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Hyungsoon Im , Kyle C. Bantz , Si Hoon Lee , Timothy W. Johnson , Christy L. Haynes , * and Sang-Hyun Oh *

Self-Assembled Plasmonic Nanoring Cavity Arrays for SERS and LSPR Biosensing

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In recent years, surface-enhanced Raman scattering (SERS) has found increased use both as a method to characterize surface molecular structure and orientation and as an analytical signal transduction mechanism in sensors. [ 1–10 ] One major challenge in widespread use of SERS lies in the materials design aspect, namely, creating noble metal substrates with reproducible and large Raman enhancements over wide areas, ideally using inexpensive, high-throughput methods. Among the various nanofabrication techniques used to produce SERS substrates, nanosphere lithography (NSL) – also called colloidal lithog-raphy – has been adopted by many researchers, because it is a facile, inexpensive, and reproducible fabrication method for the production of large-area ordered nanostructures. [ 11–17 ] In par-ticular, Ag fi lm over nanosphere (AgFON) substrates have been widely used in many SERS biosensing applications in complex media. [ 6 , 18 , 19 ] The robust AgFON structures are simply made by depositing optically thick (usually at least half the nanosphere diameter) Ag fi lms over self-assembled nanospheres. These substrates present roughness on multiple scales, including that generated by the nanosphere grating as well as the metal dep-osition-generated roughness, creating plasmonic features with improved control and tunability compared to randomly rough-ened metal surfaces. Nevertheless, previous studies have shown that only a few dominant SERS ‘hotspots’ contribute to the overall SERS signal on the AgFON substrate, [ 20 ] and thus it is desirable to control the position and density of those hotspots. Alternately to relying on metallic nanoparticles or rough sur-faces, large SERS enhancements have also been observed from molecules positioned inside thin metallic gaps and cavities.

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Dr. H. Im, [+] T. W. Johnson, Prof. S.-H. OhDepartment of Electrical and Computer EngineeringUniversity of MinnesotaMinneapolis, MN 55455, USAE-mail: sang@umn.edu Dr. K. C. Bantz, [+] Prof. C. L. HaynesDepartment of ChemistryUniversity of MinnesotaMinneapolis, MN 55455, USAE-mail: chaynes@umn.edu Dr. S. H. Lee, Prof. S.-H. OhDepartment of Biomedical EngineeringUniversity of MinnesotaMinneapolis, MN 55455, USA Prof. S.-H. OhDepartment of Biophysics and Chemical BiologySeoul National UniversitySeoul 151-747, Korea [+] These authors contributed equally.

DOI: 10.1002/adma.201204283

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Based on theoretical predictions that nanometer-thin metallic gaps will generate very large electromagnetic fi elds, [ 21–24 ] many groups have explored the controlled creation of nanometric gaps for SERS via lithographic patterning, aggregation of nano-particles, or thin-fi lm processing. [ 17 , 23 , 25–27 ] While SERS in stan-dalone gaps or on roughened nanosphere gratings have been widely investigated, it is highly desirable to build integrated structures that can combine inexpensive, large-area fabrication of AgFON substrates with large fi eld enhancements in ultrathin metallic gaps. In this work, we report a novel method for tem-plated, high-throughput fabrication of a periodic array of ring-shaped nanocavities with 10 nm gap size by combining NSL with straightforward batch processing steps, namely, atomic layer deposition (ALD) and ion milling. In the resulting hybrid nanostructure, incident light resonantly excites ring-shaped 10-nm-gap cavities formed alongside the curvature of metallic nanospheres. Compared to conventional AgFON substrates, the addition of nanoring cavities improves the SERS enhancement factor (EF) by at least an order of magnitude, and the resonance wavelength can be readily tuned by changing the size of nano-spheres used in the original templating step. After optical char-acterization of the nanoring cavity substrates, we employ them for the detection of adenine, a commonly detected molecule in intrinsic DNA sensing experiments, and demonstrate improved detection limits as well as the use of the same substrate for con-current LSPR biosensing.

The fabrication process for making the nanoring cavity sub-strate is illustrated in Figures 1 a and b. First, a AgFON sub-strate is prepared by depositing a 200 nm-thick Ag fi lm on 500-nm-diameter polystyrene nanospheres assembled on a glass substrate. For nanoring cavity formation, a thin Al 2 O 3 layer is conformally deposited on the Ag surface via ALD, fol-lowed by deposition of another 200 nm-thick Ag fi lm, forming Ag/Al 2 O 3 /Ag layers on the nanospheres. The use of ALD is critical, since the self-limiting ALD process can deposit the thin Al 2 O 3 layer with angstrom-scale thickness resolution, uni-formity, as well as nearly perfect conformality. Subsequent ani-sotropic etching of the top Ag layer using blanket ion milling reveals the underlying Al 2 O 3 layer as well as bowl-shaped Ag nanostructures formed along the curvature of the AgFON sub-strate, separated by the thin Al 2 O 3 layer. Partially removing the Al 2 O 3 layer using a buffered oxide etchant (BOE) solution with controlled etching time reveals Ag-air-Ag nanoring cavities that act as sites for localization of electromagnetic fi elds and thus, sensitive detection sites for SERS. In this scheme, the gap size is determined by the thickness of the Al 2 O 3 layer, which can be precisely controlled by the number of ALD cycles. Further-more, because the nanoring arrays are made via wafer-scale

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Figure 1 . (a) A schematic of fabrication process for plasmonic nanoring cavities using nanosphere lithography and atomic layer deposition (ALD). First ALD is used to conformally deposit an Al 2 O 3 layer on the FON substrate prepared by depositing Ag fi lms on polystyrene nanospheres dispersed on a glass slide. Another Ag layer is then deposited on the Al 2 O 3 layer, forming Ag/Al 2 O 3 /Ag layers stacked on the nanospheres. Anisotropic etching of the top Ag layer using ion-milling reveals the underlying Al 2 O 3 layer with a bowl-shaped Ag residual layer on the nanospheres. Partially removing the Al 2 O 3 layer reveals Ag/air/Ag nanoring cavities with a nanogap size defi ned by the thickness of the ALD-grown Al 2 O 3 fi lm. (b) Cross-sectional schematics of the fabrication process. (c) Scanning electron micrograph (SEM) of the nanoring cavities on the FON substrates. Scale bar: 200 nm. (d) SEM image of the nanoring cavity array formed over a 16 μ m × 10 μ m area. (e) Photograph of the nanoring cavity (FON-gap) sample. On the standard glass slide, the FON-gap structures are made in a 2 cm-wide circular area.

batch processes, a dense array of nanogaps can be readily pat-terned over cm 2 -sized areas, which cannot be accomplished by serial patterning techniques such as electron-beam lithography. Figures 1 c and d show scanning electron micrographs (SEM) of the fabricated nanoring cavity structures with a 10 nm-wide gap size. We also call these nanoring arrays formed on the fi lm-over-nanosphere (FON) substrate ‘FON-gap’ structures. Figure 1 e shows a photograph of the FON-gap sample patterned in a circular area that is 2 cm in diameter.

To characterize the SERS performance of the FON-gap sub-strate, a confocal Raman microscope with 514.5 nm excita-tion was used to measure SERS spectra after introduction of analyte molecules. The FON-gap sample was compared with the regular AgFON substrate and a FON-0 nm gap structure where the top Ag layer was deposited in the absence of the Al 2 O 3 layer. Each nanostructure was functionalized with 1 mM benzenethiol (BZT), and the signature BZT Raman band at ∼ 1000 cm − 1 shift was used to construct intensity maps, where each pixel represents the intensity of the Raman peak at the spatial position on the substrate. Figures 2 a-e show magnifi ed micrographs of the FON, FON-0 nm gap, and FON-10 nm gap structures along with confocal Raman images. For the FON-gap structures, well-defi ned 10-nm gaps are formed around each nanosphere. Notably, the nanoring cavity arrays formed on the

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FON substrate boost the Raman intensity by at least ten-fold with 514.5 nm excitation. The mean value of Raman intensity for the 10 nm gap structure (LSPR λ max = 518 nm) is improved by about 70 fold compared to the regular FON substrate (LSPR λ max = 661 nm) and over 900 fold compared to the 0 nm gap structure (LSPR λ max = 477 nm). Since the increase in the sur-face area due to the top Ag layer is no more than 15% (Table S1 in the Supporting Information), the increase in Raman intensity can be mainly attributed to the concentration of elec-tromagnetic energy in the nanoring cavities. In addition, the Raman signal from the control sample with a 0 nm gap (i.e., no Al 2 O 3 deposition) is even weaker than the regular FON sub-strate. This is likely because the deposition of the top Ag layer in the absence of the Al 2 O 3 layer fi lls sharp corners between nanospheres where strong SERS signals are usually obtained, and makes the area fl at, resulting in the reduced SERS signals. In addition, the formation of well-defi ned gap structures around nanospheres allows a structured distribution of SERS hotspots along packed nanospheres (Figure S1).

Figure 2 g shows extinction spectra of the AgFON, FON-0 nm gap, and FON-10 nm gap structures. The AgFON substrate exhibits a broad localized surface plasmon resonance (LSPR), with a maximum at 661 nm and a full-width at half-maximum (FWHM) of 122 nm. The addition of the nanoring cavity structure

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Figure 2 . SEM and scanning confocal Raman images of the (a,d) AgFON, (b,e) FON with 0 nm gap, and (c,f) FON with 10 nm gap structures. Scale bar: (a)-(c) 500 nm. Each pixel in the confocal Raman images represents Raman intensity of a distinct peak for benzenethiol around 1000 cm − 1 in 6 μ m × 6 μ m areas. (g) Measured extinction spectra of FON, FON with 0 nm gap, and FON with 10 nm gap structures. The periodicities of FON substrates, determined by the size of nanospheres used, are all 500 nm. (h) Finite-difference time-domain (FDTD) computer simulations showing the distribution of electromagnetic fi eld intensity around FONs with 0 and 10 nm gap structures. It can be seen that strong fi elds, corresponding to large enhancements of SERS signals, are confi ned within the 10 nm gap.

on the same AgFON substrate dramatically changes the optical properties, and the resulting FON-gap substrate exhibits a sharper resonance peak with a maximum of 518 nm and a FWHM of 73 nm. In contrast, when the nanoring cavities are made in the absence of Al 2 O 3 layer, i.e., FON-0 nm gap, the LSPR becomes much weaker than the one with a 10-nm gap, as explained above. To understand the physical mechanism behind electromagnetic fi eld confi nement in nanoring arrays, (3D) fi nite-difference time-domain (FDTD) calculations were done. A commercial software package was used for the FDTD calcula-tions (FullWAVE by Rsoft, Inc.). The simulation area consists of two spheres with periodic boundary conditions and a grid size of 2.6 nm graded down to 1.3 nm near the interfaces in the planes perpendicular to the substrate and perfectly-matched layer boundaries and a grid size of 10 nm graded down to 1 nm

© 2013 WILEY-VCH Verlag Adv. Mater. 2013, DOI: 10.1002/adma.201204283

in the plane parallel to the substrate. Figure S2 shows simulated extinction spectra from the FON-gap structure and FON-0 nm gap structures. Both structures show a resonance peak around 520 nm while when a 10-nm annular gap is formed, the reso-nance becomes 5-fold stronger than the 0-nm gap structure, which match well with the measured spectra, as shown in Figure 2 g. Figure 2 h shows full-fi eld cross-sectional fi eld-inten-sity maps of the FON-0 nm gap and FON-10 nm gap structures at 520 nm. Upon excitation with normally incident light, both structures show a similar plasmonic resonance on the top of the spheres partially due to their periodic nature, but in the FON-10 nm gap structures, this mode is also coupled with a resonance inside the nanoring cavity. Normally, coupling free-space light into plasmons in sub-10-nm metallic gaps suffers from low effi ciency because of a large impedance mismatch.

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Figure 3 . SEM and confocal Raman images of the fabricated FON-10 nm-gap structures. (a) and (d) were made with 400 nm nanospheres, (b) and (e) were made with 500 nm nanospheres, and (c) and (f) were made with 600 nm nanospheres. Scale bar: (a)-(c) 1 μ m. Each pixel in the confocal Raman images represents Raman intensity of a distinct peaks for benzenethiol around 1000 cm − 1 in 6 μ m × 6 μ m areas. (g) Histogram of Raman intensities for FON-10 nm gap substrates with 400, 500, and 600 nm periodicities. The portions of hotspots are obtained by normalizing the numbers of hotspots by the total pixel number in the scanned area. (h) The averaged Raman spectra of benzenethiol with various FON-gap and FON substrates. (i) LSPR extinction spectra of benzenethiol-dosed FON with 10 nm gaps with periodicities of 400, 500, and 600 nm.

In our FON-gap structure, however, the coupling effi ciency is enhanced via a two-step process, where nanosphere arrays act as an integrated receiver that converts light into plasmons and feeds the energy into surrounding nanocavities, giving rise to a very high fi eld enhancement inside the gap and large enhance-ment of SERS signal.

To achieve the maximum Raman enhancement, it is also important to position the LSPR peak close to the excitation wavelength. [ 28 , 29 ] Because different applications require different excitation wavelengths, LSPR tunability is a desirable character-istic for SERS substrates. For example, excitation with a NIR or IR source is desirable for Raman spectroscopy of biological

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samples to minimize unwanted fl uorescence background. For high-resolution Raman imaging, on the other hand, shorter wavelengths in the visible regime would be preferred. The LSPR of SERS substrates can be tuned by changing the shape, size, thickness, and material of metallic nanostructures. In the case of traditional FON substrates, the centroid resonance posi-tion can be adjusted by varying the size of nanospheres used. [ 30 ] The resonance for FON-gap structures can be tuned in a sim-ilar way. Figure 3 a-f shows SEM images of FON-gap structures made with nanospheres of 400, 500, and 600 nm in diameter and the corresponding confocal Raman micrographs. In terms of the maximum number of SERS hotspots, the FON-gap

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structure with the 400-nm-diameter nanosphere shows higher Raman signals than FON-gap structures made with 500- and 600-nm-diameter nanospheres at this excitation wavelength. The histograms of SERS intensities for FON-gap structures with different periodicities are shown in Figure 3 g. The mean values of Raman intensities for the 1000 cm − 1 shift BZT band from FON-gaps with 400, 500, and 600 nm periodicities are 11000, 8000, and 2000, respectively. These values are at least one order of magnitude higher than the regular FON and FON-0 nm gap substrates, which show mean values of 115 and 8.7, respectively (Figure S3). In addition, the normalized Raman intensities for FON-gap with 400, 500, and 600 nm periodicities are repre-sented by log-normal distribution while the regular FON and FON-0 nm gap substrates tend to tailed distributions (Figure S4). The log-normal distribution of Raman intensity with a peak position close to the mean value and only a few pixels of zero-intensities indicates a large coverage of SERS hotspots in the scanned area. Within the same size of area, however, the tailed distribution with a large portion of low-intensity pixels and a few pixels more than 10 times of mean intensity indicates only a small fraction of hotspots largely contributes the overall SERS intensity. [ 31 ] The widths of fi tted log-normal curves for 10 nm gaps with 400, 500, and 600 nm periodicities are 0.5, 0.4, and 0.7, respectively. These values are comparable to the one obtained from FIB-milled structures, showing a narrow distri-bution of Raman intensities. [ 32 ] The averaged Raman spectra for the FON-gap and control samples are shown in the Figure 3 h. The difference in Raman intensities between FON-gap sub-strates with different periodicities can be understood from their spectral resonance responses shown in Figure 3 i. The regular FON substrate with 500 nm periodicity has a broad resonance peak around 680 nm, but when the gap structures are formed, the resonance peak shifts toward shorter wavelengths, to 530 nm, and the peak become sharper as shown in Figure 2 g and Figure S5 for substrates with 400 nm periodicity. When the periodicity is reduced to 400 nm, the resonance peak shifts fur-ther to 490 nm while the peak for 600 nm periodicity FON-gap red-shifts to 690 nm. Since the 514.5 nm laser was used for con-focal Raman imaging, the FON-gap sample with 400 nm perio-dicity, exhibiting the resonance closest to the excitation wave-length, shows the highest Raman intensity. When a 532 nm excitation laser was used, however, the FON-gap sample with the 500 nm periodicity shows the highest Raman signals with an enhancement factor (EF) of almost 10 8 (Figure S6). It should be noted that this EF is calculated based on the surface area of the entire FON-gap structure rather than the local surface area only considered at the nanogap. [ 9 , 27 ] Since the near-fi eld likely contrib-utes dominantly large SERS EFs, the signals are focused at the nanogap as shown in Figure 2 h, and the local EF at the nanogap is expected to be even higher than 10 8 . While both the FON-gap sample with 600 nm periodicity and regular FON sample made with 500 nm nanospheres are off-resonance (670 nm) for the excitation laser, the FON-gap sample with 600 nm periodicity still shows one order higher SERS intensity com-pared to the FON sample. Therefore, the maximum SERS enhancement and sensitivity can be achieved by adjusting the size of the template nanospheres to match the excitation wave-length. The advantage of the FON-gap substrate is the narrow tunable plasmon, whereas the AgFON substrate has a very

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broad plasmon and therefore has a good ∼ 10 5 − 10 6 EF at many wavelengths. By further red-shifting the λ ex to match the λ max of the AgFON plasmon, we begin to lose Raman scattering because, if everything else is held constant, Raman intensity is proportional to ν 4 , where ν is the excitation frequency. Any EF gain achieved by matching the AgFON plasmon would be offset by the loss of Raman scattering at longer wavelengths.

We have used adenine to demonstrate the applicability of the FON-gap substrate as a practical biosensing platform. Ade-nine is a traditional and widely used biomolecule for intrinsic Raman and SERS sensing due to its intense purine stretch located at 731 cm − 1 shift (Figure S7). This intense stretch has facilitated detection of DNA [ 33 ] and RNA [ 34 , 35 ] during binding studies. However, due to its ubiquitous presence in biochem-ical systems, detection of the adenine stretch intensity is not only useful for DNA or RNA detection. In fact, researchers have used this spectral feature to detect the coenzymes NAD and NADP [ 36 ] or ATP, [ 37 ] and Vitol et al. used it to detect the secondary messenger NAADP in cellular extracts. [ 38 ] Adenine is an ideal molecule to test the biosensing capability of the FON-gap substrate due to the number of biomolecules that contain adenine. In Figure 4 a, both a FON-10 nm gap and AgFON sub-strates were exposed to decreasing concentrations of adenine, and the intensity of the purine stretch at 731 cm − 1 shift was used to evaluate limits of detection. The adenine signal on the FON-gap substrate was more intense than that observed on the AgFON and can be attributed to the adenine molecules expe-riencing higher electromagnetic fi elds at the surface of the FON-gap compared to those on the AgFON surface. The limit of detection of adenine on a FON-gap and AgFON were 76 nM and 524 nM, respectively. These values are much lower than previous SERS limit of detection studies, which had limits in the μ M range [ 39 , 40 ] and demonstrate that FON-gap substrates can be used to detect lower levels of analyte compared to tradi-tional sensing platforms.

The tunability and narrow linewidth of the FON-gap indi-cates that it could also be employed as a sensitive LSPR sensing platform, where a small change in the refractive index sur-rounding the substrate surface due to the binding of analytes can cause large shifts in the LSPR spectra. The bulk refractive index sensitivities were examined monitoring the LSPR while exposing the FON-gap and AgFON to solvents with increasing refractive indices; the LSPR shift was plotted versus solvent refractive index in Figure 4 b. The AgFON has a higher bulk refractive index sensitivity than the FON-gap, 481 ± 8.1 nm/RIU versus 412 ± 3.4 nm/RIU, which are signifi cantly different from each other (p < 0.0001). However, the FON-gap is more sensitive to molecules bound to its surface as indicated when, upon adsorption of benzenethiol on a FON-gap substrate, a 34 nm red-shift is observed, slightly more than double the response of the AgFON (Figure 4 c). A large LSPR shift of 31 nm was also observed when the FON-gap was exposed to 1 mM adenine (Figure S8).

We have demonstrated a hybrid SERS substrate that com-bines the tunable LSPR of AgFON substrates with a strong fi eld enhancement inside 10-nm metallic nanogaps. Using a series of batch fabrication techniques, we have produced this structure in cm 2 -sized areas with the critical dimension below 10 nm. The resonant coupling between Ag nanospheres and

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Figure 4 . (a) A limit of detection plot where the intensity of the purine stretch of adenine is plotted versus adenine concentration. The data points represent the average ± standard deviation of fi ve separate meas-urements. (b) A plot of the bulk refractive index sensitivity of an AgFON and a FON-gap substrate. (c) Extinction spectra of a AgFON substrate and an FON-gap substrate before and after exposure to 1 mM ben-zenethiol for 16 h.

the ring-shaped nanocavity in our integrated device boosts light-coupling effi ciency into the ultrathin gap and increases Raman enhancements by at least one order of magnitude compared with conventional AgFON substrates or isolated metallic nanogaps. The average enhancement factor of near 10 8 is not only higher than recently reported large-area SERS substrates, [ 41–44 ] but also improving the robust AgFON sub-

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strate toward the theoretical maximum enhancement factor of 10 9 . [ 22 , 45 ] This method is not only applicable to FON substrates, but can also be combined with other nanostructures, such as nanoholes, nanoparticles, disks, pillars, and wires. [ 27 ] In addi-tion, the increased LSPR sensitivity for surface-associated spe-cies makes the FON-gap substrate an ideal bioanalytical plat-form to combine SERS and LSPR biosensing. Considering the reproducibility and high SERS enhancement factor, popularity, and widespread usage of the conventional AgFON substrate, we believe that our results represent an important advance for the practical application of engineered SERS substrates.

Experimental Section Fabrication of FON-gap structures : AgFON substrates were made

by depositing a 200 nm-thick Ag fi lm on a packed nanosphere layer. A previously reported procedure was used to obtain a large area monodisperse nanosphere layer. [ 46 ] First, standard glass slides were cleaned with a 1:1:5 solution of NH 4 OH:H 2 O 2 :H 2 O with sonication for 30 min, followed by washing with DI water. Then, the glass slides were cleaned with a 1:1 solution of H 2 SO 4 :H 2 O 2 for 30 min. After washing with DI water, the surface was dried with a N 2 gas. A polydimethylsiloxane (PDMS) ring with 150 μ m in height and 2 cm in diameter was placed on the glass slide. The PDMS ring promotes the formation of nanosphere monolayer in a large-area and defi nes the size of nanosphere packing area. Polystyrene nanospheres (Invitrogen, USA) with the size of 400, 500, and 600 nm were diluted with ethanol and water with a 2:3:3 volume ratio and dispersed on the glass slide. Once the solution was completely dried, the PDMS ring was peeled off. A 15 nm-thick Al 2 O 3 layer was deposited using ALD at 100 ° C to improve the adhesion of FON structures on the glass slides. Then a 200 nm-thick Ag fi lm was deposited using a Temescal electron-beam evaporator with the deposition rate of 2 Å/s. To make nanogap structures on the FON substrates, a 10 nm-thick Al 2 O 3 layer was deposited by ALD at 50 ° C followed by another 200 nm-thick Ag deposition. The top metal fi lm is then anisotropically etched by an ion milling system (Technics) until the underlying Al 2 O 3 layer was exposed, leaving ring-shaped nanogaps surrounding the dome-shaped FON patterns separated by the Al 2 O 3 layer, which determines the gap size. The Al 2 O 3 was then partially removed by a buffered oxide etchant (BOE) for 15 s and washed by DI water. The etch rate for Al 2 O 3 in BOE is measured ∼ 1 nm/s. The controlled etching time is important because over-etching of the Al 2 O 3 layer will collapse the nanogap structures. The FON-gap samples are soaked in DI water for 10–15 min to remove BOE residue and dried using N 2 gas.

Raman spectroscopy : After fabrication, the FON-gap substrates or AgFON substrates were exposed to a 1 mM ethanolic benzenethiol solution for 24 h prior to SERS measurements. For confocal SERS imaging, a Witec Alpha 300R system equipped with an Omnichrome 514.5 nm argon-ion laser was used. The sample was placed on a piezo-stage that moves along x- and y-directions over a 10 μ m × 10 μ m area. The lateral imaging resolution of the system with the 514.5 nm laser was approximately 260 nm. For biosensing, the substrates were exposed to adenine dissolved in water for 15 min, the substrates were then removed from the solution and rinsed with water and allowed to air dry for 15 min prior to all SERS measurements. All macro surface-enhanced Raman measurements were performed with a Millenia Vs 532 nm excitation laser (Spectra-Physics, Mountain View, CA) with added neutral density fi lters to achieve the desired incident powers. In all cases, the laser power was chosen to avoid local heating of the sample, even with long exposure times, that would result in benzenethiol or adenine degradation and the appearance of amorphous carbon Raman bands. The laser beam was fi rst passed through an interference fi lter (Melles-Griot, Rochester, NY) and was then guided to the sample using an Al-coated prism to achieve a spot size of 1.26 mm 2 . Scattered light was collected using a 50-mm-diameter achromatic lens (Nikon, Melville, NY), and the Rayleigh

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scattered light was removed using a notch fi lter (Semrock, Rochester, NY). Detection was accomplished using a 0.5 m SpectraPro 2500i single monochromator and a Spec 400B liquid nitrogen-cooled CCD chip (both from Princeton Instruments/Acton, Trenton, NJ). In all cases, SERS spectra were measured from a minimum of fi ve randomly chosen sample areas on a given substrate.

LOD experiments : The adenine calibration curve experiments were performed by taking a FON-gap or AgFON substrate and exposing them to different concentrations of adenine in pH 7 water for 30 min. The substrates were then removed from the adenine solution and rinsed thoroughly with clean pH 7 water and allowed to dry. They were then mounted in the SERS system for measurements. For limit of detection calculations the purine stretch at 731 cm − 1 was plotted against adenine concentration, and a linear regression was fi t to the data points and extrapolated back to a signal-to-noise ratio of 3 times of the baseline, for both the AgFON and FON-gap substrates.

Calculating SERS enhancement factor : The enhancement factor (EF) for the FON-gap substrates was characterized using the intense ring breathing stretch from both liquid and surface-bound benzenethiol (BZT) at 1000 cm − 1 Raman shift and the equation EF = (N vol × I surf /N surf × I vol ), where N vol is the number of BZT molecules contributing to the normal Raman scattering signal, N surf is the number of BZT molecules contributing to the SERS scattering signal, and I surf and I vol are the intensities of the scattering band of interest for the SERS and normal Raman spectra, respectively. The packing density of BZT on Ag was assumed to be 6.8 × 10 14 molecules/cm 2 based on literature reports. [ 47 , 48 ]

LSPR biosensing : LSPR measurements were recorded using a BPS101 tungsten-halogen light source (BWTek, Newark, DE) and a USB2000 spectrometer with a 200- μ m-core diameter refl ectance probe (Ocean Optics, Dunedin, FL) from dry substrates. A fl at Ag substrate that had undergone the same fabrication process was used as a blank for the FON-gap substrates and a 200-nm-thick Ag fi lm was thermally evaporated onto a clean glass substrate for the AgFON blank in the refl ectance probe measurements. In all cases, LSPR spectra were measured from a minimum of three randomly chosen sample areas on a given substrate.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements This work was supported by grants to S.H.O. from the U.S. Department of Defense (DARPA Young Faculty Award #N66001-11-1-4152) and to C.L.H. from a Camille Dreyfus Teacher-Scholar Award. We also utilized resources at the University of Minnesota, including the Nanofabrication Center, which receives partial support from NSF through the National Nanotechnology Infrastructure Network (NNIN), and the Characterization Facility, which received capital equipment funding from NSF through the MRSEC program.

Received: October 14, 2012 Revised: December 19, 2012

Published online:

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