Localized Surface Plasmon Resonance Biosensing with Large Area of Gold Nanoholes Fabricated by Nanosphere Lithography

Localized surface plasmon resonance (LSPR) is coherent oscillation of conduction electrons confined in noble metallic nanoparticles which is excited by electromagnetic radiation [1–3]. LSPR causes two notable results that can be exploited: first, the strong extinction band (absorption and scattering) which can be used as chemical and biological molecular sensing platform [2]; second, the strong electromagnetic field near the surface of nanoparticles which is the basis for all surface-enhanced spectroscopic methods such as surface-enhanced Raman spectroscopy (SERS) [4]. The potential of LSPR as biosensing platform relies on the fact that the resonance frequency and intensity of LSPR are highly sensitive to the material, shape, size and local dielectric constant of the metal nanoparticles [5, 6]. The resonance frequency and sensitivity of LSPR is tunable by nanoparticles, and molecular adsorption or chemical bonding onto the nanoparticles causes additional resonance frequency shift which can be detected by extinction peak shift in optical transmission spectrum of the nanoparticles. Biosensing of silver nanotriangle arrays, fabricated by closely packed nanosphere lithography, has already been extensively studied [7, 8]. However, by the limit of small defect-free area on scale of μm² and instability of silver, the realization of a feasible biosensing device based on silver triangle array is difficult. Recently, LSPR biosensing based on gold nanoholes has been reported, either with randomly distributed [9–11] or periodically arranged nanoholes [12–14].

In this paper, large uniform area of gold nanoholes in cm² scale are fabricated on glass substrate by nanosphere lithography (NSL), in contrast to the smaller area of nanoparticles obtained by closely packed NSL. The morphology of the nanoholes is characterized using scanning electron microscope (SEM) and atomic force microscope (AFM), and the LSPR sensitivity of nanoholes to local refractive index is measured. Detection of bovine serum albumin (BSA) binding and biotin-streptavidin immunoassay to gold nanoholes are realized, with LSPR extinction peak red-shifts 4-fold higher than the previously reported nanohole experiments in BSA test [10]. The detection of specific biotin–streptavidin bonding demonstrates that the LSPR peak shifts 11 nm after 21.3 fMol of effective streptavidin molecules are bonded onto the biotin functionalized nanoholes, this detectable streptavidin quantity is comparable to silver triangles [7], and the sample is reusable after removal of the anchored biotin.

In our experiments, polystyrene (PS) nanospheres were purchased from Duke Scientific Ltd, biotinylated thiol was a gift from Max Planck Institute for Polymer Research in Germany, and other chemicals were purchased from Sigma–Aldrich.

A 4″ glass wafer was pre-treated with silicon ion implantation to introduce electrical charges on the glass surface for dispersing the nanospheres, then it was cleaved into 7.5 mm × 10 mm chips as substrates by Disco dicing saw. Glass substrates were cleaned by sonication in acetone for 5 min followed by DI water rinse and nitrogen gas blow-dry. Prior to deposition of PS nanospheres, the substrates were drop coated by PDDA of 2 wt%, rinsed by DI water and blow-dried by nitrogen gas. The gross electrostatic force resulted from PDDA, implanted silicon ions and PS nanospheres balances the capillary force of nanospheres, avoiding them from forming close-packed or clustered defects. Afterwards, 110-nm diameter nanospheres of 0.1% in water were drop coated and evenly dispersed onto the substrates. Gold film was perpendicularly deposited onto the substrates with nanospheres at a rate of 1 Å/s by a R-Dec thermal evaporator, and a substrate without nanospheres was deposited in the same batch and served as a reference to LSPR spectral measurement. The gold deposition thickness was 40 nm, monitored by a microbalance. The nanospheres were then removed by sonication in water for 40 s, and nanoholes were successfully fabricated. The morphology of the nanoholes was characterized by a JEOL 6700 SEM at an acceleration voltage of 5 kV and a Digital Image AFM in tapping mode.

LSPR extinction spectra were measured using an Ocean Optics fiber spectrometer with a wavelength range of 200–900 nm and a resolution of 0.33 nm. The spectrometer defines the extinction spectrum as , where I _s ,I _r and I _d are respectively the light intensities of the sample, reference and dark noise. The UV–Vis light from the deuterium and halogen source was coupled to an optical fiber, transmitted through the sample, and collected by another fiber to the spectral analyzer. Two 74-UV collimating lenses were respectively used to collimate the light from the illuminating fiber to a 5-mm diameter spot and focus it back into the collecting fiber.

In summary, we successfully fabricated uniform gold nanoholes in a large area (>cm²) of glass substrate, and characterized the morphology and the LSPR prosperities of the nanoholes. Although these nanoholes have lower sensitivity to refractive index compared to reported experiments in literatures, a higher sensitivity to biochemical molecules was demonstrated by this device due to the cone shape of the nanoholes. The nonspecific BSA binding demonstrated 4 times more wavelength shift than reported nanoholes. In biotin–streptavidin test, 21.3 fMol of effective streptavidin manifested a 11-nm wavelength redshift, this sensitivity seems satisfactory because 7.6 fMol of streptavidin is the calculated LOD of the LSPR sensor with silver nanotriangles. Since our research is based on gold and glass substrate, and the nanoholes are evenly distributed on a large glass area, our study proves the great potential for such a cost-effective and robust chip to become a specific and reliable biosensor.