Multiplexed plasmonic sensing based on small-dimension nanohole arrays and intensity interrogation

Abstract

We performed multiplexed sensing on nanohole array devices to simultaneously obtain information on molecular absorption, scattering, and refractive-index change, which were distinguished by using different array structures with distinct optical behavior. Up to 25 arrays were fabricated within a 65 μm × 50 μm area to provide real-time information of the local surface environment. The performance of multiplexed sensing was examined by flowing NaCl, Coomassie blue, bovine serum albumin, and liposome solutions that exhibit different visible light absorption/scattering properties and different refractive indices. Experimental artifacts from light source fluctuation, sample injections, and light scattering induced by aggregates in solutions were detected by monitoring superwavelength holes or nanohole arrays with different periodicity and hole diameters.

Introduction

Surface plasmons (SPs) are light-excited electron density oscillations confined at the metal–dielectric boundary (Pitarke et al., 2007). The incident light resonantly interacts with free electrons in the conduction band of metals and generates collective charge oscillations. The light-electron coupling process is typically performed with prisms (Kretschmann geometry), waveguides, or grating couplers, which increase the momentum of light to satisfy a special dispersion relation of surface plasmon waves (Raether, 1988). Because of the nature of collective charge oscillations, surface plasmons are very sensitive to any change on the refractive index around the interface of metallic and dielectric materials, such as the adsorption of biomolecules to the metal surface. This phenomenon has been extensively adopted to probe chemical and biochemical interactions. Receptive biomolecules are normally immobilized on a continuous metal film and the angular distribution, reflected spectra, or reflected intensity are measured (Homola et al., 1999). Plasmonic sensing techniques (also known as surface plasmon resonance, SPR), especially those based on prism-coupling, have been successful in performing binding affinity analysis of proteins or small molecules, and commercial instruments are available (Cooper, 2002).

The greatest attraction of plasmonic sensing is that it does not require external fluorescent labels to report the binding of a ligand to its receptor. Due to the size and the hydrophobic nature of the fluorescent compounds, the labeling step could interfere with the molecular interaction, change the binding properties, and cause serious problems like background binding and autofluorescence (Cooper, 2002). The SP phenomenon transduces chemical binding directly into an optical change, which can be measured in the far-field. In many life science fields ranging from drug discovery to systems biology, high-throughput protein microarray techniques provide new and rapidly growing platforms for analyzing a large number of proteins in parallel. Most of current high-throughput techniques are based on fluorescence readout (Sauer et al., 2005, Templin et al., 2002).

In the last decade, considerable effort has been devoted to performing high-throughput assays with prism-coupled SPR devices for label-free sensing. SPR imaging provides an alternative and potentially very powerful solution for multiplexed plasmonic sensing. It is generally an intensity interrogation technique that uses a CCD camera to monitor spatial distribution of the refractive index at the metal surface in real time (Brockman et al., 2000, Campbell and Kim, 2007). Biomolecules are normally immobilized into an array by commercial robotic spotters and simultaneous monitoring at the rate of ∼1000 interactions per run has been demonstrated. The feature size of protein microarray is reported as 100–200 μm for commercially available SPR microscopes, that typically requires ∼1 nL droplets for each spot (Campbell and Kim, 2007).

Miniaturization has been a strong trend in the development of high-throughput techniques since it significantly reduces the sample volume and increases the density of assays (Sauer et al., 2005). This is particularly important for surface-sensitive techniques like SPR, as the surface to volume ratio becomes higher through miniaturization. Although prism-coupled SPR imaging has been demonstrated to be promising on multiplexed equilibrium and kinetics measurements, it encounters a major obstacle when shrinking the array feature size down to 1 μm. The utilization of prisms limits the numerical aperture (NA) and magnification of an imaging system, resulting in difficulty in achieving the spatial resolution needed for high density multiplexed sensing. The improvement of imaging optics, including using different prism geometry (Shumaker-Parry and Campbell, 2004) and adopting the configuration of total internal refection fluorescence (TIRF) microscopy (Huang et al., 2007), is in progress to overcome this limitation.

The discovery of extraordinary optical transmission through metallic subwavelength-hole arrays (Ebbesen et al., 1998), with an efficiency that is orders of magnitude greater than that predicted by classic optical theory, has generated many exciting opportunities for plasmonic sensing. Surface plasmon polaritons (SPPs) are generally proposed to explain this phenomenon observed on periodically arrayed nanoholes: the surface nanostructure couples the incident light to SPs, which transmit through the holes to the second surface, and then re-emission from the second surface (Genet and Ebbesen, 2007). In this plasmon mode, the SP wave propagation on the metal film with nanoholes is similar to that on Kretschmann configuration. Several studies exploiting periodically arrayed nanoholes fabricated by ebeam lithography (Ji et al., 2008), soft lithography (Henzie et al., 2007, Stewart et al., 2006), and focused ion beam (Brolo et al., 2004, De Leebeeck et al., 2007, Lesuffleur et al., 2007, Stark et al., 2005, Tetz et al., 2006), have demonstrated the unique advantages when used as label-free chemical sensing devices. Compared with conventional coupling techniques, the nanohole geometry has a much smaller probing area and can be fabricated with extremely high spatial density. The transmission optical configuration allows the use of high NA optics and provides better opportunities to design compact devices integrated with microfluidics.

Although the advantages of multiplexed sensing have been well recognized, most of the recent nanohole sensing studies used polychromatic light sources and spectrometers for single-channel measurements. In order to achieve multiplex and real-time sensing capability, we used intensity interrogation with monochromatic light instead of commonly used wavelength (spectroscopic) interrogation. The change of transmission intensity of monochromatic light is defined as the sensing signal. We have shown that 30 nanohole arrays fabricated in a 48 μm × 58 μm area could provide independent sensing signals from GST/anti-GST binding (Yang et al., 2008). Each nanohole array has a feature size less than 5 μm × 5 μm, corresponding to possible ∼150 fL (femtoliter) droplets of protein solutions required for each spot. Compared with wavelength interrogation, measuring the intensity change of a single wavelength leads to a much simpler optical configuration. The spatial resolution of multiplexed measurement is essentially diffraction-limited and modern high NA optics could be used for imaging. It also enables kinetic measurements with high temporal resolution by using high-quality CCD cameras. On the other hand, since the incident light goes through the running buffer solution and the adsorbate layer before arriving the nanohole surface, any phenomenon that can change transmission intensity, including molecular absorption and Rayleigh/Mie scattering, would impose a significant disturbance on the refractive-index measurement. In this paper, we demonstrate that the advantage of multiplexed sensing is not only on increasing the throughout, namely that the simultaneous acquisition of signal from nanostructures that have resonance peaks at different wavelengths can be used to separate the contribution from light extinction (absorption + scattering) and surface refractive-index change. Small-dimension periodic nanohole arrays (<16 μm²) are used to achieve high array density. Several solutions that exhibit different visible light absorption/scattering properties and different refractive indices were used to characterize the properties of these arrays and optimize their detection performance.