Bioinspired Disordered Flexible Metasurfaces for Human Tear Analysis Using Broadband Surface-Enhanced Raman Scattering.

Flexible surface-enhanced Raman scattering (SERS) has received attention as a means to move SERS-based broadband biosensing from bench to bedside. However, traditional flexible periodic nano-arrangements with sharp plasmonic resonances or their random counterparts with spatially varying uncontrollable enhancements are not reliable for practical broadband biosensing. Here, we report bioinspired quasi-(dis)ordered nanostructures presenting a broadband yet tunable application-specific SERS enhancement profile. Using simple, scalable biomimetic fabrication, we create a flexible metasurface (flex-MS) of quasi-(dis)ordered metal-insulator-metal (MIM) nanostructures with spectrally variable, yet spatially controlled electromagnetic hotspots. The MIM is designed to simultaneously localize the electromagnetic signal and block background Raman signals from the underlying polymeric substrate-an inherent problem of flexible SERS. We elucidate the effect of quasi-(dis)ordering on broadband tunable SERS enhancement and employ the flex-MS in a practical broadband SERS demonstration to detect human tear uric acid within its physiological concentration range (25-150 μM). The performance of the flex-MS toward noninvasively detecting whole human tear uric acid levels ex vivo is in good agreement with a commercial enzyme-based assay.

Introduction

Surface-enhanced Raman scattering (SERS) has shown great promise as a technique for molecular fingerprinting because of its high sensitivity and selectivity along with its inherent simplicity.[1−3] In particular, SERS from flexible substrates has received great attention recently owing to its advantages over rigid substrates.[4−6] For instance, flexible SERS can be used on irregular surfaces for in situ biosensing thereby overcoming complex analyte extraction strategies and other sample preparation steps required while using conventional rigid substrates. However, the adoption of flexible SERS as a point-of-care diagnostic tool has been limited by a number of factors.[6] First, most structures used for this application are designed either with periodic arrangements because of their highly predictable plasmonic resonances[7,8] or with random arrangements because of their ease of fabrication.[9,10] However, periodic structures with narrowband resonance profiles are not tunable for multiplexing.[6−8] Multiplexed in situ SERS for instance would require a broadband and tunable plasmonic resonance profile to provide uniform enhancement of various Raman modes occurring at greatly differing vibrational energy states.[11−13] In the same vein, random structures do not guarantee repeatable SERS performance for a given bandwidth because of spatially varying enhancements that are not tunable.[14] Second, flexible SERS platforms are usually made of polymers that generate a considerable Raman background signal.[6] Finally, challenges in obtaining reproducible signal can also be attributed to the difficulty in homogeneously, scalable and cost effective manufacturing SERS active sites or hotspots.[15] These issues necessitate the requirement for flexible SERS approaches with spatially uniform broadband, yet tunable plasmonic resonances that are scalable and reliably manufacturable.

Inspiration can be sought from nature which boasts a plethora of biophotonic nanostructures possessing quasi-(dis)order or controlled disorder where both structural dimensions and periodicity follow unique distributions.[16,17] Such an amalgamation of short-range order (i.e., periodicity) with long-range disorder (i.e., randomization) along with variations in the structure size leads to a host of useful omnidirectional broadband, yet tunable optical properties.[18−20] In this work, using a simple biomimetic fabrication process, we realize a highly scalable and flexible plasmonic metasurface-based (flex-MS) SERS platform. The flex-MS consists of a dense quasi-(dis)ordered ensemble of gold (Au) nanodisks on nanoholes separated by a sub-10 nm silicon dioxide (SiO2) nanogap in a metal–insulator–metal (MIM) configuration. The entire configuration is created at a wafer-scale over a 400 μm-thick silicone elastomer (PDMS) layer. The MIM is designed not only for light confinement and field enhancement, but is also responsible for blocking the Raman background signal from the underlying PDMS. The fabrication approach which is spin-coating based allows for a short-range-ordered Gaussian distribution of MIM nanostructure diameters resulting in a broadband, yet tunable plasmonic resonance and corresponding |E/Eo|2 enhancement factor (EF). Through rigorous numerical simulations and comparative experimentation, we isolate the contributions of the bioinspired short-range periodicity (i.e., positional disorder) and the diameter distribution (i.e., size disorder) toward SERS EF. As a practical broadband SERS demonstration of the flex-MS, we perform the label-free detection of human tear uric acid (UA) toward the diagnosis various chronic pathologies. Our platform effectively tracks prominent peaks of UA at large Raman shifts within its physiological concentration range (25–150 μM) in human tears. Finally, we show that the performance of the flex-MS in detecting UA levels in whole human tear samples from different subjects is in good agreement with a commercial enzyme-based assay.

Results and Discussion

Flex-MS Fabrication and Characterization

The flex-MS was fabricated using a simple process consisting of three steps, as shown in Figure a. On spin-coated PDMS thin films, the first step involves a biomimetic technique that relies on nanostructuring through the lateral phase-separation of two synthetic polymers—polystyrene (PS) and polymethyl methacrylate (PMMA) co-dissolved in methyl ethyl ketone (MEK).[20,21] This approach is analogous to the formation of quasi-(dis)ordered biophotonic nanostructures on the wings and scales of birds and insects.[22] The lateral phase separation of PS and PMMA occurs under spin-coating which results in densely packed quasi-(dis)ordered distribution of circular hydrophobic PS islands in a matrix of hydrophilic PMMA (Figure S1). Furthermore, through the control of various parameters such as the spin-speeds, relative humidities, polymer weight ratios, and molecular weights, the average diameter and short-range periodicity of the PS islands can be effectively tuned.[23,24] Next, through selective dissolution of PMMA in acetic acid which leaves behind a nanopillar mask of PS, SiO2 is directionally deposited through E-beam evaporation. Finally, Au of an appropriate thickness is directionally evaporated over the SiO2 nanopillars to create a scalable flex-MS with uniform sub-10 nm thick MIM nanogaps. A photograph and SEM image of the flex-MS are shown in Figure b,c. A 2D fast Fourier transform (FFT) of the top view SEM image as shown in the inset of Figure c reveals a ring-shape distribution in the spatial domain that is characteristic of quasi-(dis)order in nature.[16,17,20] The average short-range isotropic periodicity in this case is 318 ± 45 nm. Moreover, the second inset in Figure c shows a high-magnification SEM image of a single MIM structure confirming the sub-10 nm hotspot. Additionally, we have verified the existence of the sub-10 nm gap in our prior work.[24]Figure d shows a distribution of MIM structure diameters which can be estimated by a Gaussian profile fit with a mean and standard deviation (SD) of 101 ± 49 nm.

Figure 1

(a) Simple and scalable three-step fabrication process of the flex-MS. (b) Fabricated flex-MS sample with (c) quasi-(dis)ordered MIM nanostructures. Insets correspond to the 2D FFT taken to determine a short-range periodicity of 318 ± 45 nm (left) and a single MIM nanostructure of diameter 100 nm with the sub-10 nm insulator nanogap indicated by the white arrow (right). Scale bar: 2 μm. (d) MIM structure diameter distribution with a Gaussian mean and SD of 101 ± 49 nm.

Single MIM Structure Simulations

The aforementioned dimensions were chosen through rigorous finite-difference time-domain (FDTD) simulations. First, as shown in Figure a, a single MIM structure with a 5 nm insulator gap was simulated to obtain the extinction profile and electric-field distribution. The plasmonic behaviors of the two metal layers (Au nanohole and Au nanodisk) couple with each other leading to strong electric-field confinement at the MIM junction.[24] This effect has been demonstrated for different MIM combinations toward broadband SERS applications.[25,26] Additionally, we have previously demonstrated the use of similar MIM structures with higher-order plasmonic gap modes toward plasmon-enhanced fluorescent detection of nucleic acids and suppression of fluorescence quenching.[24] A modal analysis reveals that in the vis–NIR regime, this coupling results in the formation of a dipolar mode polarized along the MIM junction (Figure b). This, in turn, produces a very tunable and large extinction cross section and a corresponding localized |E|2 enhancement. Figure c presents the maximum normalized |E/Eo|2 enhancement numerically obtained for single MIM structures ranging from 60 to 110 nm in diameter with a 5 nm gap. However, like most ordered plasmonic structures used for SERS, this mode is fairly narrowband.

Figure 2

(a) Schematic of a single MIM nanostructure on the flex-MS platform (b) Coupling of the Au nanodisk and Au nanohole across the SiO2 nanogap results in greatly enhanced electric-field environments. (c) Field profile of the nanogap shows the presence of a vertically polarized dipolar mode. (d) Normalized maximum theoretical |E/Eo|2 enhancement (solid curves) and extinction cross section (dotted curves) of a single MIM nanostructure of varying diameter between 60 and 110 nm with a fixed gap size of 5 nm.

Flex-MS Ensemble Simulations

To quantify the impact of quasi-(dis)ordering on the |E/Eo|2 enhancement, we generated an ensemble of quasi-(dis)ordered MIM structures to simulate their optical properties. Two aspects were studied: (1) short-range periodicity (i.e., positional disorder) and (2) diameter distribution (i.e., size disorder). Here, the diameters of the structures were made to obey a Gaussian distribution as with the flex-MS (diameter: 98 ± 30 nm) with short-range periodicity (319 ± 37 nm) (Figure S2). To isolate the individual contributions of (1) and (2), periodic structures (diameter: 100 nm, periodicity: 320 nm) and short-range-ordered structures of the same diameter (diameter: 100 nm) were considered. For the three categories of structures, an insulator gap of 9 nm was chosen to tune the resonance close to the laser wavelength (λL = 785 nm) (Figure S3). The effective normalized |E/Eo|2 enhancement in each case (averaged over 4000 individual hotspots) was computed not only at the laser wavelength (λL = 785 nm), but also the Raman-shifted wavelengths of relevance (λ1, λ2, and λ3 = 827, 861, and 902 nm) for this work. For periodic structures, the resonance profile is predictably governed by the collective gap-plasmon resonance of individual MIM structures that dominate any weaker in-plane lattice effects (Figure a).[27] This is evidenced by the fact that the periodic array resonance position and mode shape is identical to that of a single MIM structure (diameter: 100 nm, gap: 9 nm) (Figure S4). Next, quasi-(dis)ordered structures with the same diameter (i.e., positional disorder) were studied. The effect of the pure positional disorder retains the collective gap-plasmon resonance of individual MIM structures marked by the same resonance position and intensity as that of the periodic array given the same number of averaged hotspots (Figure b). In other words, for a periodicity that is large enough to ensure either weak or no coupling between adjacent nanostructures, the effect of the short-range order (i.e., pure positional disorder) is negligible.[28,29] Finally, the introduction of a Gaussian distribution of diameters along with the short-range order (i.e., size and positional disorder) produces a considerably more broadband |E/Eo|2 enhancement compared to the periodic structures as shown in Figure c because of the resonance of subsets of MIM structures of a given diameter (Figures d and S5).[28] The bandwidth of the |E/Eo|2 enhancement of size-disordered structures (∼219 nm) was 3.4 times larger than that of the periodic structures (∼65 nm). The fractal, yet controllable nature of the MIM ensemble with a size and positional disorder produces a broadband |E/Eo|2 enhancement that encompasses λL and λ1–3. While the periodic structures and those with a pure positional disorder provide a high effective enhancement at λL (i.e., |E/Eo|λ2) of 3358 and 3430, respectively, their enhancements at λ1–3 is considerably lower (periodic: |E/Eo|λ2 ≈ 1210, |E/Eo|λ2 ≈ 563, and |E/Eo|λ2 ≈ 285; pure positional disorder: |E/Eo|λ2 ≈ 1252, |E/Eo|λ2 ≈ 585, and |E/Eo|λ2 ≈ 292). In comparison, the introduction of the size disorder provides a more uniform enhancement with |E/Eo|λ2 ≈ 1992, |E/Eo|λ2 ≈ 1748, |E/Eo|λ2 ≈ 1305, and |E/Eo|λ2 ≈ 921.

Figure 3

Ensemble of (a) periodic MIM nanostructures and (b) those with pure positional disorder demonstrating an identical narrowband effective normalized |E/Eo|2 enhancement profile. (c) Effective normalized |E/Eo|2 enhancement profile of a quasi-(dis)ordered MIM ensemble with both positional and size disorder is broadband compared to (a,b). The |E/Eo|2 enhancement profile encompasses the excitation and the Raman-shifted wavelengths (λL, λ1, λ2, and λ3) of relevance in this work. (d) Field-map spanning the 3.5 × 3.5 μm array at λL, λ1, λ2, and λ3 shows the progressive excitation of first small and then larger MIM nanostructures with increasing wavelength. (e) Effective |E/Eo|4 enhancement from periodic structures, structures with a positional disorder only and those with both positional and size disorder. This effective enhancement was numerically computed as EF(ωL, ωR).

Broadband SERS Enhancement Factor Computation

The origin of this broadband effect can be of great benefit to SERS. As per the frequently used |E|4-approximation, SERS enhancement factor (EF) is typically expressed aswhere Eloc(ωL, m) and Einc(ωL, m) are the localized and incident electric fields at a laser excitation frequency of ωL for a Raman dipole at position m.[30] Here, a significant contribution from the radiative enhancement Mrad is overlooked in favor of electric-field enhancement Mloc.[31−33]Mrad measures the enhancement of the power radiated by a dipole in the presence of a plasmonic nanostructure. Factoring in this contribution, the EF can be more precisely expressed as[30]where ωR is the Stokes-shifted Raman scattering frequency. The |E|4-approximation assumes that ωR ≈ ωL and as a result, Mloc ≈ Mrad. While this approximation is accurate for ωL in the blue and green regime, it is inaccurate in the red and NIR regime particularly within the fingerprint region (500–1500 cm–1).[32,34] This is because for a given vibrational mode, the difference between the vibrational and excitation energies becomes more significant for lower energy excitations. In principle, for NIR-based SERS, broadband EFs are greatly desirable as they better account for Mrad.[32] Based on our ensemble simulations, the effective EF(ωL, ω1–3) for the periodic structures (EF(ωL, ω1) ≈ 4.06 × 106, EF(ωL, ω2) ≈ 1.89 × 106, and EF(ωL, ω3) ≈ 0.96 × 106) and those with a purely positional disorder (EF(ωL, ω1) ≈ 4.29 × 106, EF(ωL, ω2) ≈ 2.01 × 106, and EF(ωL, ω3) ≈ 1.00 × 106) are evidently lower for ω2 and ω3 than structures with a size and positional disorder (EF(ωL, ω1) ≈ 3.48 × 106, EF(ωL, ω2) ≈ 2.60 × 106, and EF(ωL, ω3) ≈ 1.84 × 106) (Figure e).

The effect of the bioinspired quasi-(dis)order was then experimentally verified by comparing the optical properties of the flex-MS with periodic MIM structures (diameter: 100 nm, periodicity: 320 nm, gap: 9 nm) fabricated via E-beam lithography. Using a microspectroscopic setup in dark-field (DF) mode, the scattering cross section of the two metasurfaces were measured in the NIR regime. This revealed the considerably more broadband scattering profile of the flex-MS compared to the periodic array (Figure a). Furthermore, the existence of the 9 nm gap was verified through simulation (Figure S6). The SERS performance of the two sets of structures was compared by detecting UA—a SERS-active molecule with prominent peaks at 640, 1134, and 1645 cm–1 which originate from the skeletal ring and C–N bond deformations.[35] When excited with a 785 nm laser, these peaks appear at 827, 861, and 902 nm, respectively (i.e., ωL ≠ ωR). For the same UA concentration in DI water (150 μM) and three different laser powers (0.32, 0.62, and 1.12 mW), the three peaks under consideration were uniformly enhanced by the flex-MS compared to the periodic MIM structures (Figure b,c). The 640 cm–1 peak was enhanced more significantly by the periodic structures compared to the flex-MS as this peak lies closest to ωL, where the sharp plasmonic resonance of the former is tuned. However, 1134 and 1645 cm–1 peaks that were located further away from ωL were enhanced more significantly by the flex-MS because of its broadband enhancement profile. This property is particularly useful for sensing with low power. In our case for instance, when using a power of 0.32 mW, the 1134 and 1645 cm–1 peaks were enhanced 3.1 and 5.7 fold by the flex-MS compared to the periodic structures. Finally, the background suppression property of the flex-MS was also verified through comparative experimentation (Figure S7).

Figure 4

(a) Scattering intensity of the fabricated periodic and quasi-(dis)ordered array shown as solid lines measured using a microspectroscopic setup operating in DF mode. The simulated scattering profile of a single MIM structure (gap: 9 nm) which most closely matches the experimental result is also shown as a dotted line. The SEM images of the corresponding structures are shown in the inset. (b) SERS spectra of UA measured at 0.62 mW laser power. While the periodic MIM array enhances the 640 cm–1 peak considerably, the flex-MS offers a more broadband enhancement of all three peaks. (c) Analysis performed for various laser powers.

Broadband SERS Biosensing of Tear UA

The broadband enhancement of the flex-MS platform was used to detect various concentrations of UA. Hyperuricemia (i.e., elevated levels of blood UA) has been identified as a biomarker of various diseases such as gout or gouty arthritis,[36] diabetes,[37] Parkinson’s disease,[38] renal disease,[39] and cardiovascular disease,[40] to name a few. While continual monitoring of UA in blood is hindered by the invasive nature of blood collection and sampling, human tears which are considerably less invasive to assay thereby proving to be an interesting alternative.[41] Tears are also far less complex in constituents compared to blood and have large average concentrations of UA (68 ± 46 μM).[10] Furthermore, a clear correlation between blood and tear UA levels has been established.[10] As a result, assaying tear UA levels noninvasively and in a label-free fashion via flexible SERS holds great promise. Using the flex-MS, we first tracked the 640, 1134, and 1645 cm–1 peaks for UA concentrations ranging from 25 to 150 μM in phosphate-buffered saline (PBS) (Figure a–c). Next, to better simulate the SERS profile of UA in whole tears, we prepared various concentrations of UA between 25 and 150 μM in an artificial tear buffer which consisted of various prominent tear proteins such as lysozyme, lactoferrin, albumin, and immunoglobulins, as well as electrolytes that maintain tear osmolarity such as Na+, K+, Cl–, and HCO3– (see the Methods Section in Supporting Information for additional details). As a practical demonstration of broadband SERS enhancement, the 640, 1134, and 1645 cm–1 peaks were tracked with excellent linearity being observed as shown in Figure d. These measurements from an artificial tear buffer were used as a characteristic curve to map measurements taken for whole tears. Finally, we analyzed the performance of the flex-MS toward the detection of UA levels in whole tears. Here, we tested pooled tears from eight different samples obtained from healthy subjects. As a comparison, the same measurement was made using a commercial colorimetric enzyme-based assay. The average concentration from the flex-MS for the 640, 1134, and 1645 cm–1 peaks (66, 67, and 67 μM, respectively) was in good agreement with that from the enzyme-based assay (76 μM) which demonstrates the potential of the flex-MS as an effective label-free SERS diagnostic platform (Figure e).

Figure 5

(a) Peaks of UA at 640, 1134, and 1645 cm–1 tracked between 25 and 150 μM in PBS. (b) SERS surface mapping (UA concentration: 150 μM) over a 150 × 150 μm area at 640, 1134, and 1645 cm–1 showing spatial uniformity. Scale bars: 20 μm. (c) Normalized intensity of each peak shows excellent linearity. (d) 640, 1134, and 1645 cm–1 peaks tracked between 25 and 150 μM in artificial tear buffer. (e) SERS performance of the flex-MS using all three peaks is compared for pooled whole tears consisting of 8 individual tear samples with a commercial enzyme-based assay. The two results are in good agreement.

Conclusions

In summary, using a simple biomimetic fabrication process, we have developed a cost-effective, scalable, and flexible plasmonic metasurface-based platform for label-free SERS. The flex-MS consists of a dense ensemble of closely coupled Au nanodisks and Au nanoholes separated by sub-10 nm SiO2 nanogaps in an MIM arrangement all on flexible PDMS thin films. The MIM provides a very tunable and large extinction cross section and a corresponding localized |E|2 enhancement. When considering an ensemble of such MIM structures exhibiting controlled disorder, the resonance profile of the entire system becomes broadband thereby enabling the uniform enhancement of not just the excitation wavelength (λL), but also large Stokes-shifted Raman scattering wavelengths (λR ≠ λL). Finally, using flex-MS as a broadband diagnostic platform, we demonstrate the label-free detection of UA in both artificial tear buffer as well as whole human tear samples. Additionally, we compare the performance of the flex-MS with a commercial UA measurement assay and show that they are in good agreement. As a result, we envisage that through broadband SERS enhancement, the flex-MS can prove to be a reliable and scalable label-free diagnostic platform for a variety of SERS-active molecules.

Experimental Section

Biomimetic Flex-MS Fabrication

First, a 400 μm thick PDMS layer (Sylgard 184 elastomer base mixed with curing agent in a ratio of 10:1, Dow Chemical Co., USA) was spin-coated on a 4 in. Si wafer and cured at 65 °C for 12 h. Next, poly(methyl methacrylate) (PMMA, Mw = 5090, Polymer Standards Service GmbH, Germany) and polystyrene (PS, Mw = 3250, Polymer Standards Service GmbH, Germany) were co-dissolved in MEK (Sigma-Aldrich, USA) with mass ratios of 70%:30%. The solution concentrations were 20 mg mL–1. After exposing the PDMS substrate to O2 plasma for 5 min, the polymer blend solutions were spin-coated at a spin-speed of 3500 rpm and acceleration of 2000 rpm s–1 for 30 s. Relative humidity was maintained between 40 and 50% during the spin-coating. As shown in Figure S1, the de-mixing of the blend components occurs during spin-coating because of the difference in relative solubility of PS and PMMA in MEK. First, water condensation begins at humidity levels above 35% forming a water-rich layer at the air/solution interface because of the difference in the evaporation rate between water and MEK. Water starts to condense from the air into the solution because of the evaporation of MEK decreasing the temperature on the top, below the dew point. Because of the high water concentration, a 3D phase separation occurs between PS/MEK and PMMA/MEK/water. Upon drying, a purely lateral morphology was formed with ellipsoidal PS islands in a PMMA matrix. The samples were then rinsed in acetic acid for 60 s and dried in a stream of N2 to remove the PMMA matrix leaving behind the PS islands. The resulting PS nanopillar mask then served as a template for the E-beam evaporation of a 100 ± 0.5 nm-thick SiO2 layer (CHA MK40 E-Beam Evaporation, CHA Industries, USA). Next, a 91 ± 1.1 nm-thick Au layer was deposited (CHA MK40 E-Beam Evaporation, CHA Industries, USA) over SiO2 to generate the MIM layer with ∼9 ± 1.6 nm gap. Finally, the PDMS film was peeled off the Si wafer and served as a SERS substrate for biosensing experiments.

Periodic MIM Array Fabrication

PMMA950 A4 (MicroChem, USA) E-beam resist was spin-coated at 1500 rpm for 60 s onto an Si substrate cleaned with acetone and isopropyl alcohol prior to spin-coating. The sample was then exposed at a dosage of 800 μC cm–2 (Raith EBPG 5200, Raith Nanofabrication, Germany). Following exposure, development was carried out using a 1:1 ratio of IPA/MIBK for 1 min. Next, SiO2 was directionally deposited via E-beam evaporation (CHA MK40 E-Beam Evaporation, CHA Industries, USA) after which lift-off was performed in Remover PG leaving behind SiO2 nanopillars. Finally, Au of an appropriate thickness was deposited (CHA MK40 E-Beam Evaporation, CHA Industries, USA) over SiO2 to generate the MIM layer.

High-Resolution Imaging and Statistical Analyses of the Flex-MS

SEM imaging was performed on the flex-MS using a Nova 200 Novalab Dualbeam microscope (FEI, USA) at 10 kV. ImageJ (National Institutes of Health, USA), a Java-based public-domain image processing tool was used to obtain the diameter distribution of the nanostructures. Every pixel in the acquired SEM images were converted to black or white based on a thresholding condition that was obtained by calculating the mean intensity value of all pixels of the image. Following this, the diameter distribution of the MIM scatterers was determined. Finally, the FFT analysis to determine short-range periodicity was performed using MATLAB (MathWorks, USA).

Optical Simulations of the Flex-MS

Optical simulations of a single MIM nanostructure as well as the quasi-ordered/periodic ensemble as a whole were performed using 3D finite-difference time-domain software (Lumerical Solutions, Canada). A combination of periodic and perfect matching layer boundary conditions along with a plane wave source was used. The absorption cross section was obtained in the total field region inside the source while the scattering cross section was obtained in the scattered field region outside the source. The influence of the nanogap was studied as shown in Figure S6. The |E/Eo|2 enhancement was obtained by placing a frequency-domain field monitor at the MIM junction spanning the entire MIM nanostructure or the ensemble, respectively. The field maps as well as the effective |E/Eo|2 enhancement (averaged over 4000 individual hotspots or mesh cells) in the case of the ensemble were calculated using MATLAB (MathWorks, USA).

Spectroscopic Analyses

An optical microscope operating in dark-field (DF) mode was used for the microspectroscopic investigation of the fabricated flex-MS and periodic MIM array samples. A halogen lamp was used as a light source using a 50× objective. The scattered light was collected in a confocal configuration and analyzed using a spectrometer (AvaSpec-ULS2048x64-USB2). A 400 μm core optical fiber was used to obtain a spatial resolution of 20 μm to characterize the scattering of the samples.

SERS Measurements

All SERS measurements were performed by incubating a 30 μL droplet of the reagent on the flex-MS for 30 min. Following this, the drop was dislodged from the sample surface using a stream of N2. The sample was then measured dry using a Raman microscope (inVia, Renishaw, United Kingdom) with a 50× objective lens. A 785 nm laser operated at 0.32, 0.62, or 1.12 mW for a duration of 60 s was used to take the measurements.

Artificial Tear Buffer Preparation

Artificial tear buffer was prepared using previously reported methods.[10,42] Briefly, electrolytes K+, HCO3–, Na+, and Cl– were added to DI water at concentrations of 24, 24, 130, and 130 mM, respectively. Following this, representative tear proteins such as lysozyme, lactoferrin, albumin, and IgG were added at concentrations of 2 mg mL–1, 2 mg mL–1, 0.02 mg mL–1, and 3 μg mL–1 respectively.

Whole Tear Assay

Eight individual tear samples were collected from healthy subjects and pooled for the experiment. For a comparative study, a commercial enzyme-based colorimetric UA detection kit (Sigma-Aldrich, USA) was used. The assay was performed following the manufacturer’s protocol.