Miniaturisation for chemistry, biology & bioengineering

Miniaturisation for chemistry, biology & bioengineering www.rsc.org/loc

Volume 9 | Number 3 | 7 February 2009 | Pages 361– 484

ISSN 1473-0197

Gleason Nano-adhesive for microdevices

Kaji and Nishizawa Cocultures on detachable substrates

Folch Multi-purpose perfusion system

Erickson Enhanced SERS for biomolecules

PAPER

www.rsc.org/loc | Lab on a Chip

Sub-micron resolution surface plasmon resonance imaging enabled by nanohole arrays with surrounding Bragg mirrors for enhanced sensitivity and isolation Nathan C. Lindquist, Antoine Lesuffleur, Hyungsoon Im and Sang-Hyun Oh* Received 24th September 2008, Accepted 21st November 2008 First published as an Advance Article on the web 19th December 2008 DOI: 10.1039/b816735d We present nanohole arrays in thin gold films as sub-micron resolution surface plasmon resonance (SPR) imaging pixels in a microarray format. With SPR imaging, the resolution is not limited by diffraction, but by the propagation of surface plasmon waves to adjacent sensing areas, or nanohole arrays, causing unwanted interference. For ultimate scalability, several issues need to be addressed, including: (1) as several nanohole arrays are brought close to each other, surface plasmon interference introduces large sources of error; and (2) as the size of the nanohole array is reduced, i.e. fewer holes, detection sensitivity suffers. To address these scalability issues, we surround each biosensing pixel (a 3by-3 nanohole array) with plasmonic Bragg mirrors, blocking interference between adjacent SPR sensing pixels for high-density packing, while maintaining the sensitivity of a 50! larger footprint pixel (a 16-by-16 nanohole array). We measure real-time, label-free streptavidin–biotin binding kinetics with a microarray of 600 sub-micron biosensing pixels at a packing density of more than 107 per cm2.

Introduction The gold standard for real-time, label-free measurements of biomolecular interactions is Surface Plasmon Resonance (SPR) sensing,1 wherein a local change in refractive index due to the presence of molecules binding onto a thin gold film results in a shift of the Surface Plasmon (SP) excitation wavelength or angle.2 SPs are electromagnetic surface waves coupled to the free electron plasma in metals, and confined to the interface of the metal and the surrounding dielectric.3 The SP field decays exponentially perpendicular to the interface, meaning that most of the energy is confined within "100 nm, probing the local refractive index with high sensitivity.2 The hybrid plasma-electromagnetic nature of SPs requires the use of special excitation geometries, the most common for biosensing applications being an optical prism, known as the Kretschmann configuration. The commercial BIAcore! SPR instrument, based on this principle, is capable of real-time label-free measurements of molecular binding affinity, but it can acquire data from only four channels simultaneously, and has a low spatial resolution since the focused incident light illuminates an area measuring "1.6 mm along a flow cell. For proteome-scale experiments to study protein interactions in a microarray format,4,5 high-throughput labelfree detection techniques are needed that can handle several hundred or thousand sample spots per slide. For SPR imaging,6–9 the prism-based Kretschmann configuration is combined with imaging optics to simultaneously monitor multiple sample spots on a microarray. However, this setup has several disadvantages,10 such as being unable to use high numerical aperture (NA) Laboratory of Nanostructures and Biosensing, Department of Electrical and Computer Engineering, University of Minnesota, 200 Union St. SE, Minneapolis, MN, 55455, USA. E-mail: [email protected]; Web: http://nanobio.umn.edu

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optics, with a limited depth of field, on the tilted image plane. The most advanced FlexCHIP! instrument, which utilizes a higher order diffracted mode of a grating coupler for SPR excitation, can measure binding kinetics from 400 sample spots simultaneously, but the problems of a tilted image plane still persist, making it difficult to combine both high imaging resolution and a wide field of view. Furthermore, the resolution of SPR imaging is not limited by diffraction, but by the propagation of the excited SP waves to adjacent sensing areas, causing unwanted interference. Given this, a new class of high-throughput, real-time, labelfree sensing systems is emerging based on the recent discovery11 of SP mediated and enhanced optical transmission through periodic subwavelength nanohole arrays in thin gold films.12–23 The nanohole array itself acts as a grating that directly couples the incident light to SPs,11 giving transmission peaks, for normal incidence, at specific wavelengths lpeak approximated by: rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi a0 3m 3d (1) lpeak zpffiffiffiffiffiffiffiffiffiffiffiffi i2 þ j 2 3m þ 3d

where a0 is the periodicity of the nanohole array, i and j are the grating orders, and 3m and 3d are the dielectric constants of the metal and dielectric, respectively. Since SPs play a central role in the transmission,24–26 the position of lpeak is sensitive to the refractive index changes on the surface, enabling SPR biosensing. As biomolecules bind to the gold surface or to immobilized capture molecules, 3d increases, red-shifting lpeak. This gratingcoupled excitation geometry allows for the use of simplified collinear optics and high resolution imaging, amenable to miniaturization in a microarray format.19,22,23 From an initial proof of concept,12 nanohole arrays as SPR biosensors have been shown previously in the near-infrared wavelength regime,15 with shapeenhanced sensitivity in a real-time system,17 and in a multiplex format for high-throughput SPR-imaging measurements.19,22 This journal is ª The Royal Society of Chemistry 2009

For ultimate multiplexing and miniaturization, however, several challenges exist, including: (1) as multiple nanohole arrays are brought close to one another for high-density packing, the possibility exists for neighbor-to-neighbor crosstalk and interference via the excited SP waves,27 reducing the sensitivity and introducing large sources of error, and (2) as the size of the nanohole array is reduced, i.e. fewer holes, the transmission resonance peaks are broadened,28,29 also resulting in less precision and lower sensitivity. In this paper, we demonstrate a new device architecture that uses plasmonic Bragg mirrors surrounding the nanohole arrays as a sub-micron SPR imaging pixel, addressing these resolution, scalability and isolation issues. The surrounding Bragg mirrors coherently reflect outgoing SP waves back into the nanohole array, thereby enhancing the transmission.30,31 They can also serve to isolate a single nanohole array from its closest neighbors,32 confining the excited SP waves to within the area of the nanoholes. The Bragg mirrors as additional SP optical elements33 therefore have the dual benefits of both increased transmission and sensitivity with sharper resonance peaks, and allowing for high-density packing of individually isolated sensing elements, addressing two fundamental miniaturization issues. We show the feasibility of using sub-mm2 footprint nanohole arrays with surrounding Bragg mirrors30 as an isolated, individually addressable SPR imaging pixel, without sacrificing sensitivity.

Results and discussion Fig. 1a shows a scanning electron micrograph of miniaturized and tightly packed nanohole array SPR sensing pixels in a microarray format. The samples were fabricated with focused ion beam milling on a 200 nm thick gold film on a standard glass microscope slide with a 5 nm thick chromium adhesion layer. The 3-by-3 pixel array consists of nine unique nanohole arrays spaced on a 5 mm grid, each with 49 nanoholes (7-by-7), but with a different periodicity, ranging from 370 to 450 nm, from the lower left (370 nm) to the upper right (450 nm), and each increasing by 10 nm moving left to right. The nanoholes all have a diameter of 150 nm. Surrounding each nanohole array are four Bragg mirror grooves, milled 50 nm deep and 100 nm wide. Per eqn (1), the SP waves generated by the nanohole array are momentum matched to the nanohole array’s grating orders.11 Therefore, Bragg mirror grooves with a periodicity half that of their corresponding nanohole array satisfy the Bragg condition for SP wave reflection.34 The distance between the inner Bragg mirror groove and the nanoholes is tuned to reflect the SP waves back into the nanohole array in a constructive manner, enhancing the overall transmission and the sharpness of the resonance peak.30 Fig. 1b shows a bright-field optical microscope image of the 3-by-3 pixel array with the addition of back-side (glass-side) He–Ne laser illumination, the same microscope imaging setup used to previously demonstrate multiplex SPR microarray imaging.19 A polydimethylsiloxane (PDMS) microfluidic flow cell incubates the samples with various liquids at a controllable flow rate. The He–Ne laser illumination (633 nm) samples the transmission resonances of each nanohole array. The range in SPR imaging pixel intensities seen in the image comes from the range of nanohole array periodicities. The pixels are tuned for SP enhanced transmission when incubated in water. This journal is ª The Royal Society of Chemistry 2009

Fig. 1 (A) Scanning electron micrograph of tightly packed nanohole array SPR imaging pixels, milled through a 200 nm thick gold film, each surrounded by four plasmonic Bragg mirror grooves for isolation and enhanced sensitivity. Each array has a unique periodicity, from 370 to 450 nm, lower left to upper right. (B) Bright-field microscope image of the same device, with back-side He–Ne laser illumination. The variation in pixel intensities, which passes through a minimum, is due to the nine unique periodicities. (C) Atomic force microscope scan of a single nanohole array with surrounding Bragg mirror grooves. (D) Near-field scanning optical microscope scan of the same device, with back-side He– Ne laser illumination, showing high transmission through the nanoholes, and SP wave confinement within the first few Bragg mirror grooves. The incident light was polarized vertically.

Fig. 1c shows an atomic force microscope (AFM) scan of a single isolated nanohole array with eight surrounding Bragg mirror grooves, tuned for transmission at 633 nm in air. With concurrent near-field scanning optical microscopy (NSOM), the complementary image in Fig. 1d is generated, showing bright transmission spots within the area of the nanoholes, as well as SP standing waves within the first few Bragg mirror grooves. The He–Ne laser light was again incident on the back-side (glass-side) and polarized vertically. As seen in the NSOM scan, only about four Bragg mirror grooves (distance ¼ 0.9 mm) are necessary to isolate each nanohole array, reflecting and confining the excited SP waves (that have a calculated35 propagation length of 12 mm at 630 nm on a gold–air interface), and allowing for high-density packing. Fig. 2a shows sample white-light (a tungsten-halogen lamp) transmission spectra taken from a larger 16-by-16 nanohole array, a smaller 7-by-7 nanohole array, and a 7-by-7 nanohole array surrounded with the Bragg mirror grooves. In this case, each nanohole array was separated by 50 mm, a distance much larger than the calculated35 SP propagation length of 4.6 mm at 630 nm on a gold–water interface, meaning that each nanohole array was well isolated, by distance, from its nearest neighbors. The spectra have been normalized by the spectrum of the lamp source and by the total number of holes in each array. Transmission directly through the gold film at 495 nm was subtracted out. The nanohole arrays have a periodicity of 440 nm. With the addition of the surrounding Bragg mirrors, the transmission Lab Chip, 2009, 9, 382–387 | 383

Fig. 2 (A) Transmission spectra, normalized by the number of nanoholes, for various 440 nm periodicity nanohole array devices: a 16-by-16 array (256 nanoholes), a 7-by-7 array (49 nanoholes) and a 7-by-7 array with surrounding plasmonic Bragg mirrors. The mirrors enhance and sharpen the transmission resonance, allowing the use of smaller arrays— fewer nanoholes—without sacrificing the sensitivity, or the sharpness of the slope. (B) Similar data, but for a 5-by-5 nanohole array. (C) Similar data, but for a sub-mm2 footprint 3-by-3 nanohole array. In this case, the addition of the Bragg mirrors increases the slope of the nanohole array by more than an order of magnitude ("25!). (D) Transmitted He–Ne laser intensity taken from individual 7-by-7 nanohole arrays with different periodicities, both with and without Bragg mirror grooves, and incubated in various liquids (nliquid). Each different periodicity ‘‘samples’’ the highslope region of the spectra shown in (A). The addition of the Bragg mirrors increases the sensitivity (change in intensity versus nliquid) of the smaller 7-by-7 nanohole array.

peak is slightly blue-shifted from that of the 7-by-7 nanohole array.30 For biosensing applications with He–Ne laser illumination (633 nm), as molecules bind to the gold surface and the transmission peaks shift, the high-slope regions (at 633 nm) show sharp changes in transmitted intensity.18,19,22,23 The sensitivity of the nanohole array is then characterized by both the slope of the transmission peak, and by how much the peak shifts.19 By adding the Bragg mirror grooves to a smaller 7-by-7 array, it is seen that the slope of the transmission peak is improved, and made comparable to that of the larger 16-by-16 array. The relative sensitivity (slope) enhancement is 2!. For even smaller nanohole arrays, the relative sensitivity (slope) enhancement is higher, since, for each case, the sensitivity (slope) of the 16-by-16 nanohole array is recovered. Fig. 2b shows data from a 5-by-5 nanohole array. Fig. 2c shows data from a 3-by-3 nanohole array, with a footprint of 0.77 mm2. In that case, the relative sensitivity (slope) enhancement is more than an order of magnitude ("25!). The surrounding Bragg mirrors sharpen the transmission resonance, allowing the use of smaller nanohole arrays occupying less surface area without sacrificing detection sensitivity. Fig. 2d shows the transmitted intensity at 633 nm with He–Ne laser illumination from several isolated 7-by-7 nanohole arrays in water (n ¼ 1.333) with and without surrounding Bragg mirrors, for a range of periodicities (the same range as in Fig. 1a). The periodicities are plotted in reverse order to complement Fig. 2a, 384 | Lab Chip, 2009, 9, 382–387

since, from eqn (1), the spectral features are red-shifted for larger periodicities. As the periodicity increases and the transmission spectral features are red-shifted, the fixed 633 nm laser illumination samples the ‘‘spectral’’ behavior of the nanohole arrays. The nine periodicities shown here ‘‘sample’’ a small window of the full transmission spectra shown in Fig. 2a. Each nanohole array is imaged with the CCD camera, and the total transmitted intensity is calculated by summing the CCD counts. For the nine periodicities considered, a clear trend is seen of increasing intensity towards a large peak. A minimum of transmission is also observed for a periodicity around 420 nm, consistent with the transmission image shown in Fig. 1b. To demonstrate intensity-based sensing with laser illumination, different liquids were used to characterize the sensitivity of each nanohole array SPR imaging pixel. Also shown in Fig. 2d are the intensities of the same 7-by-7 nanohole arrays incubated in a water–ethanol mixture (n ¼ 1.353). The transmission features are red-shifted and the intensity of the high-slope regions show the most change. For a 7-by-7 array without surrounding Bragg mirror grooves, the largest transmitted intensity change is 30%, whereas the addition of the Bragg mirror grooves nearly doubles the largest intensity change to 55%, consistent with the sharpened transmission peak shown in Fig. 2a. The largest intensity change for the 16-by-16 nanohole arrays was also 55%, again indicating that by including the surrounding Bragg mirrors with a smaller nanohole array, the sensitivity of the larger one is recovered. The nanohole array intensity increases at larger periodicities, since there the transmission slope has an opposite sign. The behavior of each nanohole array correlates with its periodicity, and taken together, they represent well the trends of the transmission spectra shown in Fig. 2a. The effect of the surrounding Bragg mirror grooves on suppressing neighbor-to-neighbor crosstalk is shown in Fig. 3. Devices were designed as in Fig. 1a (including some without Bragg mirrors) with center-to-center distances of 5.0, 5.5, and 6.0 mm, meaning that the edge-to-edge spacing between neighboring nanohole arrays could be as low as 2.3 mm, depending on the periodicity, and within the calculated "4.6 mm SP propagation length. The devices were incubated in water and then in the water–ethanol mixture. The resulting intensity changes were compared to those of the isolated nanohole arrays from Fig. 2d. Fig. 3a shows the changes in intensity for nine tightly packed nanohole arrays without Bragg mirror isolation. The data from nanohole arrays isolated by distance is also shown (black squares). The behavior of each nanohole array does not correlate well with its periodicity, indicating that there is a source of interference or error. For example, for a periodicity of 420 nm, an isolated nanohole array decreases in intensity, whereas the tightly packed (5.5 mm packing distance) nanohole array increases in intensity. Fig. 3b shows data from similar devices, but with the addition of the surrounding Bragg mirrors. The behavior of each nanohole array correlates well with its periodicity and with the behavior of the nanohole arrays isolated by distance (black squares), indicating that the Bragg mirror grooves act to isolate each nanohole array as its own individually unique sensing pixel, with minimal pixel-to-pixel crosstalk. This is further demonstrated in Fig. 3c, where nine SPR sensing pixels are arranged in a checkerboard pattern. By increasing the refractive index of the This journal is ª The Royal Society of Chemistry 2009

Fig. 3 (A) Relative intensity change for tightly packed 7-by-7 nanohole arrays without the surrounding Bragg mirrors for packing distances of 5.0, 5.5, and 6.0 mm. The behaviors of each different periodicity change drastically as the distance between the arrays changes, and are not consistent with the behavior of the isolated devices (black squares) considered in Fig. 2d, indicating sources of neighbor-to-neighbor interference and error. (B) Relative intensity change for tightly packed 7-by-7 nanohole arrays with the surrounding Bragg mirrors. In this case the behavior of each periodicity correlates well with that of an individual isolated device, demonstrating that the Bragg mirrors allow for higher density packing without suffering from neighbor-to-neighbor crosstalk. (C) Optical image of a tightly packed SPR imaging pixel array with Bragg mirror isolation incubated in two different liquids. The behavior of each pixel can be individually tuned, in this case to either increase or decrease with increasing index of refraction nliquid, without interference from the adjacent pixels.

surrounding liquid, five of the pixels increase in intensity, whereas the other four decrease, depending on their periodicity and designed behavior. Since the addition of the Bragg mirror grooves provides the advantages of enhanced sensitivity and pixel-to-pixel isolation, the design is amenable to a highly miniaturized nanohole array sensor in a microarray format for SPR imaging. Fig. 4a shows a ‘‘dark-field-like’’ He–Ne laser transmission image of 816 SPR sensing pixels, where the top three rows (216 pixels) are made of

the sub-array of pixels shown in Fig. 1a and 1b. The pixel-topixel pitch is 5 mm, and each sub-array is on a 20 mm grid. In this case, each sub-array, consisting of nine sensing pixels, could also be considered to be a single sensing unit, since the information gathered from each of the nine sensing pixels could be used for reducing noise, gathering ‘‘spectral’’ information, monitoring negative controls, or limiting other sources of error. The bottom three rows (600 pixels) are made up of nanohole arrays consisting of only nine nanoholes (3-by-3), each occupying less than a 1 mm2 footprint, and with a pixel-to-pixel pitch of 3 mm. The device is shown in Fig. 4b (scanning electron micrograph) and 4c (brightfield image with back-side He–Ne laser illumination). Each nanohole array has a unique periodicity (the same nine as in Fig. 1a), in increasing order from the lower left to the upper right, with the sequence repeating to fill the 25 spots. The intensity variation from pixel to pixel is clearly seen. Such an arrangement, with the Bragg mirror grooves providing neighbor-to-neighbor isolation as well as enhanced sensitivity, gives a packing density of more than 107 individual SPR sensing pixels per cm2, where each pixel occupies less than 1 mm2. To demonstrate multiplex SPR biosensing, Fig. 5 presents real-time measurements of streptavidin–biotin binding, with data taken from each SPR sensing pixel. Isolated 16-by-16 (Fig. 5a) nanohole arrays without Bragg mirrors are used to compare the sensitivities of tightly packed 7-by-7 (Fig. 5b), 5-by-5 (Fig. 5c), and 3-by-3 (Fig. 5d) nanohole arrays with surrounding Bragg mirrors. At t ¼ 150 s, streptavidin was injected, resulting in a quick change in the transmitted intensities due to the bulk refractive index variation, followed by an exponential trend characteristic of first-order binding kinetics.36 As the streptavidin–biotin reaction saturates after a few hundred seconds, the transmitted intensity stabilizes. Data is shown taken from nanohole array SPR sensing pixels that, due to their periodicities, either increase or decrease in intensity, showing that the intensity variation is from a changing local refractive index due to molecular binding, and not from another source, such as changing absorption. For the tightly packed 7-by-7, 5-by-5 and 3-by-3 nanohole arrays with the surrounding Bragg mirrors, clear binding kinetics are seen, with a signal-to-noise ratio comparable to that of a larger 16-by-16 nanohole array, consistent with the spectral data shown in Fig. 2. Without the surrounding Bragg

Fig. 4 (A) Transmission image of a large microarray with 816 SPR sensing pixels, each one isolated and enhanced by the surrounding Bragg mirrors. The top three rows (216 pixels) are made of 7-by-7 nanohole arrays, whereas the bottom three rows (600 pixels) are made of smaller 3-by-3 nanohole arrays, spaced by 3 mm, and each with a footprint less than 1 mm2. (B) Scanning electron micrograph of a tightly packed pixel array of the 3-by-3 nanohole arrays with surrounding Bragg mirrors. (C) Bright-field image with back-side He–Ne laser illumination of the 3-by-3 nanohole arrays, with the same nine periodicities as in Fig. 1a, the sequence repeating to fill all 25 spots.

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Fig. 5 (A) Specific biotin–streptavidin, real-time, label-free SPR imaging measurements from two isolated 16-by-16 nanohole arrays without surrounding Bragg mirrors, and two of each tightly packed (B) 7by-7 nanohole arrays, (C) 5-by-5 nanohole arrays and (D) 3-by-3 nanohole arrays with surrounding Bragg mirrors. The smaller nanohole arrays maintain the sensitivity of the larger 16-by-16 nanohole array. The exponential binding kinetics are clearly seen, with saturation after 600 s. The intensities are normalized to the baseline, before the injection of streptavidin at time t ¼ 150 s. The SPR sensing pixels can either increase or decrease in intensity, depending on their periodicity.

mirror grooves, the smaller nanohole arrays exhibit degraded transmission resonances and poorer sensing properties, and cannot be packed with such a high spatial density. For SPR imaging in the near-infrared regime, where the SP propagation loss is significantly reduced compared to the visible regime, the inherent tradeoff37 between sensitivity (sharper SPR peak) and lateral imaging resolution (more crosstalk) could also benefit from the nanohole arrays with surrounding Bragg mirrors platform for SPR imaging developed here.

Experimental The 200 nm thick gold films were fabricated with e-beam evaporation (CHA, SEC600) onto standard microscope glass slides with a 5 nm thick chromium adhesion layer. Focused ion beam (FEI Dual Beam Quanta 200 3D) milling (30 kV, 30 pA) was used to produce the nanohole arrays as well as the surrounding Bragg mirrors grooves. For large area sensor fabrication, emerging low-cost, high-throughput nanofabrication techniques, such as nano-imprint lithography,38 soft interference lithography,39 or colloidal templating techniques40 could also be used. Soft lithography41 with PDMS was used to fabricate the microfluidic flow cell for real-time SPR sensing. The negativetone master mold of the channel was patterned on a silicon wafer using SU-8 50 photoresist, defining 50 mm deep and 200 mm wide channels. A 10 : 1 ratio of PDMS and curing agent was degassed in vacuum and was cast 3 mm deep over the SU-8 photoresist pattern. After curing at 70 % C overnight, the PDMS flow cell was cut from the master, and inlet and outlet holes were punched for tubing connection. The PDMS channel was aligned to the nanohole arrays on a sample gold slide using a contact aligner (Karl Suss MJB3), and held in place with a mechanical clamp. For the liquid characterization measurements, for a refractive index of nliquid ¼ 1.353, a 30% by wt. mixture of water and 386 | Lab Chip, 2009, 9, 382–387

ethanol was used. For biosensing experiments, the gold slide samples were thoroughly cleaned with solvents followed by 10 min of UV ozone treatment. The samples were then incubated for 24 h in a 4 mM solution of 11-amino-1-undecanethiol hydrochloride (Sigma-Aldrich) to form a self-assembled monolayer (SAM) on the gold surface, followed by a 3 mM solution of sulfoNHS-LC biotin (Pierce, USA) for a 12 h period to covalently attach the biotin. The 200 mm wide and 50 mm tall PDMS microfluidic channel was then filled with phosphate buffered saline (PBS, pH ¼ 7.4) to establish a clean baseline. A 750 nM solution of streptavidin in PBS was then injected at t ¼ 150 s at a 4 mL h&1 flow rate with a syringe pump (Harvard apparatus PHD2000). With a standard optical microscope (50!, NA ¼ 0.55) and camera (CoolSnap! HQ2 cooled CCD), the intensity of back-side (glass-side) He–Ne laser transmission from each nanohole array SPR imaging pixel was monitored in real-time (an image taken every 5 s) with a custom made MATLAB! program for image acquisition and data analysis.

Conclusions In conclusion, we present the addition of surrounding nanohole arrays with plasmonic Bragg mirrors to provide the dual benefits of very high-density SPR imaging without pixel-to-pixel crosstalk and interference, and the use of sub-micron pixel sizes without sacrificing sensitivity. With 600 sub-micron SPR sensing pixels, we demonstrated highly multiplexed, high-resolution SPR imaging, helping to overcome the tradeoff between resolution and sensitivity. Further investigations with multiple PDMS channels, varying analyte concentrations, differential sensing, and negative controls are currently underway. As nanohole array-based SPR sensing becomes a more developed sensing platform, the additional design freedoms that SP optical elements such as Bragg mirrors provide may lead to an ultimately scaled, highly sensitive, high-throughput multiplexed system. The SPR imaging pixel developed here may also have applications beyond protein microarrays, such as in nanophotonic circuitry or displays.

Acknowledgements N. C. L. was supported by the NIH Biotechnology Training Grant #T32-GM008347, and H. I. was supported in part by the 3M Science and Technology Fellowship. S. H. O. gratefully acknowledges support from 3M Non-Tenured Faculty Award, the Minnesota Supercomputing Institute, the University of Minnesota Institute for Engineering in Medicine, and the Center for Nanostructure Applications. The authors acknowledge James Leger for helpful discussions. Device fabrication was performed at the University of Minnesota NanoFabrication Center, which receives support from the NSF National Nanotechnology Infrastructure Network (NNIN). NSOM imaging was supported by the Pennsylvania State University Materials Research Institute which receives support from NNIN.

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