Article pubs.acs.org/ac
Immobilized Nanorod Assemblies: Fabrication and Understanding of Large Area Surface-Enhanced Raman Spectroscopy Substrates Nathan G. Greeneltch, Martin G. Blaber, Anne-Isabelle Henry, George C. Schatz,* and Richard. P. Van Duyne* Northwestern University, Department of Chemistry, Evanston, Illinois 60208-3108, United States S Supporting Information *
ABSTRACT: We describe the fabrication of optimized plasmonic substrates in the form of immobilized nanorod assemblies (INRA) for surface-enhanced Raman spectroscopy (SERS). Included are highresolution scanning electron micrograph (SEM) images of the surface structures, along with a mechanistic description of their growth. It is shown that, by varying the size of support microspheres, the surface plasmon resonance is tuned between 330 and 1840 nm. Notably, there are predicted optimal microsphere sizes for each of the commonly used SERS laser wavelengths of 532, 633, 785, and 1064 nm.
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structures. The robustness and uniformity of these substrates along with their tunable LSPR are demonstrated and discussed herein. This is followed by a brief comparison of the merits of silver vs gold INRA substrates.
he combined sensitivity and analytic utility of surfaceenhanced Raman scattering (SERS) has led to diverse applications spanning both the fundamental studies of single molecule detection1,2 and reaction dynamics3 and the applied sciences of biosensing,4−6 explosives detection,7 and art conservation.8 A majority of the enhancement associated with SERS arises from the strong coupling of free space photons into the plasmon modes of a nanostructured metal surface, leading to subwavelength confinement of electromagnetic energy.9 At electromagnetic “hot-spots”, which are often associated with nanoparticle junctions, the local electric field enhancements can reach 1011 for small regions of the surface10 and 108 for a surface-averaged result.11,12 However, such high enhancements are typically difficult to reproduce over large areas.13 Recently, there has been an increase in the number and variety of nanofabrication techniques that are appropriate for highly enhancing, large area SERS substrates.14,15 SERS substrates generally fall into three categories: supported particle substrates often using nanosphere lithography (NSL)16 where typical enhancements are on the order of 106,17 structured film based substrates,18 and substrates which are lithographically patterned using techniques such as optical interference lithography,19,20 nanoimprint lithography,20,21 or multistage transfer printing.22 In general, multistage lithographic or patterning techniques have a negative impact on device cost.23 Techniques that maximize control over the nanoscale gaps that are critical to SERS while maintaining a minimal number of processing steps will lead to more cost-effective manufacturing. Here, we demonstrate that shuttering of the evaporation plume during metal deposition onto a supported microsphere array allows for the growth of radially oriented nanoscale pillars separated by small gaps. The size of the microspheres dictates the wavelength of the localized surface plasmon resonance (LSPR) of these INRA (immobilized nanorod assemblies) © 2013 American Chemical Society
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EXPERIMENTAL METHODS
Metal INRA SERS Substrate Fabrication. Glass coverslips (Fisherbrand, #1s) and polished Silicon wafers (MEMC Electronics) were used as supports for SERS substrates prepared as described in prior publications.24 Silica (Bangs Laboratories) and polystyrene (Invitrogen) microspheres were diluted to 5% by volume. Polystyrene microspheres were assembled on glass coverslips, and silica microspheres were assembled on silicon wafers. The solvent was replaced twice with Millipore (Milli-Q, 18.2 MΩ·cm−1) ultrapure H2O by a conventional centrifugation/supernatant removal procedure, followed by sonication for a minimum of 1 h. Solvent (7−10 μL) was drop-coated and manually distributed across the glass or silicon surface. The solvent was then allowed to evaporate in ambient conditions, at which time the microspheres formed a close-packed array. Silver or gold films (200 nm thick) were deposited at 200 nm thick at a rate of 3 Å·s−1 under vacuum (5.0 × 10−6 Torr) over the microsphere-covered surface using a thermal vapor deposition system (custom). The substrates were spun at 550 rpm during deposition while metal mass thickness and deposition rate were measured by a 6 MHz gold-plated quartz crystal microbalance purchased from Sigma Instruments (Fort Collins, CO). Received: November 9, 2012 Accepted: January 23, 2013 Published: January 23, 2013 2297
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Figure 1. Comparison of metal INRA substrates fabricated under two conditions: stationary (top row) and rotating (bottom row). Illustrations of each deposition condition are included in (a) and (b), respectively. Top-down (c,d) and oblique view (e,f) SEM images show the close-packed formation of the support microsphere mask on the silicon wafer surface. Nodular burls and radial nanorods are clearly visible in the side view SEM images shown in (g) and (h), respectively. All INRA samples were fabricated with 200 nm silver film deposited on 600 nm silica microsphere masks.
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Scanning Electron Microscopy (SEM). SEM images were taken on a LEO Gemini 1525 thermal field-emission gun SEM. The acceleration voltage varied between 3.0 and 3.5 kV, and the working distance varied between 2 and 3 mm. UV−Vis Spectroscopy. Absorption measurements in Figure 3 were recorded using a Perkin-Elmer LAMBDA 1050 UV/vis/NIR Spectrophotometer with Perkin-Elmer Integrating Sphere 150 mm UV/vis/NIR (InGaAs) Module. Reflectance measurements in Figure 4 were made using a Mikropack DH2000 Deuterium-Halogen light source fiber coupled (Ocean Optics QP400-2-SR and QP600-2-SR) to an Ocean Optics HR4000 high-resolution spectrometer taken with taqu = 20 ms and 103 averages. Raman Spectroscopy. The Raman spectroscopy instrument used was based on an inverted microscope (Nikon Eclipse Ti). An Innovative Photonics Solutions stabilized 785 nm diode laser was used for single frequency experiments. The detection system was an imaging spectrograph (Acton SpectraPro 2300i) with a LN2 cooled back-thinned deepdepletion CCD detector (Roper, Model Spec 10:400BR, 1340 × 400 pixels). The source was brought in through the back entrance of the microscope and then focused down to a 70 μm × 70 μm spot onto the opaque silver INRA substrates using a 20× microscope objective (wd = 2.1 mm). Theoretical Calculations. The discrete dipole approximation25−27 (DDA) was used to determine the local fields28 for a small inset of the INRA substrate. Although it is possible to simulate structures with periodic boundary conditions using the DDA,29 or finite-difference time-domain (FDTD), the simulations are prohibitively expensive for systems of this size. In the DDA, nanostructures are divided into a set of polarizable points with sufficient density so that both the surface and volume fields reproduce the fields of continuous media. The polarizabilities of the dipoles were determined using the Gutkowicz-Krusin and Draine lattice dispersion relation (GKD-LDR).30 The optical constants of Johnson and Christy31 were used for silver, including an adjustment to the electron−phonon scattering rate to include electron−interface scattering.32
RESULTS AND DISCUSSION SERS Substrate Fabrication with Nanoscale Control. Metal INRA Surface Structure. Silver films (200 nm) were vapor-deposited over 600 nm SiO2 microsphere supports. Metal film deposition normal to the sphere mask surface produced rough metal nanostructures strewn across a mountain-like terrain. SEM images of the SERS substrates are presented in Figure 1. The substrates were exposed in one of two conditions during the deposition step: stationary (Figure 1a) and spinning (Figure 1b). In the spinning case, the spinplate rotated concentric and normal to the metal plume. The spin plate was held offset from the plume so that only one-half of it was exposed to metal at any given time. The silicon wafers were attached to the outermost diameter of the spin radius so that, upon spinning, they would move in and out of the plume at a predictable rate, mimicking a fast-action shutter. This technique also ensures uniform metal film deposition across all substrates present by exposing each one to the identical portion of the plume for an equal amount of time. The top down SEM images (Figure 1c,d) show the apparently indistinguishable film structure of the two types of substrates. However, oblique (Figure 1e,f) side-on (Figure 1g,h) high-resolution SEM images reveal a difference between the two substrates. Metal protrusions were found standing up from the sphere surface. Substrates held stationary during deposition were coated with nodular burls everywhere except for areas in the microsphere junctions, where pillars formed. In contrast, spinning of the substrate during deposition yielded well-defined pillar structures further up the faces of underlying sphere structure; i.e., the pillars were not restricted only to the junctions. The described growth features are similar to those reported using the technique of glancing-angle deposition (GLAD).33,34 In GLAD studies, randomly distributed pillars of varying porosity are grown parallel to the deposition direction. More oblique deposition angles result in larger induced shadowed areas adjacent to initial nucleation sites. In addition, lower substrate temperature promotes less grain coalescence during film growth, resulting in higher film porosity at later growth stages.35−37 The substrates were spun at a rate corresponding to 1 metal atom adsorption per surface site for every 8−10 revolutions. This condition reduces heat build-up at the surface because 2298
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each adsorption site has multiple cooling laps between landings and is ultimately positioned outside of the metal plume for half of the deposition. We believe that this heat dissipation is the active mechanism for promoting less metal diffusion and increased nanopillar segregation when the substrate is spun during deposition. The contribution of rotational motion to the pillar growth can be interrogated using the kinetic theory of gases to estimate atomic speed relative to substrate speed. The average speed of an atom in a sufficiently dilute gas is: c̅ =
8RT πM
where R is the ideal gas constant, T is temperate in K, and M is atomic mass. The average speed of a silver atom at 1300 K is 504.7 m·s−1. The substrates were spun about the circumference of a circle at a rate of 8.8 m·s−1. This two order-of-magnitude disparity in speeds suggests that the substrates were essentially stationary from the traveling silver atoms’ frame of reference, indicating that any shadowing effect is less impactful on pillar formation than substrate cooling. The resulting effect is a successful multiscaled fabrication with control of microscale surface curvature as well as nanoscale surface pillar structure. It is the separate control of fabrication at these two length scales that makes this substrate valuable for sensing applications. Theoretical Model of Hot Spot Location. Nanoscale gaps and sharp points in metal nanostructures are the primary features needed to produce the highest possible enhancement in Raman scattering by small molecules. The local electric field decays as r−3 where r is the molecule−metal separation, so close proximity of molecules to hot spots is essential for high enhancement. To determine the approximate gain in enhancement that can be attributed to the nanopillars, we have used DDA simulations of dimers of 540 nm diameter microspheres, coated with 200 nm of silver, with and without the nanopillar inclusions. Figure 2 shows the simulated structures and the local field enhancement. In the absence of nanopillars in the gaps between microspheres, the maximum field enhancement reaches 106 at a wavelength of 820 nm. The bridging of the crevice with the pillars leads to further localization of the local field in the crevice and increases the field enhancement by an order of magnitude to 107 at a wavelength of 805 nm. It should be noted that these field enhancements are peak values, rather than the surface averaged values represented by the experiments. This indicates that the simulations underestimate the peak field enhancement due to limitations in the discretization of the computational grid for nanostructures of this size. Here, the grid is discretized into cubes with an edge length of 2 nm. Localized Surface Plasmon Resonance (LSPR) Tunability. The wavelength of the LSPR is very sensitive to the ratio of the nanostructure diameter to the interstructure gap.38,39 For touching structures, the radius of curvature at the point of contact is the dominant factor in determining the LSPR position.11 In the spun case, there are two levels of structure, that of the nanopillars and their associated gaps and that of the microspheres and their structure and radius of curvature. The average nanopillar diameters scale roughly with the size of the support microsphere (see Supporting Information Figure S1) but include large spreads from the mean, while the absolute position of the nanopillars is random due to the nature of the growth mechanism. The resulting range of interpillar gaps leads to essentially broadband resonance.38 However, we show below
Figure 2. Electric field enhancement for a dimer of silica microspheres coated with 200 nm of silver (a) without pillars at a wavelength of 820 nm and (b) with pillars at a wavelength 805 nm, calculated using the DDA. The pillars localize the electric field to small gap regions and their tips, increasing the overall enhancement factor.
that the LSPR maximum (λmax) of the INRA substrate is determined primarily by the radius of curvature of the gaps between the microspheres rather than the interpillar gaps. Since radius of curvature is a function of the radius of the microspheres and the thickness of the deposited metal, the λmax position is a function of the microsphere diameter. To elucidate the details of the relationship between sphere diameter and LSPR wavelength, silver INRA substrates were fabricated with 200 nm of metal over support microspheres of varying composition and diameter. Far-field plasmon resonance spectra were recorded using diffuse reflectance. Both silica and polystyrene spheres were used in order to show the tunability of the LSPR using commercially available products. The results of these measurements are shown in Figure 3. The substrates exhibit tunable LSPR with λmax values ranging from 330 nm (with 160 nm support spheres) to 1838 nm (with 1490 nm spheres) (Figure 3). The combination of LSPR tunability with nanoscale metal pillars in the junctions between microspheres is ideal for the enhancement of Raman scattering. The electromagnetic mechanism of SERS predicts that maximum field enhancement occurs when the λmax lies between the Raman excitation and scattered photon wavelengths.12,40,41 On the basis of this criteria, the substrates predicted to perform best for common SERS laser wavelengths (532, 633, 785, and 1064 nm) are denoted with blue stars in Figure 3b. In addition, the linear slope of data points in Figure 3b allows for easy prediction of the λmax. The slope of the λmax as a function of support sphere diameter (both values in nm) plot is 1.19 with an intercept of 120.36 nm. Comparison of Silver and Gold Substrates. Representative LSPR and SERS (λex = 785 nm) spectra from a 200 nm metal INRA substrate over 600 nm silica spheres are shown in Figure 4a,b respectively. The red-shift associated with the transition 2299
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associated with silver nanostructures will always be higher than gold structures (assuming the two structures have the same λmax). The larger polarizability in silver systems is caused by a combination of a sharp onset of interband transitions between occupied sd-hybrid bands and unoccupied hybrid-sp bands43 and a lower electron−phonon scattering frequency in silver compared to gold. The difference in the SERS enhancement factor between silver and gold is predicted by QSPP2 to be approximately 1 order of magnitude at 785 nm (Figure 4d). Figure 4b shows that in practice only a factor of 5 is measured. This can be rationalized by noting the difference in the plasmon resonance between the silver and gold substrates and the possibility that there are differences in surface coverage between the metals. Characterization of the Metal INRAs. Figures of Merit. Two methods were used to quantify the enhancement of the SERS substrates. The first, called the fundamental enhancement factor (EF), is appropriate for fundamental research and describes the Raman intensity increase per molecule. The second, called the analytic enhancement factor (aEF), is better suited for field work and describes the decrease in the limit of detection imparted on a system by the SERS substrate. Although both methods of measuring INRA substrate enhancement returned values of 10−100 million, they are two completely independent quantifications and are each ideal for different research situations. Fundamental Enhancement Factor (EF) and Uniformity. For fundamental research into the SERS effect, a figure of merit is required that takes into account the Raman intensity increase per molecule. Comparison of experiments to theoretical calculations would otherwise not be possible. The EF is a ratio of SERS scattered photons to normal (i.e., nonenhanced) Raman scattered photons of the same molecule, corrected for the number of the molecules illuminated and is given by:45,46
Figure 3. (a) Sample LSPR spectrum of INRA substrate fabricated using 600 nm silica sphere mask. (b) Plot of LSPR maximum (λmax) reflectance as a function of sphere mask diameter. The substrates predicted to perform best for common SERS laser wavelengths (532, 633, 785, and 1064 nm) are denoted with blue stars. The point marked with a red square represents the data presented in panel (a). All INRA samples were made with 200 nm silver film deposited while spinning the masks. The masks consisted of silica or polystyrene microspheres on a polished silicon wafer. A silver mirror was used as a 100% reflectance standard.
EF =
from silver to gold INRA substrates can be explained by examining the dispersion characteristics of the dielectric functions of silver and gold (Figure 4c). For a given nanostructure, the position of the resonance is dictated by the real part of the dielectric function for that structure. For example, individual nanospheres have a resonance when the real part of the dielectric function is approximately −2. For the silver INRA in Figure 4a, the resonance appears at 800 nm, which corresponds to a real part of the dielectric function of approximately −30.5 (Figure 4c). This indicates that, for the same nanostructure made from gold, the resonance should appear at 865 nm, which compares favorably with the resonance for the fabricated gold nanostructure in Figure 4a. Outgoing Raman shifted photons associated with benzenethiol stretches in the 1000−1600 cm−1 frequency region are at 851−898 nm. Therefore, maximum SERS enhancement is predicted with a λmax of 818−841 nm. The substrates interrogated in Figure 4a represent the closest achievable λmax optimization for each metal and allow for a direct comparison of their resulting SERS intensities. In Figure 4d, we present a quality factor, QSPP = εr2/εi (where εr and εi are the real and imaginary parts of the dielectric function of the metal) that describes the polarizability of the metal as a function of the LSPR position for both silver and gold.42−44 QSPP is proportional to E2 when the laser wavelength is tuned to the optimum wavelength, and therefore, QSPP2 demonstrates that, for a given LSPR wavelength, the enhancement factor
ISERS/NSERS IRS/NRS
where ISERS and IRS are the SERS and solution Raman intensity, respectively, and N is the number of molecules excited. The 1076 cm−1 peak of benzenethiol was chosen for analysis, and a fundamental EF was measured for each fabricated substrate using the 785 nm excitation wavelength. All measured intensities were normalized for power and acquisition. An INRA sample (Figure 5a,b) was analyzed for SERS EF and uniformity. A Kel-F mask with nine interrogation wells (Figure 5c) was used to cover the INRA substrate. Each well was pretreated with 100 μM ethanolic benzenethiol (SigmaAldrich) for use as a probe molecule. The method of measurement and results are shown in Figure 5d. For each well (labeled 1−9), five rows (labeled A−E) of 10 spots were analyzed. The rows were spaced 20 μm apart in the y-direction, and the spot-to-spot spacing within rows was 10 μm. For each row and well, the mean and standard deviation of the 10 spots are shown in Figure 5d. Average EFs are reported for each well and together produce a complete average EF for the entire substrate of 4.23 (±8.6%) × 107. These results suggest the use of these substrates for studies where reproducibility over a large area or across multiple measurements is desired. Examples might include studies of multiple nonidentical molecules in different areas of the substrate,47,48 remote/roadside detection of chemical warfare agents49−51 or illicit drugs,52−54 and large illumination area electrodes for plasmonic fuel cells.55−57 A 2300
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Figure 4. (a) Localized surface plasmon resonance and (b) surface-enhanced Raman spectra of benzenethiol on gold (red) and silver (black) INRA substrates. (c) Dispersion characteristics of gold and silver: for a fixed geometry, the resonance condition is described by the real part of the dielectric function. The difference in LSPR wavelength between silver and gold is due to the difference in dispersion. (d) Plots of quality factor, a generic metric for the field enhancement associated with the LSPR: Silver is a more polarizable metal over all wavelengths, leading to larger field enhancements. All INRA samples were fabricated with 200 nm silver or gold film deposited on 600 nm silica sphere masks. The SERS spectra in (b) were recorded with 207 μW of 785 nm excitation and an acquisition time of 5 s.
isotherm, and the concentration (CSERS) corresponding to 25% of saturation peak ratio was chosen. A linear fit of the nonenhancing Raman data was used to find the concentration (CNES) needed to produce the same peak ratio. The aEF is then given by:58
macro-scale and SEM image of a successfully fabricated uniform substrate is also included in Figure 5 for reference. Visible large scale green iridescence indicates areas of high enhancement and uniformity. A 10.2 cm (4 in.) substrate was chosen to show the scalability of this fabrication method and is included in Figure 5a. Analytical Enhancement Factor (aEF). For field studies where often untrained, nonscience personnel may be required to carry out measurements, a figure of merit is needed to quantify the overall boost in signal provided by a substrate in a given environment. An analytical enhancement factor (aEF) therefore should be based on practical, empirical observations. The aEF utilized in this study is a comparison of the Raman signal produced in the presence of the enhancing substrates to that of the nonenhancing condition. The resulting values of the measurement are a description of how much the analytical limit of detection of the system has been lowered. In this work, the nonenhancing condition was generated by flipping the substrate over and exposing the nonenhancing silicon support side of the wafer to excitation. The target analyte was ethanolic BPE (trans-1,2-bis(4pyridyl)ethylene, Sigma-Aldrich). The 1203 cm−1 Raman peak of BPE was divided by the internal standard 888 cm−1 peak of ethanol to produce a ratio of peaks. This ratio was measured at different concentrations ranging from 1nM to 1 μM for the SERS condition and up to 7.5 M for the nonenhancing case. The SERS data was then fit to a Langmuir
aEF =
CSERS C NES
Results for the analytical enhancement factor (aEF) measurements are shown in Figure 6. A representative plot showing the Raman peaks of interest is shown in the Supporting Information. Twenty-five percent of the saturation level from the Langmuir isotherm corresponded to a ratio of 1.881203 cm−1 BPE/888 cm−1 EtOH. This intensity ratio correlates to concentrations of 52.9 nM in the SERS fit and 0.63 M in the linear nonenhancing fit. The aEF of the silver INRA substrate was therefore found to be 1.21 × 107. It is important to restate that this metric for measuring substrate enhancement does not take into account the molecular surface coverage. Coupled with the large scale uniformity and tunability of metal INRAs, this aEF value proves the usefulness of this robust substrate in analytical as well as nonlaboratory based studies. 2301
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These substrates were demonstrated to return uniform (
