Directional Nanoplasmonic Antennas for Self-Referenced

Article pubs.acs.org/JPCC

Directional Nanoplasmonic Antennas for Self-Referenced Refractometric Molecular Analysis Martin Wersal̈ l,† Ruggero Verre,† Mikael Svedendahl,† Peter Johansson,†,‡ Mikael Kal̈ l,† and Timur Shegai*,† †

Department of Applied Physics, Chalmers University of Technology, 412 96 Göteborg, Sweden School of Science and Technology, Ö rebro University, 701 82 Ö rebro, Sweden

‡

S Supporting Information *

ABSTRACT: Localized surface-plasmon resonance (LSPR) sensors are typically based on tracing resonance peak shifts that precisely follow changes in the local refractive index. Such measurements usually require a spectrometer, a stable light source, and an accurate LSPR position tracing technique. As a simple but efficient alternative, we investigated a selfreferenced single-wavelength sensing scheme based on angle-dependent and highly directional radiation patterns originating from a monolayer of asymmetric gold nanodimers. We found that one could easily trace a model biotin−neutravidin recognition reaction as well as minute bulk refractive index changes, by measuring the intensity ratio between the light scattered in two different directions with respect to the dimers. The refractometric resolution of the methodology was estimated to be on the order of Δn ≈ 10−5 RIU. These results may be particularly useful for label-free biosensing applications that require a combination of simple and cost-effective optical readout with a reasonable sensitivity.

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INTRODUCTION The possibility to detect low concentrations of target molecules is of great importance within several research and industrial areas, including medical diagnostics, analytical chemistry, biochemistry, material science, and many others.1−4 For example, revealing certain diseases at an early stage often requires techniques with the ability to detect small amounts of specific biomolecules in the patient’s blood or urine.4,5 Small metal nanoparticles are regarded as important ingredients in a wide range of promising sensing schemes due to their unique capability to sense minute changes in the ambient dielectric environment.6−10 Several optical sensing methods, including tracking of the localized surface-plasmon resonance (LSPR) peak through optical extinction or scattering spectroscopy, have been demonstrated with competitive performance.9,11 LSPR refractometric detection limits down to the order of Δn ≈ 10−6 RIU (refractive index units), and even single molecules in the case of single nanoparticle measurements,10,12−14 have been reported, evincing the future potential of plasmonic nanoparticles for a variety of molecular analysis applications. The readout in LSPR biosensing is traditionally performed by following resonance peak shifts, a procedure that often requires rather complex and expensive instrumentation such as a spectrometer, spectrally and temporally stable light source, and accurate referencing to achieve reasonable signal-to-noise levels.15 The performance of LSPR-based sensors is also largely dependent on other factors, such as surface chemistry, analyte diffusion and binding, molecular specificity, ability to operate in crowded realistic fluids such as serum or blood, parallel readout possibilities, etc.16−19 Motivated by the possibility to reduce © 2014 American Chemical Society

both complexity and expense associated with LSPR-based sensors, we have previously demonstrated that Pd−Au heterodimers can be used for hydrogen sensing using a selfreferenced optical readout based on directional scattering arising from the built-in material asymmetry of the nanostructures.20 Here, we show that this measurement scheme can be further simplified and applied to molecular detection based on biorecognition reactions on the surface of monometallic but asymmetric gold nanodimers. The approach demonstrated here enables single-wavelength and self-referenced biosensors that require neither a spectrometer nor a microscope. The key factor in the presented sensing scheme is interference between the scattering fields of the individual components comprising the plasmonic nanodimer. The scattering pattern is directional due to the asymmetry of the dimer. Therefore, not only the plasmon resonance but also directional scattering pattern is a function of the electromagnetic environment around the nanostructures. Changes in the dielectric environment can then be monitored by simply measuring the ratio between the light scattered in two different directions with respect to the dimers. This approach may be advantageous in comparison to tracing the LSPR peak shift because intensity fluctuations of the light source can be efficiently canceled out in real time by dividing the signals from the two channels. The device can also operate in a single-wavelength mode, provided the directional ratio contrast at this wavelength is strongly responsive to the Received: June 30, 2014 Revised: August 13, 2014 Published: August 19, 2014 21075

dx.doi.org/10.1021/jp5064929 | J. Phys. Chem. C 2014, 118, 21075−21080

The Journal of Physical Chemistry C

Article

Figure 1. (a) SEM image of asymmetric Au dimers manufactured using hole-mask colloidal lithography. The inset shows a side view of a single dimer. (b) Sketch of the working principle of the Fourier imaging concept. (c) Color microscope Fourier plane images for dimers in air and water, respectively. The asymmetry in the images is due to directional scattering, while the central bright spot is due to directly transmitted light.

environment change. We here demonstrate that LSPR biosensing can be performed in a very simple and cost-effective manner by building an optical setup consisting of cheap optical components, such as a laser diode and a pair of photodiodes. Keeping in mind that the asymmetric dimer pairs used in this study were fabricated using an inexpensive self-assembly technique,21 our sensors represent a balanced compromise between a simple detection and reasonable sensitivity, an approach that may prove useful for LSPR-based label-free biosensing.

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EXPERIMENTAL SECTION Sample Fabrication. The Au dimer samples were fabricated using hole-mask colloidal lithography21 by sequentially depositing gold disks of different height on a glass substrate. This was achieved by tilting the sample with respect to the evaporation direction. Note that prior to the optical measurements, the samples have been chemically treated to remove any possible contaminants (20 mL of distilled water, 5 mL of concentrated H2O2, and 5 mL of ammonia, boiled at 150 °C for 3 min) and further annealed at 250 °C for 10 min to improve the nanodisks’ crystal quality. Microscope Back-Focal Imaging. The back-focal plane images (shown in Figures 1 and 2) were collected using a polarized white light incidence loosely focused on the sample by a low-magnification microscope objective (air 10×, NA = 0.3). The transmitted and diffusely scattered light was then collected by the ultrahigh numerical aperture TIRF objective (oil 60× , NA = 1.49) which ensures nearly maximal collection efficiency. The Fourier images were measured by the SLR camera (Nikon D300s) using a 4f correlator. A 650 nm (40 nm bandwidth) filter was used for the color image shown in Figure 2. Macroscopic Spectrometer-Free Setup. The incident white light was linearly polarized perpendicular to the dimer axis and delivered to the sample by means of a fiber and a collimating lens. The spot size (∼5 mm) at the sample was controlled by a tunable iris aperture. The sample was in this case mounted inside a flow cell (chamber volume 0.5 mL). The collection optics consisted of either a hemispherical or rightangle prism attached to the outer surface of the flow cell using index matched oil. The light scattered at angles greater than critical from both sides of the prism was further collected by two fibers (400 μm core) and simultaneously imaged on a CCD chip (Andor). A 650 nm (40 nm bandwidth) color filter was installed in front of the sample to mimic a simple spectrometer-free performance. The intensity of the light at the

Figure 2. (a) Extinction spectrum (green) with incident light polarized perpendicular to the dimer axis. The ratio spectrum (blue) exhibits a maximum in the spectral region between the resonance peaks. The measurement was conducted with air as ambient dielectric medium. (b) Greens’ function calculations showing scattering and ratio spectra for single isolated dimer on the air−glass interface. Geometrical parameters of the dimer in simulations are D1 = 105 nm, H1 = 25 nm, D2 = 100 nm, H2 = 60 nm, and gap = 15 nm. Insets in both (a) and (b) show Fourier plane images of scattering radiation patterns measured at 650 ± 20 nm and calculated at 636 nm.

sample (after passing the filter) was 0.5 mW. In further simplification of the optical setup a laser diode illumination (intensity = 10 mW, λ = 660 nm) and Si photodetectors (Taos, TSL 252R) were used. The data are shown in Figure 5. 21076



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Figure 3. (a) An illustration of the macro-optical setup. (b) Data from bulk refractometric sensing, performed by measuring ratios of left/rightscattered intensities, for nanodimers located in six distinct dielectric environments. In the notation EGx in the legend, x refers to ethylene glycol concentration in %. (c) Image displaying the directional scattering effect from the dimer sample positioned on top of a semispherical prism. Paper sheets are placed in front of each detector to visualize how different colors with varying intensities scatters in opposite directions along the dimer axis. (d) Spectrometer-free macroscopic (650 ± 20 nm) bulk sensing data performed by immersing the samples in water−EG mixtures.

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RESULTS AND DISCUSSION A Pair of Detuned Point Dipoles. We start by considering the elastic scattering from an asymmetric gold nanoparticle dimer. If the nanodisks comprising the dimer are much smaller than the wavelength, they may be approximated by a pair of point dipoles.22 In our previous works, we have shown that the radiation pattern arising from such an asymmetric pair can be highly directional as a result of the dipole−dipole interference.20,23 The ratio of intensities scattered to the right and left of the dimer along its axis is given by R=

opposite directions. When the local dielectric environment around the nanodisks change, for example due to the adsorption of protein molecules, all the parameters defining the interference pattern also change, which can be directly observed by tracing the directivity ratio contrast R. Importantly, the ratio is insensitive to fluctuations in the incident light intensity (see Supporting Information Figure S1 for a clear demonstration of this effect). Microscope Back-Focal Plane Imaging. Figure 1a shows a scanning electron microscopy (SEM) image of the Au dimer sample fabricated using colloidal lithography. The dimer symmetry is broken by sequentially depositing disks of different heights (see Figure 1), which ensures that the plasmon resonances of the disks are detuned. The image processing of the SEM data reveals the dimer dimensions as follows: D1 = 104 ± 5 nm for the lower disk (height H1 = 26 nm), D2 = 101 ± 6 nm for the taller disk (H2 = 62 nm), and an intermediate gap of d = 12 ± 3 nm. The angular distribution of scattered light can be directly monitored in an optical microscope by imaging the objective’s back-focal plane, also known as the Fourier plane. This imaging principle is illustrated in Figure 1b. Each pixel in the Fourier plane corresponds to a particular solid angle, and therefore, the image represents an angle-resolved scattering pattern projected on a plane by means of the microscope objective. A variation of

|p1 |2 + |p2 |2 + 2|p1 ||p2 | cos( −kL + Δϕ) |p1 |2 + |p2 |2 + 2|p1 ||p2 | cos(kL + Δϕ)

(1)

Here, |pi| is the magnitude of the ith dipole moment, Δϕ is the intrinsic phase lag between the dipoles along the dimer axis, L is the distance between the dipoles, and k is the wavevector. The numerator and denominator in eq 1 describe the total intensity scattered to the left and right, respectively. The first two terms in both expressions correspond to scattering of each individual disk, while the third term describes the interference between the dipoles in the corresponding direction. The optimal ratio occurs in a situation when |p1| = |p2|, Δϕ = π/2, and L = λ/4n, corresponding to constructive and destructive interference in 21077



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Figure 4. Model biosensing reaction using microscope- and spectrometer-free directional scattering setup. At the beginning of experiment the dimers reside in PBS buffer. Then bBSA molecules (100 μg/mL) are added, causing a decrease in ratio. After that neutravidin molecules (10 μg/mL: blue; 100 μg/mL: green) are added, which is accompanied by a further ratio drop. Local refractometric sensing exploiting changes in ratio due to specific binding of neutravidin to biotin-conjugated bovine serum albumin (bBSA). The measurement was conducted with a 650 ± 20 nm optical filter placed in front of the CCD detector.

i.e., the glass, at angles exceeding the critical.25−28 Moreover, the exact distribution of radiation over the solid angle depends on the distance between the radiating dipole and the interface. For dipoles located far away from the surface, scattering at large angles is very small because the evanescent components of the field do not reach the interface. For this reason the macrooptical setup presented in Figure 3 is able to efficiently discriminate between the transmitted light as well as light scattered by impurities (for example, protein aggregates or dust particles) located far away (at distances large compared to the wavelength) from the interface, while scattering from a quasirandom array of directional dimers appears at angles greater than critical. Figure 3b shows bulk refractometric sensing data obtained using the macroscopic configuration. The figure displays the ratio between left- and right-scattered light intensities as a function of wavelength for six different dielectric environments. The ratio spectrum was first measured in air, and then the samples were immersed into ethylene glycol (EG)−water mixtures of variable concentration. A 10% increase in the EG concentration corresponds to a refractive index increase of about ∼0.01. After each measurement, a new EG−water mixture was introduced as an ambient medium using a flow cell. The acquired data clearly demonstrate a change in spectral ratio caused by the change in refractive index. This justifies further simplification of the readout using detection exclusively at the spectral region where the ratio change is maximal. With this in mind, we modified our measurement setup to read out the directional signal directly on the photodetector without the spectrometer. Further details about the setup are given in the Experimental Section. Bulk refractive index sensing data using EG−water mixtures in a spectrometer-free macroscopic configuration are shown in Figure 3d. As the refractive index around the dimers is increased, the ratio decreases in a steplike fashion. Sensitivity values for this particular configuration of detectors was about s = 37 RIU−1, which together with the noise levels of σR ≈ 1.86 × 10−3 gives a refractometric detection limit of Δn = σR/s ≈ 5 × 10−5 RIU. Sensitivity (s) and noise level (σR) are defined as s = ∂R/∂n and standard deviation of the ratio σR = σ(R/n),

the local dielectric environment around the dimers will therefore be possible to observe as a change in angular distribution of the scattered light. This is illustrated in Figure 1c for Fourier images measured in air and water. Experimentally measured extinction spectrum of the same asymmetric dimer sample as shown in Figure 1 is shown in Figure 2a. It is evident that two overlapping but detuned plasmon resonance peaks are present. These peaks are dominated by the plasmon resonances of each individual disks comprising the dimer. The highest directionality is expected in the spectral region between the two peaks, as dictated by the phase retardation condition described above. The measured directional ratio (blue curve in Figure 2a) indeed peaks in between the two detuned resonances. Therefore, spectroscopic and angular-resolved radiation pattern data presented in Figures 1 and 2 demonstrate that we have fabricated an array of plasmonic gold dimers that scatters light in a highly directional manner. This is additionally supported by the asymmetry in the radiation pattern measured using a 650 nm band-pass filter (see Figure 2a inset) and numerical simulations for a single isolated nanodimer using the Greens’ function method24 (see Figure 2b). In general, there is a good qualitative agreement between the experimental observations and the numerical simulations. The quantitative differences mainly arise because of inhomogeneous spectral broadening effects and a stray light background present in the experiments, as discussed in detail in ref 20. This discrepancy, however, does not strongly affect the arguments put forward in the following discussion, as is shown below. Macroscopic Single-Wavelength Biosensing. Directional emission from fabricated asymmetric Au pairs is so pronounced that, in fact, it is possible to observe it with the naked eye! A sketch of a macro-optical setup together with a color photograph demonstrating the asymmetric scattering profile is shown in Figure 3a,c. Instead of a microscope objective, we here use a prism to collect the scattered light. Two detectors are positioned in opposite directions along the dimer axis at an angle exceeding the critical angle. It is wellknown that an oscillating dipole located close to a planar interface radiates preferentially into the denser optical medium, 21078



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respectively. Additional details are given in the Supporting Information. The collection time for the data shown in Figure 3d was t = 20 s/frame. To demonstrate label-free biosensing using the asymmetric gold dimers, we performed a proof-of-principle experiment based on the well-known biotin−neutravidin recognition reaction (see Figure 4). The experiments were conducted by first injecting 0.5 mL of PBS−buffer solution and measuring the ratio over a time span of 500 s. Thereafter, 0.5 mL of a 100 μg/ mL biotin-conjugated bovine serum albumin (bBSA) solution was added, and the ratio was traced for another 60 min. During this process, bBSA molecules bind to the Au surface of the disks, causing a slight change in the local refractive index. Finally, 0.5 mL of 10 or 100 μg/mL neutravidin solution was added in order to study binding to bBSA. In order to observe all possible binding events between neutravidin and bBSA (as well as possible unspecific binding), a time interval of 60 min was chosen. After injecting neutravidin, binding events can be seen as a continuous decrease toward saturated values of R10 ≈ 4.44 for 10 μg/mL and R100 ≈ 4.18 for 100 μg/mL. These ratio values are nearly optimal in terms of signal-to-noise as described in more detail in the Supporting Information. From time of injection until the end of the measurement, a decrease in signal of ΔR10 ≈ 0.19 and ΔR100 ≈ 0.45 occurs for the corresponding neutravidin concentrations. With a standard deviation of σR = 8.15 × 10−4 for the saturated ratio value in this region, we end up with signal-to-noise values of ΔR10/σR ≈ 233 ans ΔR100/σR ≈ 552. The measurements were performed with a t = 100 s/frame temporal resolution using the same light intensity as in the case of bulk sensing. So far we have utilized a white light source, a band-pass filter, and a CCD detector in our measurements. The data obtained using this equipment indicate that label-free biosensing is possible to do in a spectrometer-free macroscopic fashion; however, the complexity and cost of these optical components were still relatively high. To demonstrate that it is feasible to perform biosensing experiments using much simpler alternatives, we repeated the bulk refractive index measurements using a laser diode as a light source and two cheap silicon photodetectors to monitor the scattering ratio. This is shown in Figure 5. In panel a, photographs of the experimental setup under laser diode illumination taken from the right and the left with respect to the dimer orientation are shown. A strong directional scattering contrast produced by the dimer array is evident. Panel b shows the change in scattering ratio as the sample is immersed into various EG−water mixtures. Clearly, this inexpensive setup is sensitive enough to monitor ∼0.005 refractive index changes on every step of the sensing ladder. In fact, the signal-to-noise shows refractometric resolution on the level of Δn ≈ 2.33 × 10−5 RIU (laser diode intensity 10 mW at the sample, time resolution 0.4 s/frame, σR = 8.86 × 10−4, and sensitivity s = 38 RIU−1). It should be pointed out, however, that the exact value of the directional contrast turned out to be sensitive to the exact position of the photodetectors relative to the sample. Nevertheless, once they were aligned and rigidly fixed, the ratio signal was robust and stable. The alignment is likely to be the reason for differences between the ratio contrasts shown in Figures 3d and 5b.

Figure 5. Refractive index sensing using a red laser diode and cheap Si photodetectors. (a) Color photographs taken from the left and right of the right-angle prism attached to the flow cell with mounted directional dimer nanoantennas using index matched oil. The contrast in scattering is easily visible by a naked eye. (b) Ratio between left and right detectors measured as a function of time as directional dimers are immersed into water−EG mixtures of variable content. Numbers in the figure indicate percentage of EG used.

refractometric sensing experiments in a self-referenced spectrometer-free manner. We demonstrated both bulk and local refractive index sensing reaching a refractometric detection limit of about 10−5 RIU. This reasonably high number was reached despite using simple and low cost optical components. By considering that no optimization of the setup, no symmetric stray light correction and no optimization/ simplification of directional plasmonic arrays was performed, we conclude that the self-referenced approach presented in this study is an interesting alternative to traditional LSPR and SPR sensing schemes.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

Additional figures and text describing self-referencing and noise analysis. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*E-mail: [email protected] (T.S.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Swedish Research Council (VR) and Knut and Alice Wallenberg Foundation.

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CONCLUSION We fabricated arrays of asymmetric gold dimers and experimentally studied their directional scattering properties. The directionality was further utilized as a tool to perform

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Directional Nanoplasmonic Antennas for Self-Referenced

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