A plasmonic gold prism array for digital surface-enhanced raman spectroscopy sensing

Abstract

A method of fabricating plasmonic gold prism arrays for digital surface enhanced Raman spectroscopy, a plasmonic gold prism array arranged on a substrate for digital surface enhanced Raman spectroscopy, and a method of using a plasmonic gold prism array that is arranged on a substrate for digital surface enhanced Raman spectroscopy are disclosed. The method of fabricating plasmonic gold prism arrays for digital surface enhanced Raman spectroscopy includes depositing more than one layers of chromium and gold onto a plasmonic substrate comprising a monolayer of polystyrene beads and removing the monolayer of polystyrene beads under sonication in ethanol.

Description

Attorney Docket No. 0184.0323-PCT Client Reference No. C18391_P18391-02A Plasmonic Gold Prism Array for Digital Surface-Enhanced Raman Spectroscopy SensingCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 683,292 filed on August 15, 2024, the contents of which are hereby incorporated by reference in its entirety.GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant 1R35GM149272 awarded by the National Institute of Health. The government has certain rights in the invention.FIELD OF THE DISCLOSURE

[0003] The present disclosure is directed to digital surface-enhanced Raman spectroscopy sensing, and in particular methods and systems for plasmonic gold prism array for digital surface-enhanced Raman spectroscopy sensing.BACKGROUND

[0004] While surface-enhanced Raman spectroscopy (SERS) has proved itself as a powerful tool in the analytical domain, its capability hinges on, and therefore, is governed by the interaction between molecules and plasmonic hotspots on nanostructured metallic substrates. Yet, the highly dynamic and complex molecule-metal interactions give rise to pronounced SERS signal fluctuations, compromising quantitative analysis at ultralow molecular concentrations. Rather than overcoming SERS signal fluctuations, the recently introduced digital SERS approach takes full advantage of their stochastic nature and allows digital visualization of SERS events at the single pixel level based on a predefined signal threshold, significantly pushing down the lowest detectable molecular concentration. Nevertheless, extending digital SERS for routine molecular analysis requires superior two-dimensional plasmonic substrates.Attorney Docket No. 0184.0323-PCT Client Reference No. C18391_P18391-02SUMMARY

[0005] According to examples of the present disclosure, a plasmonic gold prism array for digital SERS sensing is disclosed. As opposed to nanostructured metallic arrays with similar geometry where the vertex and edge modes dominate, the gold prism array displays strong scattering as well as pronounced surface roughness and nanoparticle-like surface features, thus supporting additional plasmonic enhancements beyond the vertex and edge modes. Using 4- aminothiophenol as a model molecule, the counterintuitive distribution of SERS hotspots at the center of a gold prism instead of at its vertices was first verified, and digital SERS sensing at the femtomolar level was demonstrated. The gold prism array can serve as a superior plasmonic platform for a wide range of digital SERS applications in diagnostics, environmental monitoring, and food safety.

[0006] According to examples of the present disclosure, a method of fabricating plasmonic gold prism arrays for digital surface enhanced Raman spectroscopy is disclosed. The method comprises depositing more than one layers of chromium and gold onto a plasmonic substrate comprising a monolayer of polystyrene beads; and removing the monolayer of polystyrene beads under sonication in ethanol. The polystyrene beads can have a diameter of about 5 pm. The polystyrene beads can have a diameter of about between 2 p and 5 pm. The plasmonic substrate can comprise quartz. Each layer of chromium can be 5 nm in thickness. Each layer of gold can be 500 nm in thickness. The plasmonic substrate can have lateral dimensions ranging from tens to hundreds of nanometers. Each gold prism of the plasmonic gold prism arrays can have a length dimension at the base of about 2000 nm, a length dimension at the top of about 1200 nm, and a heigh dimension of about 500 nm. The chromium can be deposited between adjacent polystyrene beads of the monolayer of polystyrene beads and onto a top surface of the plasmonic substrate. The gold can be deposited onto a top surface of each polystyrene bead of the monolayer of polystyrene beads. A center-to-center distance between adjacent plasmonic gold prisms in the plasmonic gold prism array can be about 5 pm. Each plasmonic gold prism in the plasmonic gold prism can provide plasmonic hotspots that are distributed near a center of each gold prism.

[0007] According to examples of the present disclosure, a plasmonic gold prism array arranged on a substrate for digital surface enhanced Raman spectroscopy is disclosed, wherein each gold prism of the plasmonic gold prism arrays have a length dimension at the base of about 2000 nm, a length dimension at the top of about 1200 nm, and a heigh dimension of about 500 nm, a center-to-center distance between adjacent plasmonic gold prisms in theAttorney Docket No. 0184.0323-PCT Client Reference No. C18391_P18391-02 plasmonic gold prism array is about 5 pm, and each plasmonic gold prism in the plasmonic gold prism provides plasmonic hotspots that are distributed near a center of each gold prism.

[0008] According to examples of the present disclosure, a method of using a plasmonic gold prism array that is arranged on a substrate for digital surface enhanced Raman spectroscopy is disclosed, wherein each gold prism of the plasmonic gold prism arrays have a length dimension at the base of about 2000 nm, a length dimension at the top of about 1200 nm, and a heigh dimension of about 500 nm, a center-to-center distance between adjacent plasmonic gold prisms in the plasmonic gold prism array is about 5 pm, and each plasmonic gold prism in the plasmonic gold prism provides plasmonic hotspots that are distributed near a center of each gold prism.BRIEF DESCRIPTION OF THE FIGURES

[0009] FIG. 1A, FIG. IB, FIG. 1C, FIG. ID, FIG. IE, and FIG. IF show a nanosphere lithography fabrication of gold prism arrays according to examples of the present disclosure. The dimensions specified in the inset of FIG. ID are approximated based on scanning electron microscope (SEM) characterizations in FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E, and FIG. 2F.

[0010] FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E, and FIG. 2F show SEM characterization of fabricated gold prism arrays according to examples of the present disclosure, where FIG. 2A, FIG. 2B, and FIG. 2C show top views and FIG. 2D, FIG. 2E, and FIG. 2F show titled views of gold prism arrays with different magnifications. FIG. 2G and FIG. 2H show FDTD simulation of electric field distribution under horizontal and vertical|E|4polarization, respectively, where the SERS enhancement factor is defined as (■ — -) at 785 nm.1^0 IThe color bar i

[0011] FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D show Raman and dark-field spectroscopy characterization according to examples of the present disclosure, where FIG. 3A shows a representative SERS spectrum collected on the gold prism array, which was incubated for two hours into the ethanolic solution of 4-aminothiophenol (4- ATP) with a concentration of 10 pM, FIG. 3B shows dark-field image of the gold prism array under Raman microscope, FIG. 3C shows a 3D view and FIG. 3D shows a top view of Raman mapping of the green enclosed region in FIG. 3B. For FIG. 3C and FIG. 3D, the Raman mapping was conducted over an area of about 20 pm x 20 pm with a pixel size about 1 pm with a total of 21 x 21 spectra collected.Attorney Docket No. 0184.0323-PCT Client Reference No. C18391_P18391-02 The peak of 4- ATP at about 1074 cm1was used to reconstruct the images in FIG. 3C and FIG. 3D.

[0012] FIG. 4A shows a background Raman spectra of a bare Au prism array by directly taking Raman spectroscopy measurements without using any Raman molecules and FIG. 4B shows Raman imaging reconstructed based on the integrated Raman intensity in the wavenumber range from 1800 cm'1to 2000 cm'1, as indicated by the grey region in FIG. 4A according to examples of the present disclosure. The bolded red and blue spectra in FIG. 4A were collected from point ‘ 1’ and ‘2’ in FIG. 4B.

[0013] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 5G, and FIG. 5H show digital SERS mapping of 4-ATP molecules with varying concentration on Au prism array substrates according to examples, where FIG. 5A shows digital SERS ‘on’ criteria based on Is— x > 5<J, where Isis the SERS peak intensity at 1074 cm'1, and (x, <J) represent the mean and the standard deviation of the maximum intensity between 1020-1040 cm'1(grey region), and FIG. 5B show digital SERS ‘off criteria based on Is— x < 5<J. FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 5G, and FIG. 5H show representative digital SERS images with the 4-ATP molecule concentration of 100 nM in FIG. 5C, 10 nM in FIG. 5D, 10 pM in FIG. 5E, 10 fM in FIG. 5F, 1 fM in FIG. 5G. and percentage of digital count in relation to the concentration of 4- ATP in FIG. 5H.

[0014] FIG. 6 shows a plot of a regression analysis based on digital SERS sensing of 4-ATP molecules on Au prism array substrates from FIG. 5H according to examples of the present disclosure.DETAILED DESCRIPTION

[0015] Surface-enhanced Raman spectroscopy (SERS), since discovered 50 years ago,1has dramatically advanced our fundamental understanding of light-matter interactions at the nanoscale.2'5Owing to its high sensitivity,6'7molecular specificity,8'10and nondestructiveness,11'12SERS has proved itself as a powerful analytical tool for molecular analysis,13'15biomedical diagnostics,4, 16-17and environmental monitoring.18-20Nevertheless, the capability of SERS hinges on, and therefore, is governed by the interaction between molecules and plasmonic hotspots on nanostructured metallic substrates, being either colloidal metallic nanoparticles or metallic nanostructured arrays.21-23Particularly, recent singlemolecule SERS studies have revealed a highly dynamic process underpinning the complex molecular-metal interactions, which gives rise to pronounced SERS signal fluctuations.21-22While SERS analysis of molecules at a high concentration is barely affected owing to theAttorney Docket No. 0184.0323-PCT Client Reference No. C18391_P18391-02 ensemble effect, the intrinsic SERS signal fluctuations could compromise quantitative analysis of molecules at ultralow concentrations.

[0016] Rather than countering single-molecule SERS intensity fluctuations, Brolo et al. recently harnessed such unique SERS effects and introduced a pixel-based digital SERS protocol for chemical analysis at ultralow molecular concentrations.24In digital SERS, each pixel is derived from a SERS spectrum. Owing to the stochastic nature of single-molecule SERS, a large number of SERS spectra are often collected in order to statistically and meaningfully account for the positive digital SERS events, which are increasingly rare to occur at a lower molecular concentration. The positive digital SERS signals can be determined based on a predefined signal threshold, which filters background and removes false positives.18The percentage of positive digital SERS events in relation to the studied molecular concentration can be utilized to generate a calibration curve in the same manner as the mean SERS intensitybased traditional approach. The unique advantage of digital SERS is that it allows digital visualization of SERS events at the single pixel level, significantly pushing down the lowest detectable molecular concentration.17’24In contrast, for the mean SERS intensity -based traditional approach, the positive SERS signals may be either too rare to be captured based on limited spectral sampling or obscured by the background after averaging. Given its extraordinary capability, the digital SERS has recently been leveraged to develop a digital colloid-enhanced Raman spectroscopy by single-molecule counting.18Such a colloidal digital SERS approach, limited only by the Poisson noise of the measurement process, allows reproducible detection of various molecular analytes in liquid form at ultralow concentrations.

[0017] Despite the apparent success of the colloidal digital SERS approach, conducting routine digital SERS-based molecular analysis is contingent upon the availability of superior two-dimensional plasmonic substrates, which can provide a more versatile platform free of nanoparticle aggregation issues under harsh or physiologically relevant conditions for a wide range of applications.23, 25-26While dried gold nanoparticles-based substrates were utilized to demonstrate the concept of digital SERS for various sensing applications,17, 24, 27-28plasmonic nanostructured arrays with a well-defined geometry are more desirable, as they are not only compatible with industrially relevant silicon-based cleanroom fabrication process, but also can provide better controlled hotspots for SERS enhancement.

[0018] Herein, a two-dimensional plasmonic platform for digital SERS sensing based on a plasmonic gold prism array is disclosed. As opposed to nanostructured metallic arrays with similar geometry where the vertex and edge modes dominate the plasmonic response,29-31the gold prism array displays strong scattering because of its microscale dimensions, as well asAttorney Docket No. 0184.0323-PCT Client Reference No. C18391_P18391-02 pronounced surface roughness and nanoparticle-like surface features, thus supporting additional plasmonic enhancements beyond the vertex and edge modes. Using 4- aminothiophenol (4-ATP) as a model Raman molecule, the counterintuitive distribution of SERS hotspots at the center of an Au prism instead of its vertices or edges was first verified. Furthermore, digital SERS sensing at the femtomolar level was demonstrated by digitizing each SERS spectrum based on a predefined signal threshold.

[0019] Results and Discussion

[0020] FIG. 1A, FIG. IB, FIG. 1C, FIG. ID, FIG. IE, and FIG. IF show a nanosphere lithography fabrication of gold prism arrays according to examples of the present disclosure. The dimensions specified in the inset of FIG. ID are approximated based on scanning electron microscope (SEM) characterizations in FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E, and FIG. 2F.

[0021] FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E, and FIG. 2F show SEM characterization of fabricated gold prism arrays according to examples of the present disclosure, where FIG. 2A, FIG. 2B, and FIG. 2C show top views and FIG. 2D, FIG. 2E, and FIG. 2F show titled views of gold prism arrays with different magnifications. FIG. 2G and FIG. 2H show FDTD simulation of electric field distribution under horizontal and vertical|E|4polarization, respectively, where the SERS enhancement factor is defined as (■ — -) at 785 nm.1^0 IThe color bar i

[0022] Fabrication and characterization of plasmonic gold prism arrays. Nanosphere lithography,32'34as a well-established and versatile nanofabrication method, was adopted to create the gold prism arrays. Currently, most of the fabricated plasmonic substrate by nanosphere lithography are based on small polystyrene beads, typically with a diameter of or less than 2 pm.32This is because of the ease of performing monolayer patterning of smaller polystyrene beads. As a result, the obtained plasmonic substrates have typical lateral dimensions ranging from tens to hundreds of nanometers. To create gold prism arrays with microscale lateral dimensions, herein, large polystyrene beads with a diameter of 5 pm is used. Following the experimental protocol that was optimized previously,4, 31, 35'37, monolayer polystyrene beads-based templates were first created through the dip-coating process on cleaned quartz slides (FIG. 1 A and FIG. IB). After successive deposition of chromium (Cr, 5 nm, which helps improve the adhesion between quartz and gold) and gold (Au, 500 nm), followed by removal of the polystyrene beads under sonication in ethanol, Au prism arrays were obtained (FIG. 1C, FIG. ID, FIG. IE, and FIG. IF). Given the versatility of nanosphereAttorney Docket No. 0184.0323-PCT Client Reference No. C18391_P18391-02 lithography, a wide spectrum of parameters, such as the deposited layer thickness, periodicity, and deposition angle, can be adjusted to tune geometry of the fabricated Au prism arrays.

[0023] The fabricated Au prism arrays display well-defined prism structures arranged in a hexagonal fashion (FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E, and FIG. 2F), as revealed by scanning electron microscope (SEM) characterizations. Each individual gold prism has a nominal height of about 500 nm. The top and bottom edge length was approximated to be 1212 nm and 2078 nm, respectively, as indicated in FIG. IF. With such a large dimension in the microscale, the Au prism array can support strong scattering and is thus well suited for SERS applications.

[0024] Finite-difference time-domain (FDTD) simulations were employed to investigate the electric field distribution on the Au prism array under an excitation wavelength of 785 nm, to be consistent with experimental studies. Each Au prism was modelled based on the experimentally measured dimensions (FIG. IF) without considering surface roughness or any other fine features on the surface. The FDTD simulation results suggest that the electric field enhancement primarily arises from light confinement at the vertices and edges of the prism with a maximum SERS enhancement factor estimated to be ~107(FIG. 2G and FIG. 2H).Such an estimation is likely to be an underestimate, as the FDTD simulations didn’t take the surface roughness and other nanoparticle-like features into account. By closely examining the surface of an Au prism (FIG. 2C and FIG. 2F), it can be reasonably inferred that the surface roughness and nanoparticle-like features could support plasmonic coupling, and can provide additional SERS enhancements not fully captured by FDTD simulations.

[0025] FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D show Raman and dark-field spectroscopy characterization according to examples of the present disclosure, where FIG. 3A shows a representative SERS spectrum collected on the gold prism array, which was incubated for two hours into the ethanolic solution of 4-aminothiophenol (4- ATP) with a concentration of 10 pM, FIG. 3B shows dark-field image of the gold prism array under Raman microscope, FIG. 3C shows a 3D view and FIG. 3D shows a top view of Raman mapping of the green enclosed region in FIG. 3B. For FIG. 3C and FIG. 3D, the Raman mapping was conducted over an area of about 20 pm x 20 pm with a pixel size about 1 pm with a total of 21 x 21 spectra collected. The peak of 4- ATP at about 1080 cm1was used to reconstruct the images in FIG. 3C and FIG. 3D.

[0026] SERS mapping of an Au prism array. To evaluate the SERS performance of the Au prism array, correlative characterization was conducted by combing Raman spectroscopy andAttorney Docket No. 0184.0323-PCT Client Reference No. C18391_P18391-02 dark-field imaging. In Raman spectroscopy measurements, 4-ATP was selected as the Raman molecule (FIG. 3 A, inset). Under white light illumination, the structure of an Au prism array can be well resolved by the dark-field imaging (FIG. 3B). This suggests strong light scattering from each Au prism. To perform Raman spectroscopy imaging, the Au prism array substrate was first incubated at room temperature for two hours in the ethanolic solution of 4-ATP with a concentration of 10 pM. During incubation, 4-ATP molecules became covalently functionalized onto the Au prism surface through Au-S covalent bonding. Excessive and physically adsorbed 4-ATP molecules were washed away by rinsing using ethanol. Subsequently, Raman mapping was conducted over an area of about 20 pm x 20 pm with a total of 21 x 21 spectra collected under an excitation wavelength of 785 nm. The studied area was indicated by the green enclosed region in the dark-field image (FIG. 3B). The SERS peak intensity of 4-ATP at about 1074 cm'1after background removal was used to reconstruct 3D and 2D images of the studies area (FIG. 3C and FIG. 3D). The reconstructed images provided a detailed SERS intensity distribution over the studied area. Counterintuitively, the highest SERS intensity predominately occurred at the center of an Au prism, instead of its vertices (FIG. 3C and FIG. 3D). The discrepancy between FDTD simulations of the plasmonic hotspots (FIG. 2G and FIG. 2H) and experimental measurements (FIG. 3C and FIG. 3D) highlights the dominant contributions of surface roughness and plasmonic coupling among nanoparticle-like features on an Au prism to SERS enhancement, which was not accounted for in FDTD simulations.

[0027] FIG. 4A shows a background Raman spectra of a bare Au prism array by directly taking Raman spectroscopy measurements without using any Raman molecules and FIG. 4B shows Raman imaging reconstructed based on the integrated Raman intensity in the wavenumber range from 1800 cm'1to 2000 cm'1, as indicated by the grey region in FIG. 4A according to examples of the present disclosure. The bolded red and blue spectra in FIG. 4A were collected from point ‘ 1’ and ‘2’ in FIG. 4B.

[0028] Digital SERS sensing. Prior to performing digital SERS studies of 4-ATP molecules, the background Raman spectra was first collected from a bare Au prism array substrate without any Raman molecules used (FIG. 4A). Owing to the strong scattering of an Au prism as compared to the bare quartz substrate, the collected background Raman spectra can be utilized to map the scattering intensity distribution (FIG. 4B). The reconstructed background Raman scattering image of an Au prism array is largely similar to that obtained based on mapping the SERS intensity distribution of 4-ATP molecules as shown in FIG. 3C and FIG. 3D, validating the counterintuitive distribution of plasmonic hotspots at the center of an Au prism. This alsoAttorney Docket No. 0184.0323-PCT Client Reference No. C18391_P18391-02 suggests that the background Raman scattering image can provide a reference frame to pinpoint each positive digital SERS signal in the ensuing digital SERS studies of 4-ATP molecules on the Au prism array.

[0029] To perform digital SERS molecular sensing, 10 mL of ethanolic solutions containing different concentrations of 4-ATP molecules were prepared. To study a given concentration of 4-ATP, an Au prism array substrate was incubated in the corresponding 4-ATP ethanolic solution for 2 hours. After removal of excessive and nonspecifically adsorbed 4-ATP molecules, the substrate was dried by compressed air and then studied by Raman mapping over an area of about 20 pm x 20 pm with a total of 21 x 21 spectra collected under an excitation wavelength of 785 nm. This gives each pixel a dimension of approximately 1 pm x 1 pm. While increasing the number of SERS spectra collected over a given area could theoretically improve the resolution of each pixel, and thus improving the accuracy of detection, this is limited by the intrinsic spatial resolution of the Raman microscope. In the present disclosure, a confocal Raman microscope (XploRA PLUS from Horiba) with an objective lOOx (NA=0.9, WD=0.21mm) was used to perform SERS spectral collection at an excitation wavelength of 785 nm. This provides a theoretical spatial resolution of about 532 nm based on r=0.61k / NA, where r is the spatial resolution, is the excitation wavelength, and NA is the objective lens numeric aperture. However, the practical resolution of a Raman microscope is often less than the theoretical diffraction limit due to factors like optical aberrations, noise, and sample properties that degrade signal quality. Additionally, environmental vibrations, focus stability, and detector limitations can further reduce the effective resolution in real-world applications.

[0030] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 5G, and FIG. 5H show digital SERS mapping of 4-ATP molecules with varying concentration on Au prism array substrates according to examples, where FIG. 5A shows digital SERS ‘on’ criteria based on Is— x > 5<J, where Isis the SERS peak intensity at 1074 cm’1, and (x, <J) represent the mean and the standard deviation of the maximum intensity between 1020-1040 cm’1(grey region), and FIG. 5B show digital SERS ‘off criteria based on Is— x < 5<J. FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 5G, and FIG. 5H show representative digital SERS images with the 4-ATP molecule concentration of 100 nM in FIG. 5C, 10 nM in FIG. 5D, 10 pM in FIG. 5E, 10 fM in FIG. 5F, 1 fM in FIG. 5G. and percentage of digital count in relation to the concentration of 4- ATP in FIG. 5H.

[0031] A representative SERS spectrum of 4-ATP molecules after background removal was shown in FIG. 5 A. The characteristic peak occurs at about 1074 cm’1(pink highlight) with anAttorney Docket No. 0184.0323-PCT Client Reference No. C18391_P18391-02 origin from the C-S stretching mode.38This peak is defined to have an intensity of Is. To assign a digital SERS ‘on’ signal, Is— x > 5<J (FIG. 5A) is used, (x, <J) represent the calculated mean and standard deviation of the maximum intensity over the spectral window from 1020 to 1040 cm’1(grey region) from the spectra collected on the same Au prism array substrate, as this corresponds to the baseline of the 1074 cm’1peak. Otherwise, the digital SERS is assigned an ‘off signal (FIG. 5B). The selection of a relatively high threshold (Js— x > 5<J) for a positive digital SERS signal was made based on the consideration of achieving low false positives.

[0032] Using the above digital SERS ‘on’ criteria, the distribution of 4-ATP molecules with various concentrations can be accurately pinpointed over the background Raman scattering image of an Au prism array (FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, and FIG. 5G). The percentage of digital count of SERS ‘on’ signals was found to increase roughly linearly in relation to the logarithmic concentration of 4-ATP molecules from 1 fM to 10 nM, as shown in FIG. 5H, where each studied concentration was repeated at least three times on independent samples to generate the error bars. Regression analysis confirmed the strong correlation between the digital SERS count and the logarithmic concentration of 4-ATP with R2=0.95, as shown in FIG. 6. With a further concentration increase, as the single-molecule SERS behaviors became overshadowed by ensemble SERS signals, the digital count of SERS ‘on’ signals increases dramatically.

[0033] It is important to note that the observed digital SERS ‘on’ signals at 10 nM and less (FIG. 5D, FIG. 5E, FIG. 5F, and FIG. 5G) could either be missed because of limited sampling on the Au prism array or obscured by the negative digital SERS signals and the background after averaging if the mean SERS intensity-based traditional approach was implemented. This highlights the distinct advantage of utilizing the pixel-based digital SERS protocol for molecular analysis at ultralow molecular concentrations.

[0034] FIG. 6 shows a plot of a regression analysis based on digital SERS sensing of 4-ATP molecules on Au prism array substrates from FIG. 5H according to examples of the present disclosure.

[0035] Details of chemicals, instruments and characterizations, FDTD simulations and regression analysis (FIG. 6) based on digital SERS sensing of 4-ATP molecules on Au prism array substrates.

[0036] ChemicalsAttorney Docket No. 0184.0323-PCT Client Reference No. C18391_P18391-02

[0037] 4-Aminothiophenol and ethanol were purchased from Sigma-Aldrich. Polystyrene microspheres (5 micron, 4 wt% dispersion in water) were purchased from ThermoFisher Scientific. Quartz slides were purchased from Technical Glass Products.

[0038] Instrumentations and characterizations

[0039] Mira 3 Tesscan scanning electron microscopy (SEM) with an acceleration voltage of 10 kV was uszed to characterize Au prism arrays. Raman spectroscopy characterizations were conducted by an XploRA PLUS Raman microscope (HORIBA Instruments Inc., Edison, NJ, USA) with an excitation laser wavelength of 785 nm and an objective of 100* with an output power approximately to be 0.4 pW. Dark-field image was obtained on the same Raman microscope with an objective of 100 x.

[0040] FDTD simulations

[0041] Ansys Lumerical FDTD (release: 2023. R2.1) was used for all numerical simulations. A plane wave was implemented as the input light source with a wavelength of 785 nm. A mesh size of 5 nm was used. Perfectly matched layer boundary conditions were imposed in the direct vertical to the Au prism array. Periodic boundary conditions were imposed in the direction parallel to the Au prism array. The 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.39

[0042] In this disclosure, an gold prism array substrate with microscale lateral dimensions based on nanosphere lithography for digital SERS applications is disclosed. The fabricated Au prism array was found to display counterintuitive behaviors, with the plasmonic hotspots predominantly distributed at the center of an Au prism instead of the vertices and edges, which was confirmed by both the reconstructed Raman image using the characteristic peak intensity of 4-APT at about 1074 nr1and the reconstructed background Raman scattering image of a bare Au prism array. Furthermore, the digital SERS sensing protocol was implemented and digitalized SERS detection of 4-APT molecules down to the femtomolar level was demonstrated. This study paves the way for the Au prism array to be extended as a superior plasmonic platform for a wide variety of digital SERS sensing applications in chemistry and biology.

[0043] In one or more embodiments, the functions described can be implemented in hardware, software, firmware, or any combination thereof. For a software implementation, the techniques described herein can be implemented with modules (e.g., procedures, functions, subprograms, programs, routines, subroutines, modules, software packages, classes, and so on) that perform the functions described herein. A module can be coupled to another module or aAttorney Docket No. 0184.0323-PCT Client Reference No. C18391_P18391-02 hardware circuit by passing and / or receiving information, data, arguments, parameters, or memory contents. Information, arguments, parameters, data, or the like can be passed, forwarded, or transmitted using any suitable means including memory sharing, message passing, token passing, network transmission, and the like. The software codes can be stored in memory units and executed by processors. The memory unit can be implemented within the processor or external to the processor, in which case it can be communicatively coupled to the processor via various means as is known in the art.

[0044] Further, the steps in the processing methods described herein may be implemented by running one or more functional modules in information processing apparatus such as general purpose processors or application specific chips, such as ASICs, FPGAs, PLDs, or other appropriate devices. These modules, combinations of these modules, and / or their combination with general hardware are all included within the scope of protection of the invention.

[0045] The examples set forth herein represent the necessary information to enable those skilled in the art to practice the embodiments and illustrate the best mode of practicing the embodiments. Upon reading the description in light of the accompanying drawing figures, those skilled in the art will understand the concepts of the disclosure and will recognize applications of these concepts not particularly addressed herein. It should be understood that these concepts and applications fall within the scope of the disclosure and the accompanying claims.

[0046] It will be understood that, although the terms first, second, etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. For example, a first element could be termed a second element, and, similarly, a second element could be termed a first element, without departing from the scope of the present disclosure. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0047] It will be understood that when an element such as a layer, region, or substrate is referred to as being “on” or extending “onto” another element, it can be directly on or extend directly onto the other element or intervening elements may also be present. In contrast, when an element is referred to as being “directly on” or extending “directly onto” another element, there are no intervening elements present. Likewise, it will be understood that when an element such as a layer, region, or substrate is referred to as being “over” or extending “over” another element, it can be directly over or extend directly over the other element or intervening elements may also be present. In contrast, when an element is referred to as being “directly over” or extending “directly over” another element, there are no intervening elements present.Attorney Docket No. 0184.0323-PCT Client Reference No. C18391_P18391-02It will also be understood that when an element is referred to as being “connected” or “coupled” to another element, it can be directly connected or coupled to the other element or intervening elements may be present. In contrast, when an element is referred to as being “directly connected” or “directly coupled” to another element, there are no intervening elements present.

[0048] Relative terms such as “below” or “above” or “upper” or “lower” or “horizontal” or “vertical” may be used herein to describe a relationship of one element, layer, or region to another element, layer, or region as illustrated in the Figures. It will be understood that these terms and those discussed above are intended to encompass different orientations of the device in addition to the orientation depicted in the Figures.

[0049] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises,” “comprising,” “includes,” and / or “including” when used herein specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms used herein should be interpreted as having a meaning that is consistent with their meaning in the context of this specification and the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

Claims

Attorney Docket No. 0184.0323-PCT Client Reference No. C18391_P18391-02What is Claimed is:

1. A method of fabricating plasmonic gold prism arrays for digital surface enhanced Raman spectroscopy, the method comprising: depositing more than one layers of chromium and gold onto a plasmonic substrate comprising a monolayer of polystyrene beads; and removing the monolayer of polystyrene beads under sonication in ethanol.

2. The method of claim 1, wherein the polystyrene beads have a diameter of about 5 pm.

3. The method of claim 1 , wherein the polystyrene beads have a diameter of about between 2 p and 5 pm.

4. The method of claim 1, wherein the plasmonic substrate comprises quartz.

5. The method of claim 1, wherein each layer of chromium is 5 nm in thickness.

6. The method of claim 1, wherein each layer of gold is 500 nm in thickness.

7. The method of claim 1, wherein the plasmonic substrate has lateral dimensions ranging from tens to hundreds of nanometers.

8. The method of claim 1, wherein each gold prism of the plasmonic gold prism arrays have a length dimension at the base of about 2000 nm, a length dimension at the top of about 1200 nm, and a heigh dimension of about 500 nm.

9. The method of claim 1, wherein the chromium is deposited between adjacent polystyrene beads of the monolayer of polystyrene beads and onto a top surface of the plasmonic substrate.

10. The method of claim 1, wherein the gold is deposited onto a top surface of each polystyrene bead of the monolayer of polystyrene beads.Attorney Docket No. 0184.0323-PCT Client Reference No. C18391_P18391-0211. The method of claim 1 , wherein a center-to-center distance between adj acent plasmonic gold prisms in the plasmonic gold prism array is about 5 pm.

12. The method of claim 1, wherein each plasmonic gold prism in the plasmonic gold prism provides plasmonic hotspots that are distributed near a center of each gold prism.

13. A plasmonic gold prism array arranged on a substrate for digital surface enhanced Raman spectroscopy, wherein each gold prism of the plasmonic gold prism arrays have a length dimension at the base of about 2000 nm, a length dimension at the top of about 1200 nm, and a heigh dimension of about 500 nm, a center-to-center distance between adjacent plasmonic gold prisms in the plasmonic gold prism array is about 5 pm, and each plasmonic gold prism in the plasmonic gold prism provides plasmonic hotspots that are distributed near a center of each gold prism.

14. The plasmonic gold prism array of claim 13, wherein the substrate comprises quartz.

15. A method of using a plasmonic gold prism array that is arranged on a substrate for digital surface enhanced Raman spectroscopy, wherein each gold prism of the plasmonic gold prism arrays have a length dimension at the base of about 2000 nm, a length dimension at the top of about 1200 nm, and a heigh dimension of about 500 nm, a center-to-center distance between adjacent plasmonic gold prisms in the plasmonic gold prism array is about 5 pm, and each plasmonic gold prism in the plasmonic gold prism provides plasmonic hotspots that are distributed near a center of each gold prism.

16. The method of claim 15, wherein the substrate comprises quartz.

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