Open Access

Austin Journal of Biosensors &

Bioelectronics

Editorial

Nanohole Biosensor-Origin and Application as Multiplex

Biosensing Platform
Shibsekhar Roy1* and Joseph O’Mahony2 of transmitted light through an aperture in a metal sheet (necessarily
1
Pharmaceutical and Molecular Biotechnology Research opaque) should be inversely proportional to the hole-area. This notion
Centre, Waterford Institute of Technology, Ireland was built upon some interesting optical experiments performed
2
School of Engineering, Waterford Institute of
by Bethe et al during 1930s [2]. In this work, he showed that, if the
Technology, Ireland
hole diameter is smaller than wavelength of transmitted light, then
*Corresponding author: Shibsekhar Roy,
transmittance, T α (d/λ)4 (d=hole diameter and λ= wavelength). This
Pharmaceutical and Molecular Biotechnology Research
Centre, Waterford Institute of Technology, Cork Road classical aperture theory was based on a single hole in an infinitely
Campus, Waterford, Ireland thin slab and was further extended to real metals and hole arrays.
Received: June 22, 2015; Accepted: June 23, 2015; However, it took the physicists nearly six decades to discover
Published: June 25, 2015 something very different. In 1998, Ebbesen’s research group
performed a pioneering experiment on the transmission of light
through nanohole arrays fabricated on Gold and Silver thin films [3].
Editorial They observed a significant deviation from the classical aperture theory
One of the primary challenges faced by the present day diagnostic as the amount of transmitted light was observed to be significantly
industry is to develop novel strategies for analysing multiple samples larger than that predicted for particular wavelengths. They also
in parallel without compromising the target specificity and Limit of observed that the noble metal thin film had become more transparent
Detection (LOD). This requirement has led to the emergence of assay than predicted. This is indicative of more light transmission than the
platforms that can efficiently analyse multiple samples or to put it amount impacted on the hole. This highly unpredicted observation
into context, a sample containing multiple bio-markers with a single was termed as Extraordinary Optical Transmission (EOT). However,
test. These new generations of assay methods are commonly known the effect gradually wore off when noble metals were replaced by
as multiplex bioassays. Clinical diagnostic industries embracing this other metals. The maximum transmission peaks were found to be a
multiplex platform are capable of offering simultaneous analysis of function of the nanohole periodicity. From the outset, EOT looked
up to 100 biomarkers within a single sample. The target analytes are like a potential game-changer in optical instrumentation, especially
more often customizable and include (but not limited to) metabolic, for high precision sensing devices (Figure 1).
immune/auto-immune, isotyping, cell/tissue-typing, cytokines, and Understanding EOT- Many Key Players
genotyping.
The next big step was to understand the phenomena properly to
The modes of detection for multiplexed bioassays mainly are make maximum use of EOT in device fabrication. Before divulging
absorbance, fluorescence, Raman scattering, and Surface Plasmon
Resonance (SPR). However, one bottleneck for various multiplex
techniques occurs when multiple targets are present in an analyte
with a highly disproportionate ratio. The presence of trace amounts of
key analytes may remain undetected or more commonly get masked
by some abundant co-analytes. Hence, very often vital information
may be potentially lost as a key component remains undiagnosed.
This limits the very basic requirement of multiplex bio-assays, which
is to detect all the target analytes efficiently from a mixture.
To address this limitation, scientists have come up with an
approach, which can significantly improve the detection efficiency
for trace analytes by enhancing the optical signals (transmission) by
introducing an assay platform involving a specifically patterned noble
metal nano surface. This platform, which is an extension of traditional
SPR methodologies, is known as Enhanced Optical Transmission
(EOT) and the patterns formed on the noble metal surfaces are arrays
of nanoholes with defined diameter and periodicity. This generation
of assay platform or sensors is known as ‘Nanohole Biosensors’ [1].
EOT-Breaking the Dogma Figure 1: Nanohole biosensor is a high throughput optical platform for
In aperture theory an old school of thought was that the amount improving state of the art bio-assay sensitivity.

Austin J Biosens & Bioelectron - Volume 1 Issue 3 - 2015 Citation: Roy S and O’Mahony J. Nanohole Biosensor-Origin and Application as Multiplex Biosensing Platform.
ISSN : 2473-0629 | www.austinpublishinggroup.com Austin J Biosens & Bioelectron. 2015;1(3): 1012.
Roy et al. © All rights are reserved
Shibsekhar Roy Austin Publishing Group

into the theories behind EOT through nanohole arrays, it is essential visualization of target physiological phenomena and their sensitive
to understand the Surface Plasmon Polariton (SPP) or in short, quantification. Fortunately enough, EOT provides both. The most
Surface Plasmon (SP) itself. It will also be important to understand sensitive mode of light microscopy (UV/vis to IR range) to date is
how differently a single nanohole interacts with the SP compared to considered to be an optimum combination between fluorescence
the nanohole array. microscopy and dark field microscopy providing higher range
of fluorescence signal to noise ratio as well as lower background
SPPs are described as collective charge oscillations produced by the
compared to conventional fluorescence microscopy. Techniques like
resonant interaction between free electrons and light at the interface
confocal microscopy, fluorescence lifetime imaging microscopy used
of dielectric materials and metallic surface. It was mathematically
to be a benchmark for this type of detection approach. The inclusion
derived by solving Maxwell’s equations under the condition where
of EOT provides nano-antenna structures with grating capabilities
the in-plane vector, k that is continuous at the interface between
to further improve the resolution paving the way towards a super-
semi-infinite metal surface having complex di-electric function (di-
resolution microscopy platform. Concomitant with improved
electric constant εm) and the di-electric medium (di-electric constant
detection, the assay performance has improved further with 10
εd) displays the interaction between refraction and absorption. This is
to 15 times enhancement of fluorescence signal due to EOT [7].
given in equation 1.
1/2
An additional three-fold increase of assay sensitivity was achieved
ω ε ε  by optimizing the angle of incident light on the sample. Further
k=  m d  = k SP improvement was achieved when fabrication of single-hole nano-
c  εm + εd  arrays have been graduated to double-hole nano-arrays as shown by
It is quite evident here that the dielectric functions, εm and εd must Lesuffleur et al. [8].
be of opposite sign if ‘k’ needs to be a real component assuming the
strong conduction behaviour of εm. In this situation, the magnitude
Fabrication of Nanohole Array- Lithography
of kSP will always be larger than the photon (kL= ω/c) - making the
Approaches
coupling between light and SP not feasible. Hence, the primary The recent advances in nanofabrication techniques have
requirement would be to increase the value of wave vector of photons revolutionized our ability to design and create a patterned
to be equal to the value of kSP. nanosurface with desired optical properties. Depending on the
detection requirements, both the
Two theories were proposed to aid the coupling [4]. The first one
is based on Attenuated Total Reflection (ATR) theory based on the ‘close-ended’ (for flow-over operation mode) as well as ‘through’
Kretschmann–Raether geometry. Under this optical condition, the (for flow-through operation mode) nanoholes are being fabricated
dielectric function of the dielectric medium increases significantly for the last two decades using a variety of lithographic techniques.
over that of the air, increasing the photon momentum leading to Some of these lithographic approaches are worth mentioning to give
(angularly modulated) coupling between the SP and photon. The an idea of the state-of-the-art nanofabrication [9].
second theory is based on the coupling of grating momentum.
Colloidal (micro/nano) lithography
Grating structures are created by the presence of periodic structures
(i.e. periodic arrays of nanohole in this case). This grating momentum This simple and relatively economic technique requires colloidal
is inversely proportional to the lattice parameter of the system, which nano or micro particles (polystyrene, silica etc) as mask. At first a
corresponds to the measure of periodicity of the nanohole arrays. monolayer of the beads are achieved (by spin coating). Then it
Hence, for an optimized small value of array periodicity, photonic undergoes controlled metal coating using thermal evaporation.
and plasmonic vectors can undergo efficient coupling leading to Finally, beads are peeled off using adhesive tapes or controlled
EOT. In reality, it is probably the interplay of both the mechanisms sonication to expose metallic hole-arrays as a footprint of the ‘up-
that results in EOT, where the optical configuration determines rooted’ beads. However, this method very often suffers from the lack
which mechanism dominates. Here also lies the difference between of a sufficiently large coverage of hexagonally close packed monolayer
the optical behavior of nanohole arrays compared to that of the single distribution.
holes. Single holes or rather isolated holes are devoid of such grating Focussed ion beam (FIB)
momentum coupling due to the unavailability of any periodicity.
This high resolution, mask less and direct writing technique
Hence, only the first mechanism (ATR) is operative here. However,
uses a low energy ion beam (ion sources as Ga, Au-Si-Be) as the
a detailed description of the electromagnetic coupling phenomena
milling tool. The beam removes material from the metal surface by
around a single elliptical aperture in an optically thick metal was
collision thereby creating holes. The hole diameter, depth and shape
provided by Zakharian [5]. This theory was further modified by
is controlled by tuning the beam intensity.
several groups by extending the phenomena to LSP (localized surface
plasmon)-photon coupling, LSP-SPP coupling and its polarization Electron beam lithography (EBL)
dependent nature are strongly supported by electro-dynamic This writing technique is associated with the SEM (scanning
simulation approaches [6]. electron microscopy) and utilizes a focussed electron beam for the
purpose of milling. EBL can either be a direct writing method or it
EOT Mediated Improvement of Bio-
may follow lift-off procedures. This technique is usually hailed as
Diagnostics
more versatile and better suited for high-density nanohole array
The two most important aspects of clinical diagnostics are the fabrication.

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Shibsekhar Roy Austin Publishing Group

Nanoimprint lithography (NIL) References

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method uses a negative mould with nanostructures as the stamp on
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gradually being introduced to the nano-fabrication industry. elliptical apertures. Opt Express. 2004; 12: 2631-2648.

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Conclusion apertures. Opt. Commun. 2005; 255: 338-348.

Nanohole biosensors are potential game changers in the 7. Wang Y, Wub L, Zhou X, Wong TI, Zhanga J, Baib P. Incident-angle
multiplexed biosensing industry. They are capable of developing very dependence of fluorescence enhancement and biomarker immunoassay on
gold nanohole array. Sensors and Actuators B. 2013; 186: 205– 211.
high resolution microscopy techniques as well as bio-assay platforms
with multi-fold improved assay sensitivity. Conventional as well as 8. Lesuffleur A, Kumar LKS, Brolo AG, Kavanagh KL, Gordon R. Apex enhanced
Raman spectroscopy using double-hole arrays in a gold film. J Phys Chem
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Austin J Biosens & Bioelectron - Volume 1 Issue 3 - 2015 Citation: Roy S and O’Mahony J. Nanohole Biosensor-Origin and Application as Multiplex Biosensing Platform.
ISSN : 2473-0629 | www.austinpublishinggroup.com Austin J Biosens & Bioelectron. 2015;1(3): 1012.
Roy et al. © All rights are reserved

Submit your Manuscript | www.austinpublishinggroup.com Austin J Biosens & Bioelectron 1(3): id1012 (2015) - Page - 03