.6 ng / mL, and 5.2 pg / mL, respectively. As a comparison, the normal concentrations for these three cardiac biomarkers are 0.3 to 4 ng / mL, 50 ng / mL or less, and 40 pg / mL or less. These results validate the potential for utilizing the frequency shift in SERS on the plasmonic metasurface for separate detection of serum cardiac biomarkers.

[0031] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, and FIG. 5E show multiplexed detection of cardiac biomarkers in serum, according to examples of the present disclosure, where FIG. 5A shows functionalization of the plasmonic metasurfaces. FIG. 5B shows experimental protocol for multiplexed detection of cardiac biomarkers. SERS spectra depicting the frequency shift of the peak of interest (top panel) and the regression analysis for the relative frequency shift with respect to the concentration of the detected cardiac biomarker (lower panel) for detection of (FIG. 5C) CK-MB, (FIG. 5D) Mb, and (FIG. 5E) cTnl. Error bars indicate the standard deviation of the mean frequency shifts for the studied concentration.

[0032] Multiplexed detection of cardiac biomarkers in serum is now discussed. Studies performed by the inventors went on to examine multiplexed detection of these three serum cardiac biomarkers on the plasmonic metasurface. While the quartz-based plasmonic metasurfaces for separate cardiac biomarker detection have a dimension of 12 mm by 12 mm or larger, these for multiplexed detection are silicon-based and were diced into smaller substrates with a dimension of about 5 mm by 5 mm. The plasmonic metasurface 502, 512, 522 was then fabricated onto these smaller silicon substrates following the fabrication protocol in FIG. 1A, FIG. IB, FIG. 1C, FIG. ID, FIG. IE, FIG. IF, FIG. 1G, FIG. 1H, and FIG. II. Subsequently, they were functionalized 506, 516, 526 and 510, 520, 530 with three types of Raman molecules (MBA 504, DTNB 514, TG 524) and monoclonal antibodies (CK-MD Ab 508, Mb Ab 518, cTnl Ab 528), respectively, as schematically shown in FIG. 5 A. In the meanwhile, 3D printing was used to create a compartmentalized biosensing platform 532, as shown in FIG. 5B, with sectors measuring 6 mm by 6 mm, so that each compartment could hold a functionalized plasmonic metasurface substrate as shown in FIG. 5 A. The functionalizedAttorney Docket No. 0184.0334-PCT Client Reference No. Pl 8519-02 plasmonic metasurfaces were thereby integrated, at 534, into the 3D printed biosensing platform, where each row had the same type of monoclonal antibody functionalization, at 536. Subsequently, serum samples containing a mixture of cardiac biomarkers including equal portions of CK-MB, Mb, and cTnl were pipetted onto the multiplexed biosensing platform, at 538. After incubation at 37 °C for 20 minutes at 540, excessive reagents were washed away using PBS buffer and dried with compressed air. The integrated multiplexed biosensing platform was then characterized, at 52, using Raman spectroscopy with a total of 5 / 5 spectra acquired over areas of 20 pm x 20 pm under an excitation wavelength of 785 nm, at 544.

[0033] As each row was functionalized with the same type of monoclonal antibodies, it was thus capable of capturing the corresponding cardiac biomarker in a highly specific manner despite the co-existence of other two types of nonspecific biomarkers in the sample. The obtained SERS spectra from each row were averaged and plotted with a vertical offset in FIG. 5C, FIG. 5D, FIG. 5E for detecting CK-MB, Mb, and cTnl, respectively. Corresponding SERS spectra with an extended wavenumber range were presented in FIG. 7A, FIG. 7B, FIG. 7C. The visually discernible frequency shifts for the studied peak of interest with respect to varying concentrations of cardiac biomarkers confirmed the specificity for the multiplexed biosensing method. This is reinforced by the coefficient of determination R2based on the regression analysis for the correlation between the frequency shift and the cardiac biomarker concentration, which are 0.97, 0.90, and 0.98 for detecting CK-MB, Mb, and cTnl, respectively. Notably, the linear detection ranges from 0.3 to 33.3 ng / mL, 3.3 to 1333 ng / mL, and 10 to 9009 pg / mL obtained in the multiplexed manner also covered the cutoff values for these three serum cardiac biomarkers. Additionally, the LOD for multiplexed detection of CK- MB, Mb, and cTnl was estimated to be about 0.05 ng / mL, 3.8 ng / mL, and 7.0 pg / mL, respectively.

[0034] Specificity tests were also performed for detecting each of the targets by utilizing mixtures of serum samples containing several paired concentrations of the other two cardiac biomarkers introduced as interfering agents. For example, to test the specificity of detecting CK-MB, we mixed in paired portions of serum samples containing Mb (concentrations are: 0, 50, 200, 800, 2000 ng / mL) and cTnl (concentrations are: 0, 30.7, 144, 567, 2293 pg / mL). The mixed serum samples were marked as M0 to M4. M0 contained a mixture of paired portions of Mb and cTnl, both without any biomarkers. Ml contained a mixture of paired portions of Mb with a concentration of 50 ng / mL and cTnl with a concentration of 30.7 pg / mL. In the same manner, M4 contained a mixture of paired portions of Mb with a concentration of 2000 ng / mL and cTnl with a concentration of 2293 pg / mL. M0 to M4 were then pipetted onto the row ofAttorney Docket No. 0184.0334-PCT Client Reference No. Pl 8519-02 the multiplexed biosensing platform that were functionalized with CK-MB monoclonal antibodies (FIG. 5B). Following the same incubation and detection protocol as presented in FIG. 5B, the SERS spectra were obtained and averaged, which, along with the relative frequency shifts, were plotted in FIG. 8A. The relative frequency shift Av was defined as Av = v0— vi, where Vj is the frequency measured for the sample(i = 1, 2, 3, 4) and v0is that for the sample Mo. Detailed protocol for the specificity tests for detecting Mb and cTnl are summarized below. Likewise, the SERS spectra and frequency shifts obtained in specificity tests for detecting Mb and cTnl are presented in FIG. 8B and FIG. 8C, respectively.

[0035] It was observed that, for the specificity test of detecting CK-MB (FIG. 8A), the mixture of Mb and cTnl biomarkers initially induced a slight frequency shift. The frequency shift plateaued with further interferent concentration increases. This suggested a limited interference from the mixture of Mb and cTnl biomarkers. For the specificity test for detecting Mb (FIG. 8B) and cTnl, while slight frequency shifts were initially induced, no further consistent frequency shifts were observed. By comparing FIG. 8 A, FIG.8B, FIG. 8C with FIG. 5C, FIG, 5D, FIG. 5E, it can be reasonably deduce that the observed frequency shifts in FIG. 5C, FIG. 5D, and FIG. 5E are more likely to be induced by the captured biomarkers through specific antibody-antigen interaction rather than the interfering agents.

[0036] It is also noted that the multiplexed biosensing method features a series of unique advantages. Specifically, it combines spatially extended and weakly wavelength-dependent EM field profiles on the plasmonic metasurface for SERS enhancement while using the singleantibody strategy to capture cardiac biomarkers in a specific and cost-effective manner. The nanomechanical perturbation-induced frequency shift for signal readout is not subjected to the SERS intensity fluctuations, whereas the three different Raman molecules used can transduce spectrally differentiated SERS peaks for multiplexing without spectral interference. The 3D printing method used to create a compartmentalized biosensing platform significantly increases the detection efficiency on the same substrate. A comparison between our study and the prevailing SERS methods for detection of cardiac biomarkers, as summarized in Table 1, further highlights that our integrated SERS frequency-shift method provides a comprehensive and innovative strategy based on SERS frequency shifts, rather than SERS intensity, for studying a panel of serum cardiac biomarkers with practically useful and clinically relevant performance.

[0037] In one non-limiting examples, the following chemicals can be used. 4- Mercaptobenzoic acid (99%), 5,5 ’-Dithi obis (2-nitrobenzoic acid) (>98%), and 6-ThioguanineAttorney Docket No. 0184.0334-PCT Client Reference No. Pl 8519-02(>98%) were purchased from Sigma- Aldrich*. Creatine Kinase MB (CK-MB) Monoclonal Antibody and Myoglobin (Mb) Monoclonal Antibody were purchased from Scripps Laboratories*. Cardiac Troponin-I (cTnl) Monoclonal Antibody and PBS buffer solution (l x, pH 7.4, Catalog number: 10010023) were purchased from ThermoFisher Scientific*. CK-MB, Mb, and cTnl with varying concentrations in serum were provided by Beckman Coulter Inc*.

[0038] As discribed above, the fabrication of plasmonic metasurfaces, as schematically laid out in FIG. 1 A, FIG. IB, FIG. 1C, and FIG. ID, begins with cleaning the quartz substrate. This was done by immersing the quartz substrates into acid piranha under heating at 90 °C for 2 hours. Afterwards, the quartz substrates were rinsed in D.I. water and sonication in ethanol and D.I. water, respectively. Prior to patterning of polystyrene (PS) beads onto the cleaned quartz slides, they were redispersed with a weight percentage of 10% in the mixture of water and ethanol with a volume ratio of 1 : 1. The PS beads (1 pm in diameter) were then transferred using dip coating to the cleaned quartz substrates and left to dry naturally. Afterwards, gold and silica thin layers (20 nm and 10 nm in thickness, respectively) were alternately deposited by e-beam evaporator to fill the gaps defined by the hexagonally patterned polystyrene beads. A total of five thin-film layers of gold and four layers of silica were alternately deposited. Because of the poor adhesion between the metal and dielectric, a thin layer of chromium with a nominal thickness of 5 nm was deposited as the initial step, and then an ultrathin layer of chromium with a nominal thickness of 2 nm was deposited between each subsequent gold and silica deposition. After removal of the PS beads by sonication in ethanol led to the formation of the pyramidal plasmonic metasurfaces.

[0039] Instrumentations and characterizations are now discussed. Mira 3 Tesscan* scanning electron microscopy (SEM) with an acceleration voltage of 10 kV was used to characterize the fabricated plasmonic metasurfaces. Raman spectroscopy characterizations were performed using an XploRA PLUS Raman microscope (HORIBA Instruments Inc.*, Edison, NJ, USA) with an excitation laser wavelength of 785 nm and an objective of 50*. The output power was measured to be about 0.4 pW.

[0040] 3D printing is now discussed. A FDM* 3D printer (CR-10S Pro V2) was employed to perform 3D printing. Thermoplastic polyurethane (TPU) filament was used and heated to 228 °C at the extrusion nozzle for 3D printing. In FIG. 4B, TPU grids were printed on the surface of each of the three plasmonic metasurfaces. The printed compartments have a dimension of about 3 mm by 3 mm and are separated by a TPU wall with a thickness of about 1 mm or less. In FIG. 5B, a compartmentalized TPU-based platform was 3D-printed and consists of an array of sectors with each measuring 6 mm by 6 mm (FIG. 5B), so that eachAttorney Docket No. 0184.0334-PCT Client Reference No. Pl 8519-02 compartment could hold a functionalized plasmonic metasurface substrate as shown in FIG. 5A. The functionalized plasmonic metasurfaces were thereby integrated into the 3D printed TPU-based biosensing platform, where each row had the same type of monoclonal antibody functionalization.

[0041] Specificity test are now discussed. To perform specificity tests for detecting each of the targets by utilizing mixtures of serum samples containing several paired concentrations of the other two cardiac biomarkers introduced as interfering agents. For example, to test the specificity of detecting CK-MB, we mixed in paired portions of serum samples containing Mb (concentrations are: 0, 50, 200, 800, 2000 ng / mL) and cTnl (concentrations are: 0, 30.7, 144, 567, 2293 pg / mL). The mixed serum samples were marked as M0 to M4. M0 contained a mixture of paired portions of Mb and cTnl, both without any biomarkers. Ml contained a mixture of paired portions of Mb with a concentration of 50 ng / mL and cTnl with a concentration of 30.7 pg / mL. In the same manner, M4 contained a mixture of paired portions of Mb with a concentration of 2000 ng / mL and cTnl with a concentration of 2293 pg / mL. M0 to M4 were then pipetted onto the row of the multiplexed biosensing platform that were functionalized with CK-MB monoclonal antibodies (FIG. 5B). After incubation at 37 °C for 20 minutes, excessive reagents were washed away using PBS buffer and dried with compressed air. That particular row was then characterized using Raman spectroscopy with a total of 5 / 5 spectra acquired over areas of 20 pm x 20 pm under an excitation wavelength of 785 nm. The obtained averaged SERS spectra along with the relative frequency shifts are plotted in FIG. 8A. The relative frequency shift Av was defined as Av = v0— Vj,is the frequency measured for the sample(i = 1, 2, 3, 4) and v0is that for the sample Mo.

[0042] Likewise, to perform the specificity test of detecting Mb, we mixed in paired portions of serum samples containing CK-MB (concentrations are: 0, 3, 10, 30, 100 ng / mL) and cTnl (concentrations are: 0, 30.7, 144, 567, 2293 pg / mL). The mixed serum samples were also marked as M0 to M4. To perform the specificity test for detecting cTnl, we mixed in paired portions of serum samples containing CK-MB (concentrations are: 0, 3, 10, 30, 100 ng / mL) and Mb (0, 50, 200, 800, 2000 ng / mL). The mixed serum samples were similarly marked as M0 to M4. The obtained SERS spectra and relative frequency shifts are presented in FIG. 8B and FIG. 8C, respectively.

[0043] FDTD numerical simulations are now discussed. Ansys Lumerical FDTD* (release: 2021. R2; version: 8.26.2717) was utilized for numerical simulations. A total-field scattered- field (TFSF) was implemented as the input light source from 700 nm to 1000 nm. A mesh size of 1 nm was used. Perfectly matched layer boundary conditions were imposed in all directions.Attorney Docket No. 0184.0334-PCT Client Reference No. Pl 8519-02The background refractive index was set at 1.0, whereas the refractive index for quartz substrates was set at 1.45. The dielectric function for gold was extracted from Johnson and Christy.

[0044] FIG. 6A, FIG. 6B, and FIG. 6C show extended wavenumber range for SERS spectra collected for separate detection of each type of serum cardiac biomarkers according to examples of the present disclosure, where FIG. 6A shows SERS spectra of CK-MB monoclonal antibody-conjugated MBA after capturing CK-MB antigens with various concentrations, FIG. 6B shows SERS spectra of myoglobin monoclonal antibody-conjugated DTNB after capturing myoglobin antigens with various concentrations, and FIG. 6C shows SERS spectra of troponin- I monoclonal antibody-conjugated MP after capturing troponin-I antigens with various concentrations.

[0045] FIG. 7A, FIG. 7B, and FIG. 7C shows extended wavenumber range for SERS spectra collected for multiplexed detection of serum cardiac biomarkers according to examples of the present disclosure, where FIG. 7A shows SERS spectra of CK-MB monoclonal antibody- conjugated MBA after capturing CK-MB antigens with various concentrations, FIG. 7B shows SERS spectra of myoglobin monoclonal antibody-conjugated DTNB after capturing myoglobin antigens with various concentrations, and FIG. 7C shows SERS spectra of troponin- I monoclonal antibody-conjugated MP after capturing troponin-I antigens with various concentrations.

[0046] FIG. 8A, FIG. 8B, and FIG. 8C show specificity test for detecting according to examples of the present disclosure, where FIG. 8A shows CK-MB, FIG. 8B shows Mb, and FIG. 8C shows cTnl. The upper panel showed the averaged SERS spectra; the lower panel showed the corresponding frequency shifts. The relative frequency shift Av was defined as Av = v0is the frequency measured for the sample(i = 1, 2, 3, 4) and v0is that for a sample Mo. The definition of Ml to M4 can be found herein.

[0047] Table 1 shows a comparison between our current study and prevailing SERS methods for detection of cardiac biomarkers. It is important to note that the normal concentrations for CK-MB, Mb, and cTnl biomarkers are 0.3 to 4 ng / mL2, 50 ng / mL or less3, and 40 pg / mL or less2. “NA” means being not applicable.

[0048] Table 1Attorney Docket No. 0184.0334-PCTClient Reference No. Pl 8519-02

[0049] In summary, a hexagonally periodic plasmonic metasurface using nanosphere lithography was designed and fabricated. Each constituent element within the array was featured by a nanopyramidal meta-atom comprising alternately layered gold-silica thin films. FDTD numerical simulations revealed that the plasmonic metasurface supported spatially extended and overall weakly wavelength-dependent SERS enhancement. Such superior SERS performance was used to first create a biosensing platform for separate detection of three types of serum cardiac biomarkers, including CK-MB, Mb, and cTnl. Functionalized plasmonic metasurfaces were further integrated with a 3D printed biosensing platform for multiplexed detection of these three serum cardiac biomarkers in a specific manner from sample mixtures. Given the adaptability of this multiplexed biosensing platform, it can be extended as a general in-vitro optical sensing platform for high-throughput multiplexed analysis of a wide range of chemical and biological species, such as small molecules, proteins, peptides, and nucleic acids.

[0050] Reference herein to "one example" means that one or more feature, structure, or characteristic described in connection with the example is included in at least one implementation. The phrase "one example" in various places in the specification may or mayAttorney Docket No. 0184.0334-PCT Client Reference No. Pl 8519-02 not be referring to the same example. As used herein, a system, apparatus, structure, article, element, component, or hardware "configured to" perform a specified function is indeed capable of performing the specified function without any alteration, rather than merely having potential to perform the specified function after further modification. In other words, the system, apparatus, structure, article, element, component, or hardware "configured to" perform a specified function is specifically selected, created, implemented, utilized, programmed, and / or designed for the purpose of performing the specified function. As used herein,"configured to" denotes existing characteristics of a system, apparatus, structure, article, element, component, or hardware which enable the system, apparatus, structure, article, element, component, or hardware to perform the specified function without further modification. For purposes of this disclosure, a system, apparatus, structure, article, element, component, or hardware described as being "configured to" perform a particular function may additionally or alternatively be described as being "adapted to" and / or as being "operative to" perform that function.

[0051] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the embodiments are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all sub-ranges subsumed therein. For example, a range of "less than 10" can include any and all sub-ranges between (and including) the minimum value of zero and the maximum value of 10, that is, any and all sub-ranges having a minimum value of equal to or greater than zero and a maximum value of equal to or less than 10, e.g., 1 to 5. In certain cases, the numerical values as stated for the parameter can take on negative values. In this case, the example value of range stated as “less than 10” can assume negative values, e.g. -1, -2, -3, - 10, -20, -30, etc.

[0052] Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.” As used herein, the phrase “one or more of’, for example, A, B, and C means any of the following: either A, B, or C alone; or combinations of two, such as A and B, B and C, and A and C; or combinations of A, B and C.The foregoing description, for purpose of explanation, has been described with reference to specific embodiments. However, the illustrative discussions above are not intended to beAttorney Docket No. 0184.0334-PCT Client Reference No. Pl 8519-02 exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in view of the above teachings. Moreover, the order in which the elements of the methods are illustrated and described may be re-arranged, and / or two or more elements may occur simultaneously. The embodiments were chosen and described in order to best explain the principles of the invention and its practical applications, to thereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications as are suited to the particular use contemplated.

Claims

Attorney Docket No. 0184.0334-PCTClient Reference No. Pl 8519-02What is claimed is:

1. A biosensing system comprising: a functionalized plasmonic metasurface; a biosensing platform that is integrated with the functionalized plasmonic metasurface; and a detection system for detecting one or more chemical and / or biological species introduced to the functionalized plasmonic metasurface.

2. The biosensing system of claim 1, wherein the functionalized plasmonic metasurface comprises a top surface with a plurality of hexagonally arranged periodic structures formed using nanosphere lithography.

3. The biosensing system of claim 2, wherein each of the plurality of hexagonally arranged periodic structures comprise a nanopyramidal meta-atom comprising alternately layered goldsilica thin films.

4. The biosensing system of claim 1, wherein the one or more biological species comprise one or more serum cardiac biomarkers.

5. The biosensing system of claim 1, wherein the one or more serum cardiac biomarkers comprise creatine kinase-myocardial band (CK-MB), myoglobin (Mb), and cardiac troponin-I (cTnl).

6. The biosensing system of claim 1, wherein the one or more biological species comprise one or more small molecules, proteins, peptides, or nucleic acids.

7. The biosensing system of claim 1, wherein the detection system comprises an in-vitro optical sensing platform that provides a high-throughput multiplexed analysis of a wide range of chemical and biological species.

8. The biosensing system of claim 1, wherein the functionalized plasmonic metasurface is a surface-enhanced Raman spectroscopy (SERS)-active plasmonic metasurface.Attorney Docket No. 0184.0334-PCT Client Reference No. Pl 8519-029. A biosensing method comprising: applying one or more chemical and / or biological species to a biosensing platform that is integrated with a functionalized plasmonic metasurface; and detecting, with a detection system, the one or more chemical and / or biological species that was introduced to the functionalized plasmonic metasurface.

10. The biosensing method of claim 9, wherein the functionalized plasmonic metasurface comprises a top surface with a plurality of hexagonally arranged periodic structures formed using nanosphere lithography.

11. The biosensing method of claim 10, wherein each of the plurality of hexagonally arranged periodic structures comprise a nanopyramidal meta-atom comprising alternately layered gold-silica thin films.

12. The biosensing method of claim 9, wherein the one or more biological species comprise one or more serum cardiac biomarkers.

13. The biosensing method of claim 9, wherein the one or more serum cardiac biomarkers comprise creatine kinase-myocardial band (CK-MB), myoglobin (Mb), and cardiac troponin-I (cTnl).

14. The biosensing method of claim 9, wherein the one or more biological species comprise one or more small molecules, proteins, peptides, or nucleic acids.

15. The biosensing method of claim 9, wherein the detection system comprises an in-vitro optical sensing platform that provides a high-throughput multiplexed analysis of a wide range of chemical and biological species.

16. The biosensing method of claim 9, wherein the functionalized plasmonic metasurface is a surface-enhanced Raman spectroscopy (SERS)-active plasmonic metasurface.

17. A biosensing device comprising: a functionalized plasmonic metasurface; a biosensors that is integrated with the functionalized plasmonic metasurface; andAttorney Docket No. 0184.0334-PCT Client Reference No. Pl 8519-02 an optical detector for detecting one or more chemical and / or biological species introduced to the functionalized plasmonic metasurface.

18. The biosensing device of claim 17, wherein the functionalized plasmonic metasurface comprises a top surface with a plurality of hexagonally arranged periodic structures formed using nanosphere lithography.

19. The biosensing device of claim 18, wherein each of the plurality of hexagonally arranged periodic structures comprise a nanopyramidal meta-atom comprising alternately layered gold-silica thin films.

20. The biosensing device of claim 17, wherein the one or more biological species comprise one or more serum cardiac biomarkers.

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