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Apr 30, 2015 - ABSTRACT: We present a novel blurring-free stencil lithography patterning technique for high-throughput fabrica- tion of large-scale ar...
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Large-Scale Arrays of Bowtie Nanoaperture Antennas for Nanoscale Dynamics in Living Cell Membranes Valentin Flauraud,† Thomas S. van Zanten,‡ Mathieu Mivelle,‡ Carlo Manzo,‡ Maria F. Garcia Parajo,*,‡,§ and Jürgen Brugger*,† †

Microsystems Laboratory, Institute of Microengineering, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland ICFO-Institut de Ciencies Fotoniques, Mediterranean Technology Park, 08860 Castelldefels, Barcelona, Spain § ICREA-Institució Catalana de Recerca i Estudis Avançats, 08010 Barcelona, Spain ‡

S Supporting Information *

ABSTRACT: We present a novel blurring-free stencil lithography patterning technique for high-throughput fabrication of large-scale arrays of nanoaperture optical antennas. The approach relies on dry etching through nanostencils to achieve reproducible and uniform control of nanoantenna geometries at the nanoscale, over millimeter-sizes in a thin aluminum film. We demonstrate the fabrication of over 400 000 bowtie nanoaperture (BNA) antennas on biocompatible substrates, having gap sizes ranging from (80 ± 5) nm down to (20 ± 10) nm. To validate their applicability on live cell research, we used the antenna substrates as hotspots of localized illumination to excite fluorescently labeled lipids on living cell membranes. The high signal-to-background afforded by the BNA arrays allowed the recording of single fluorescent bursts corresponding to the passage of freely diffusing individual lipids through hotspot excitation regions as small as 20 nm. Statistical analysis of burst length and intensity together with simulations demonstrate that the measured signals arise from the ultraconfined excitation region of the antennas. Because these inexpensive antenna arrays are fully biocompatible and amenable to their integration in most fluorescence microscopes, we foresee a large number of applications including the investigation of the plasma membrane of living cells with nanoscale resolution at endogenous expression levels. KEYWORDS: Optical nanoantennas, fluorescence correlation spectroscopy, fluorescence-burst analysis, shadow mask, nanostencil lithography, cell membrane dynamics

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processes at physiological expression levels. Unfortunately, extending this technology to live cell research is challenged by the inherent variability of living cells, requiring the development of large-scale antenna arrays while maintaining nanoscale control of their geometries. Importantly, for realistic applications, fabrication methods should become scalable and cost-efficient. Electron beam lithography (EBL) and focused ion beam (FIB) milling are the prototyping workhorse technologies for patterning metal nanostructures and nanoapertures with excellent precision,15 but they are both costly and slow in throughput. Alternative fabrication techniques are currently being developed to overcome these limitations such as bottomup methods that rely on capillary- or DNA-based assembly of optically functional colloids.16−19 Post processing of nonfunctional particle monolayers via tilted evaporation or hole-mask

ingle-molecule detection has become a powerful tool to study biological processes both in vitro and in living cells at diluted sample concentrations.1−3 However, traditional single molecule methods rely on diffraction-limited optics, which pose a challenge to the study of individual dynamic events at relevant physiological concentrations.4 Advances in the field of nanophotonics are starting to provide elegant strategies to reduce the excitation volume beyond diffraction allowing in vitro detection of single molecules at nearly physiological concentrations (μM range).4,5 For instance, zero-mode-waveguides (ZMW) consisting of nanoapertures (∼150 nm in size) on metallic substrates6,7 have been used in a large number of single molecule in vitro assays6−11 and in some live cell experiments.12,13 More recently, single molecule analysis in solution at 20 μM sample concentrations has been impressively demonstrated using plasmonic optical antennas, which synergistically combine large enhancement and confinement of the light down to nanoscale volume.14 The application of these devices to live cell research would indisputably open up new possibilities to investigate in real time multimolecular © XXXX American Chemical Society

Received: April 9, 2015 Revised: April 27, 2015

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Figure 1. Fabrication of BNA antenna arrays. (a) Schematic representation of etching through stencil. The substrate to be patterned consists of a glass coverslip (150 μm thick) covered with a 50 nm-thick Al layer. A 50 nm-thick SiO2 layer is sputtered on top of the Al layer. The bowtie nanoaperture (BNA) patterns are transferred to the SiO2 layer by RIE through the stencil. After RIE, the stencil is removed for further use. (b) Fabrication of SiN stencils. BNA antenna patterns are defined on the front side of the chip using EBL and RIE. Backside dry etching and KOH release are performed to obtain SiN membranes with thickness of 100 nm and 1 × 1 mm2 in size. Stencil membranes are protected with a 10 nmthick Al2O3 layer. (c) Final transfer of the BNA pattern onto the Al-coated glass substrates by chlorine RIE. The residual SiO2 layer after Al etching is approximately 10 nm thick as determined by scanning electron microscopy (SEM) inspection and etch rate measurements.

schemes20−23 also offer the combined advantage of low cost and scalability.24 Although satisfying specific applications, these methods generally suffer from a lack of design flexibility and reproducible geometrical control at the nanoscale. Nanoimprint (NIL) and stencil lithography (SL) are other scalable methods for metallic nanostructure fabrication.25 While well suited to the indentation of metal films,26,27 NIL struggles to produce isolated metal structures or residue-free apertures at sub-20 nm accuracy over large areas. Localized metal deposition by SL has been successfully used for the fabrication of different antenna geometries on various substrates28−32 down to 50 nm resolution.33 Nevertheless, metal deposition through shadow masks is ultimately limited by pattern blurring and aperture clogging.34 Here, we report on a novel blurring-free SL-based patterning technique that relies on localized reactive ion etching (RIE)35 to fabricate large arrays of nanoaperture-based antennas. The method is based on metal removal by RIE to create nanoapertures, as opposed to local metal deposition, avoiding traditional blurring and dramatically increasing geometry control to the nanometer range over large areas. Together with scalability and throughput, stencils can be reused multiple times, further reducing fabrication costs. We demonstrate the reproducible fabrication of chips containing 12-antenna arrays for a total of over 400 000 antennas, with features controlled down to 20 nm. We further show the applicability of these large-scale antenna arrays for biological research by recording for the first time the diffusion of individual lipids on living cell membranes in regions as small as 20 nm in size. Our results demonstrate that thru-stencil etching of metal nanostructures represents a cost-effective and scalable alternative for the fabrication of large arrays of photonic antennas fully compatible with life science applications. Results and Discussion. The process of pattern transfer via RIE through nanostencils is based on the fabrication and protection of shadow masks. The concept is illustrated in Figure 1, panel a. A SiN stencil prepatterned with the nanoaperture antennas of interest is brought in close contact with the underlying substrate. RIE is then performed through the stencil nanoapertures to selectively etch material away, transferring the desired pattern to the substrate. This approach has several advantages as compared to the normally performed

metal deposition through shadow masks. Indeed, during metal deposition, stress gradients build-up and lead to displacement of the membrane due to buckling. Thereby, the geometry of the nanoapertures on the stencil membrane can be strongly distorted and ultimately also clogged by in-plane material growth on the shadow mask. Finally, the quality of the deposited metal is affected by geometrical blurring and diffusion of the evaporated material on the target substrate.34 In the approach introduced here, stress and clogging issues are circumvented by using the stencil as an etch mask instead of a deposition mask. Furthermore, the directionality of anisotropic RIE and the subtractive nature of the method result in minimal pattern distortion and enlargement. We focused on the optimization of the nanopatterning process of bowtie nanoaperture (BNA) antenna arrays on Alcoated glass substrates. BNAs consist of two triangle openings faced tip-to-tip and separated by a small opening gap. Previous reports have demonstrated that these nanostructures provide a superconfined spot at the gap region together with broadband response in the visible regime.36,37 Stencils with the BNA patterns were fabricated in a low-stress SiN layer deposited on a Si wafer (Figure 1b). Freestanding SiN membranes were obtained by backside dry etching of the Si substrate followed by KOH treatment to release the membrane from the remaining Si. Finally, atomic layer deposition (ALD) of Al2O3 was used to conformaly cover the stencil. This thin layer effectively protects the stencil from erosion during the subsequent RIE process, extending its lifetime and allowing its reutilization multiple times. If the protection layer starts eroding by RIE, it can easily be removed in KOH and redeposited at will, restoring the shadow mask to its original state. The conformality of ALD furthermore minimizes deformations of the stencil membrane while allowing precise tuning of the BNA gap on the stencil. The actual pattern transfer of BNAs on the Al-coated glass substrates was performed in two RIE steps to improve the quality and uniformity of the entire patterning process (Figure 1c) (see Methods in the Supporting Information). We sputtered a 50 nm-thick SiO2 layer onto the Al-coated glass substrates and used it as a hard mask for the first RIE step. The pattern was transferred through the stencil onto this sacrificial SiO2 mask using fluorocarbon-based RIE. After displacement of the stencil, chlorine-based RIE was then used to finally transfer B

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Figure 2. Fabrication process from nanostencil to the final BNA antennas on Al substrates. (a) Stencil details: (I) photograph of the chip containing 12 different 1 × 1 mm2 SiN membranes supported by a Si frame; (II) SEM micrograph taken from the backside of the stencil; (III) enlarged SEM image of a 45° tilt view of the patterned stencil membrane; (IV) detail of a single BNA on the stencil with a sub-20 nm gap after Al2O3 protection. (b) BNA pattern transferred into SiO2 hard mask after RIE through the stencil. (c) (I, II) SEM images of two different BNAs etched into aluminum. (III) Resulting BNA array fabricated on Al-coated glass substrates.

Figure 3. Applicability of BNA arrays to measure lipid diffusion on living cell membranes. (a) Transmission optical image of a 1 × 1 mm2 antenna array etched into 50 nm-thick Al layer. (b) Overlay of a detailed view of the antenna array together with a fluorescence image of CHO cells (red) cultured over 40 h on the antenna array substrate. (c) Schematic description of the experimental configuration. Diffusing labeled lipids on the cell membrane are excited by the confined field emanating from the BNA. The BNA is excited in the far field using an inverted confocal microscope. (d) Typical fluorescence time trace as recorded with a BNA antenna of 20 nm nominal gap.

more chemical-based etching when nanostructuring the Al. Moreover, the residual thin SiO2 layer (∼10 nm thick) that remains after Al etching has a dual function. It protects the metal from further degradation, and most importantly, it creates

the BNA pattern from the SiO2 layer into the Al. The use of this intermediate SiO2 etching through the stencil significantly improved pattern uniformity by combining a more physicalbased etching when in contact with the stencil (for SiO2) and a C

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Figure 4. Lipid diffusion on living cell membranes as detected by BNA arrays. (a) Averaged and normalized autocorrelation functions of the fluorescence time traces from BNA antennas with 20 nm gap sizes compared to confocal illumination. (b) τD1 (left) and τD2 (right) values retrieved from a two-component fit of the ACF curves as a function of BNA gap sizes. (c,d) Correlation plots of burst intensity versus burst length from multiple fluorescence trajectories recorded with a BNA of (c) 20 nm gap and (d) 80 nm gap. The open circles correspond to individual fluorescence bursts, while the filled squares denote average values. The continuous lines are guides to the eye. Histograms of the burst length and burst intensity are shown in the upper horizontal and right axes, respectively.

spontaneously attach on the BNA arrays for 42−48 h. Prior to investigation, phosphoethanolamine (PE) lipids labeled with the fluorescent dye Atto647N were incorporated in the cell membrane at concentrations varying between 100 and 500 nM (see Methods in the Supporting Information). After washing, the chip was placed in a heating stage at 37 °C on top of an inverted confocal microscope. Standard fluorescence imaging showed that the cells fully attach and spread over different BNA arrays (Figure 3b) having morphologies that are similar to CHO cells directly cultured on glass. These first results validate the biocompatibility of the BNA arrays for live cell experiments. For direct observation of single-molecule diffusion events, Atto647N-PE was excited by focusing the light (λ = 640 nm, transversal polarization) onto individual BNAs using a 1.2 NA water immersion objective (Figure 3c). Since the BNA antennas are fabricated in an opaque metal film, only lipids that diffuse on the plasma membrane above the BNA illumination area are efficiently excited, reducing the background by preventing direct excitation of molecules away from the BNA and maximizing signal-to-background discrimination of individual molecules. Fluorescence fluctuations arising from Atto647N-PE diffusing in the cell membrane above the BNA were collected via the same objective, spectrally separated from the excitation light, and detected using a single photon counting avalanche photodiode (APD) detector. A representative fluorescence trajectory of diffusing PE lipids obtained with a BNA antenna of nominal 20 nm gap size is shown in Figure 3, panel d. The BNA antenna was excited using transversal polarization (i.e., across the gap) for maximum confinement and enhancement of the field at the gap region.36 Remarkably, individual fluorescence bursts with emission rates around 5 × 104 counts/s are clearly discriminated in excitation

a biocompatible surface that improves the attachment and spreading of live cells on the arrays. Figure 2 shows detailed images of the fabrication process and geometry quality of the resulting BNA antenna arrays. BNA stencils typically contain 12 different SiN membranes of 1 × 1 mm2 in size each, supported by a Si frame (Figure 2a). Each SiN membrane in turn contains arrays of BNAs at a desired pitch and with an impressive geometry control of the BNA gap down to 20 nm (Figure 2a). The quality of the BNA geometry is faithfully maintained after transfer of the pattern to the SiO2 mask via RIE through the stencil (Figure 2b) and after final transfer to the Al-coated glass substrates (Figure 2c). By using this methodology, we have been able to fabricate multiple substrates with 12 arrays of millimetric footprint. Each area containing a total of over 40 000 BNAs was designed to obtain gap sizes covering the range from 80 nm down to 20 nm. Remarkably, the gap sizes resulted extremely uniform over the entire arrays with variations of only ±5 nm for the 50 and 80 nm gap antennas and ±10 nm for the smallest antenna gaps, as determined by scanning electron microscopy (SEM). The resolution limit of the process is mainly associated with the morphology of the polycrystalline Al thin film, causing variations in the etch rate between the metal grains and the boundaries (see Methods in the Supporting Information). Further optimization of the etching process or the use of single crystalline metals could lead to even higher control of the gap size. To investigate the applicability of these BNA arrays for live cell research, we examined the nanoscale dynamics of individual lipids on the plasma membrane of living CHO cells. The chip containing multiple BNA arrays (Figure 3a) was mounted on a cell culture dish, and CHO cells were allowed to grow and to D

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Figure 5. FDTD simulations of the near field intensity distribution for different BNA gaps. (a−c) 2D normalized near-field intensity at z = 20 nm away from the BNA antennas with (a) 20 nm, (b) 50 nm, and (c) 80 nm gap sizes for transversal excitation polarization. (d) Normalized intensity as a function of z for different antenna gaps. (e) Effective illumination area provided by the BNA as a function of z for different antenna gaps.

τD2 as a function of gap size clearly demonstrates the strong lateral confinement of the optical field at the BNA gap region. The two different τD values indicate that lipids on the cell membrane possibly experience different illumination volumes as they transit through the entire BNA. Since ACF curves solely provide average values of the transient times, and given the high signal-to-background of the recorded fluorescence, we performed single burst analysis on multiple trajectories (see Methods in the Supporting Information). Histograms of the burst length and burst intensity for the 20 and 80 nm BNA gaps are shown in Figure 4, panels c and d together with the respective correlation plots. The histograms show a broad distribution of burst lengths, with transient times that vary from 10−4−10−1 s and burst intensities ranging from 104−105 counts/s irrespective of the BNA gap size. Interestingly, two main regions are identified in the correlation plots, that is, a first region where the burst intensity inversely, and sharply, correlates with burst length (τburst < ∼1 ms), and a second region containing a larger number of occurrences, for τburst > ∼1 ms, where the burst intensity weakly correlates with the burst length. We ascribe these two regions to the different intensity profiles of the near-field emanating from the BNAs. The region with τburst < ∼1 ms corresponds to excitation of the lipids within the gap region. Here, the field is highly confined and enhanced so that the higher the confinement, that is, shorter burst lengths, the higher the intensity. Additionally, this effect is more pronounced for the 20 nm gap-BNA compared to the 80 nm gap, with measured burst lengths as short as 10−4s and signals of 105 counts/s on the 20 nm gap-BNA. On the other hand, the region of τburst > ∼1 ms most probably corresponds to the excitation of lipids transiting through the large triangle aperture regions. Although the near-field here is considerably weaker as compared to the field at the gap,36 it spatially extends over the BNA arms, weakly exciting multiple

regions of only 20 nm in size. This large emission rate together with background suppression and light confinement brought about by the BNA allows measuring the nanoscale dynamics of individual PE lipids in living cells at 500 nM labeling conditions. To provide more quantitative information on the fluorescence trajectories, we generated autocorrelation functions (ACF) from multiple BNA antennas of different gap sizes over different CHO cells. ACF curves were averaged and normalized as shown in Figure 4, panel a. The resulting curve for 20 nm gap-BNAs shows a clear shift toward shorter timelags as compared to confocal illumination. Indeed, the average transient time τD defined at G(0,0) = 0.5 is 8 ms for BNA excitation, and τD = 22 ms for confocal excitation. The shortening of τD is a clear signature of the reduced excitation volume,38−40 demonstrating that the BNA effectively confines the light to subdiffraction volumes. Nevertheless, considering that the diffusion of PE on CHO cell membranes is ∼0.5 μm2/ s,39,40 the τD value obtained under BNA excitation is significantly longer than expected for the lipid diffusing over an excitation area of 20 nm. To gain more insight into this apparent discrepancy, we fitted the ACF curves using a twocomponent function as derived from Vobornik et al.,41 which assumes a sharp step-like illumination profile, well-suited to subwavelength evanescent illumination. ACF curves from BNA structures with gap sizes of 80, 50, and 20 nm were analyzed. The fitting rendered two different τD values: τD1, corresponding to long transit times and contributing to ∼75% of the fitting, showed a weak dependence on the BNA gap size, reducing from 16.0 ms for an 80 nm gap to 13.1 ms for the 20 nm gap antennas (Figure 4b). Remarkably, the short transit times τD2, contributing to ∼25% of the fitting, reduced dramatically with decreasing gap size from 14.7 ms for an 80 nm gap down to 1.1 ms for the 20 nm gap (Figure 4b). This marked dependence of E

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In summary, we have demonstrated the reproducible fabrication and characterization of large arrays of BNA antennas on biocompatible substrates using a SL-based patterning technique that relies on RIE through the stencil. The method has a high throughput, which allows the fabrication of thousands of nanoaperture antennas on a single chip with geometrical control below 20 nm. Moreover, stencils are reusable, which further reduces fabrication costs. We demonstrate the compatibility of these antenna-substrates for live cell research by measuring the diffusion of individual lipids in living cell membranes over nanoscale regions of 20 nm in size. With further shortening of the axial distance separation between the cell membrane and the BNA end, it should be possible to further increase the lateral confinement beyond the values reported here and to decrease the effect of the residual field at the BNA arms. We foresee that these engineered substrates would become inexpensive, powerful tools to investigate the plasma membrane of living cells with nanoscale resolution at endogenous expression levels.

lipids over this large area and contributing to a weak increase of the burst intensity. Thus, the larger the illumination area, the longer the transient times and the increased burst intensity, resulting in a direct but weak correlation between these two parameters, as experimentally observed. It should be also mentioned that the probability of observing lipid diffusion through the small gap region of the BNA is much lower than that of many lipids diffusing over the arms, which explains why the occurrence of longer bursts is higher than that of short burst events. Finally, to link the measured transient times of the diffusing lipids to the effective illumination volume provided by the BNAs, we performed finite difference time domain (FDTD) simulations (see Methods in the Supporting Information). We calculated the normalized total near-field intensity (Etot2/E02) of the BNA as a function of the axial distance z away from the BNA end, where Etot2 corresponds to the total intensity at the end of the BNA, and E02 is the intensity of the far incoming field to the BNA. Thus, Etot2/E02 directly estimates the degree of field intensity enhancement of the BNAs under transversal excitation conditions. Representative results at z = 20 nm are shown in Figure 5, panels a−c for 20, 50, and 80 nm gaps, respectively. As expected, for a given z distance away from the BNA, higher field enhancement and lateral confinement are obtained for the smaller antenna gaps. The normalized average intensity as a function of z for the three different gap sizes is shown in Figure 5, panel d. Clearly, the near-field emanating from the BNA decays exponentially as a function of z, with shorter decay lengths for the smaller gaps. Considering that the BNA arrays are covered with a 10 nm SiO2 layer and that the separation distance between the cell membrane and the array substrate is somewhere between 5 and 10 nm, the actual z distance between the diffusing lipids and the BNA could extend up to 20 nm. At these distances, the field intensity is comparable for all three-gap sizes, a result that is fully consistent with the similar intensity bursts experimentally obtained on the 20 and 80 nm BNA gap antennas (Figure 4c,d). The lateral confinement areas provided by the different BNAs as a function of z are shown in Figure 5, panel e. The illumination area has been considered to span a region between the intensity maximum Imax and 0.5 × Imax. For z = 20 nm, the effective areas are 1.9 × 103 nm2, 5.9 × 103 nm2, and 1.3 × 104 nm2 for the 20, 50, and 80 nm gaps, respectively. Considering the diffusion coefficient of PE on the surface of CHO cells,39,40 the estimated transient times of PE diffusing in these nanoscale regions become 1, 3, and 6.5 ms for 20, 50, and 80 nm gaps, respectively. These transient times are remarkably similar to the τD2 values extracted from the ACF data (Figure 4b) and close to the τb values obtained from the burst analysis on the region where the near-field at the gap dominates (Figure 4c,d). The excellent agreement between experimental and simulation data demonstrates that the measured diffusion of PE is performed in nanoscale regions of the cell membrane brought about by the lateral confinement provided by the BNA antennas. Outside the gap region, the near-field contribution extends to the triangle apertures with effective areas between 2.7 × 104 nm2 and 3 × 104 nm2 depending on the field intensity so that the transient times of PE range from 13.5−15 ms. Once more, these values are remarkably close to the longer τD1 values obtained from the AFC data and from the burst analysis at longer times, confirming that these transient times stem from excitation of the lipids by the field in the BNA arms.

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ASSOCIATED CONTENT

S Supporting Information *

Methods and RIE processing. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.5b01335.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: juergen.brugger@epfl.ch. *E-mail: [email protected]. Present Address

(T.S.v.Z.) National Centre for Biological Sciences (TIFR), Bellary Road, Bangalore 560 065, India Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the staff of the Center of Micro/ Nanotechnology (CMI) EPFL for the valuable discussions and support. This research was funded by the European Commission (FP7-ICT-2011-7, NANO-VISTA, under Grant Agreement No. 288263).

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DOI: 10.1021/acs.nanolett.5b01335 Nano Lett. XXXX, XXX, XXX−XXX