PAPER www.rsc.org/loc | Lab on a Chip

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Multifunctional plasmonic sensors on low-cost subwavelength metallicnanoholes arrays

Valentin Canpean* and Simion Astilean

Received 15th July 2009, Accepted 14th September 2009

First published as an Advance Article on the web 15th October 2009

DOI: 10.1039/b914235e

Localized surface plasmon resonance (LSPR) sensing is combined with surface enhanced Raman

scattering (SERS) detection on periodic arrays of subwavelength metallic nanoholes for the first time.

LSPR sensors provide detection of molecular adsorption in both transmission and reflectivity modes

with sensitivity greater that 300 nm/RIU and a spectral shift of 16 nm in the case of binding

a monolayer of p-aminothiophenol (p-ATP) molecules. Subsequent SERS analysis enables

identification of the adsorbed p-ATP molecule, its structure and orientation on the metal surface.

This synergistic LSPR–SERS approach on low-cost metallic films perforated with periodic arrays of

subwavelength nanoholes opens a route for molecular dual-modal detection to be integrated in

lab-on-chip systems to increase the reliability of biological detection.

Introduction

The development of highly selective and sensitive sensors is of

foremost importance for medical diagnostics, environmental

protection, drug screening or food safety.1 Much sensor research

has been devoted to the evaluation of various signal transduction

methods including optical, radioactive, piezoelectric, and

magnetic. Strategies for the development of sensing devices that

can detect the analyte species without labelling, i.e. label-free

detection, are of significant interest. Optical sensors based on

plasmonic transducers are fast becoming the method of choice in

non-labelling analysis of biomolecular interaction.2,3 In

conjunction with plasmonic transduction based on excitation of

surface plasmons on planar gold surface in conventional surface

plasmon resonance (SPR) devices (see ref. 4 and references

therein), recently there has been an increasing interest in optical

nano-sensors based on localized surface plasmon resonance

(LSPR) in nanometer-sized metallic structures.5–8 On the other

hand, surface-enhanced Raman scattering (SERS), with its great

ability to detect single molecules, has been proved to have great

potential in label-free ultrasensitive biomolecule detection.9,10 In

recent years the interest in LSPR and SERS-based sensors was

completely revived, mainly because the accumulated experi-

mental data has clearly demonstrated the huge potential of both

methods in surface science, analytical and environmental

applications, biomedicine, biophysics and biochemistry.

The main optical signature of metallic nanoparticles is a strong

UV-VIS-NIR extinction band that is not present in the spectrum

of the bulk metal. Such optical response results when the

frequency of the incident photon is resonant with the collective

oscillation of the conduction electrons, and is known as localized

surface plasmons resonance. The peak extinction wavelength

(lmax) of the LSPR spectrum is strongly dependent upon the size

Babes-Bolyai University, Faculty of Physics and Institute forInterdisciplinary Experimental Research, 42 Treboniu Laurian, 400271Cluj-Napoca, Romania. E-mail: vcanpean@phys.ubbcluj.ro;Fax: +40(264) 591906; Tel: +40(264)454554/119

3574 | Lab Chip, 2009, 9, 3574–3579

and shape of the nanoparticle, as well as the interparticle spacing

and dielectric function of the local environment.11,12

The LSPR sensing mechanism is based on the modification of

the electromagnetic field decay length when adsorbate molecules

come in contact with the surface of metal. In the case of nano-

particles, the local electromagnetic field operates in a manner

analogous to propagating surface plasmon-polaritons (SPP) on

a flat metallic film and enables the transduction of chemical

binding events into a measurable wavelength shift of the extinc-

tion peak. Although many effective nanoparticle-based LSPR

biosensors have been demonstrated at laboratory level13,14 the

practical implementation of LSPR instrumentation still requires

large improvement regarding reproducibility, spatial resolution

and monodispersity in size and shape of nanoparticles. However,

similar plasmonic response can be devised from much more stable

and reproducible metallic nanostructures which are continuous

metallic films perforated with regular arrays of subwavelength

nanoholes, rather than assemblies of nanoparticles. In contrast to

conventional SPR devices, a continuous metallic film perforated

with regular arrays of subwavelength nanoholes can operate

without requiring a prism for plasmon coupling, requiring only

simple reflection or transmission configurations. In this case

a UV-Visible microspectrometer can be efficient enough to obtain

the signal and, therefore, the experimental setup can be more

easily integrated into portable, low-cost devices for rapid bio-

analytical measurements.15,16 Furthermore, short-range ordered

nanoholes in thin metal films can be inexpensively fabricated with

colloidal lithography techniques over large area.17,18

Besides many interesting optical features,19–22 the excitation of

surface plasmon modes in nanohole arrays exhibits a very

sensitive response to the refractive index of a thin molecular layer

located within a few nm of the metal. In particular when metal

films are sufficiently thin (films become semitransparent even in

the absence of holes), in addition to exciting surface plasmon-

polariton (SPP) modes by two dimensional grating scattering,

there is also the possibility to excite localised modes (LSPR)

associated with the hole cavities.23 A hole in thin film actually

This journal is ª The Royal Society of Chemistry 2009

Fig. 1 AFM image of metallic film perforated with an array of holes.

The scale bar is 1 mm.

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exhibits a resonant scattering spectrum that is qualitatively

similar to a particle of approximately the same dimensions and

such resonance can be also assigned to LSPR.24 Notably, the

optical excitation of the LSPR associated with the hole cavity

results in not only a UV-VIS extinction band, but also strong

enhancement of the local electromagnetic field for operation in

SERS. As a result a noble metallic film perforated with sub-

wavelength nanohole arrays are attractive substrates for both

plasmonic and SERS sensing. Indeed, many examples of nano-

hole arrays have been previously studied with specific regard to

either LSPR or SERS operation. Pang et al. demonstrated the

applicability of periodic arrays of metallic nanoholes as plas-

monic sensors by monitoring protein–protein specific bonding.25

Brolo and co-workers demonstrated the enhanced-Raman

scattering from molecules adsorbed on arrays of nanoholes in

noble-metal thin films.26

To the authors’ knowledge, work aiming to demonstrate that

both sensing abilities, namely plasmonic (LSPR) and SERS

detection, can be integrated onto the same periodic array of

nanoholes, has not been reported so far. However, the combining

of LSPR sensing with SERS molecular identification can expand

the range of applications, allowing analyte identification and

improvement of the detection limits, especially for low molecular

weight molecules. Moreover, metallic films perforated with peri-

odic arrays of subwavelength nanoholes can be easily integrated

with microfluidics for portable lab-on-a-chip applications.16,27

The present work implements a synergistic LSPR–SERS

approach on short-range ordered nanoholes in a thin metal film,

inexpensively fabricated with colloidal lithography techniques.

Primarily, the LSPR bulk sensitivity of the substrate is assessed

by measuring important shifts in transmission and reflectivity as

function of the refractive index changes when the film was

immersed in different liquids. Secondly, the LSPR surface

sensitivity of the substrate is demonstrated by detecting a single

molecular layer of para-aminothiophenol (p-ATP) adsorbed

onto the film surface. Subsequently the SERS method is

employed to elucidate the structure and orientation of p-ATP on

the metal surface.

Experimental

The method for experimentally fabricating the array of nano-

holes is explained in detail elsewhere.18 Briefly, our approach

consists of four steps: (1) convective assembly of a monolayer of

polystyrene spheres of 450 nm diameter on a microscope slide;

(2) reactive ion etching of the substrate in oxygen plasma to tailor

the polystyrene spheres to the desired size (diameter 320 nm);

(3) gold film evaporation at chosen thickness (here 40 nm) onto

the previously etched substrate and (4) removal of the poly-

styrene spheres by sonication in toluene to leave behind a regular

distribution of nanoholes in the deposited Au film. Fig. 1 shows

a typical AFM image of the metallic nanohole arrays. The film in

Fig. 1 exhibits some patterning ‘‘defects’’ which were transferred

to the metallic structure from the crystallization imperfections

(gaps between crystalline domains or missing spheres) which

existed in the initial colloidal mask. Typically for a very good

quality sample such defects represent only a small percentage of

the area of the highly ordered close-packed crystal. Film thick-

ness measured by atomic force microscopy (alpha 300 AFM

This journal is ª The Royal Society of Chemistry 2009

module from Witec) was found to agree closely with thicknesses

measured during evaporation. In this study a film thickness of

40 nm, an array period of 450 nm and a hole diameter of 320 nm

were chosen in a trade-off between direct transmission efficiency,

dynamic range of our spectrometer and wavelength of laser line

available for Raman excitation. For transmission and reflection

measurements, a drop of liquid was inserted between two

microscope slides, one of them sustaining the metal film with the

array of sub-wavelength holes. The optical spectra were

measured at normal incidence and unpolarized light with

a miniature spectrometer (Ocean Optics USB4000 UV-VIS). The

spectrometer was equipped for transmission with two QP100-2-

VIS/BX optical fibers with core diameters of 100 mm (one fiber

serving for sample illumination and one for reading the trans-

mitted light) and with a QR200-7-VIS/BX reflection optical fiber

probe consisting of a tight bundle of 7 optical fibers of 200 mm

diameter (6 illumination fibers around 1 read fiber) for back

reflectivity measurement. The spectra were normalized against

a reference sample consisting of bare glass slides for transmission

and an optically thick gold film for reflectivity measurements.

For Raman measurement, the sample consisting of metallic

nanohole arrays deposited on microscope slide was first immersed

in a 10�3 M p-ATP solution in methanol for 3 h, then rinsed to

ensure one molecular layer was adsorbed on the metallic surface.

Raman spectra were obtained in backscattering geometry with

a WiTec CRM 200 Confocal Raman Microscope equipped with

a Raman Spectroscopy System UHTS 300 charge coupled device

(CCD) operating at �60 �C, using a 632.8 nm He–Ne laser as

excitation source. All measurements were conducted using

a 20� microscope objective with a numerical aperture of 0.4.

Results and discussion

Fig. 2 depicts the normalized optical transmission and reflectivity

of a perforated metallic film recorded in air (reference spectrum)

Lab Chip, 2009, 9, 3574–3579 | 3575

Fig. 2 (a) Transmission and (b) reflectivity spectra of a metallic array of nanoholes at various refractive indices: (A) 1, (B) 1.333, (C) 1.343, (D) 1.357,

(E) 1.371, (F) 1.385, (G) 1.399, (H) 1.414, (I) 1.428. The spectra were vertically translated for clarity.

Fig. 3 Dependence of the plasmon resonance wavelength on the

refractive index in transmission and reflectivity modes.

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and in liquids of different refractive indices. Water–glycerol

mixtures of varying volume ratios were used to tune into different

effective refractive indices from 1.333 (for pure water) to 1.473 (for

pure glycerol), according to the Lorentz–Lorenz formula.28 While

a flat film exhibits the well-known characteristics of semi-

transparent thin films (transparency in the green at 510 nm and

high reflectivity at longer wavelengths), the optical spectra of the

perforated film of the same thickness appear to be significantly

altered. For normal incidence investigation the structure’s dif-

fractive modes cut-off at the wavelength given by eqn (1):

lmax ¼ 2pnef/Gij (1)

where the first evanescent modes (SPP propagative modes) occur

at the metal/liquid interface.

Here

Gij ¼�

4p=ffiffiffi3p

D� ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

i2 þ j2 þ ijp

(2)

is the reciprocal vector of the grating, integers i and j are for the

SPP first-order (0, �1), nef is the effective index of water–glycerol

solution and D is the diameter (450 nm) of the polystyrene sphere

which determines the grating periodicity. The above relation

gives cut-off wavelengths (lmax) ranging from 520 to 553 nm

when the effective index varies between 1.333 and 1.428. Each of

the spectra presented in Fig. 2 exhibit maxima and minima of

interest at wavelengths at which the grating is non-diffractive,

namely the perforated film behaves like a zero-order grating and,

consequently, no diffraction orders into the far-field are possible.

Therefore we have only to analyse light that is transmitted and

back reflected into the zeroth orders.

We can attribute the strong wavelength dependent modula-

tions between 550 and 800 nm to excitation of surface plasmon

modes although it is not clear how much contribution comes

from localized (LSPR) and propagative (SPP) plasmon reso-

nances. It is important to note that the position of transmission

and reflection maxima/minima does not coincide with the reso-

nances provided by the grating equation because of coupling

between propagating SPPs and LSPR. Moreover, in a previous

study we have shown that the position and intensity of trans-

mission maxima varies with the hole diameter,18 which is also

explained by the coupling between LSPR and SPPs.

3576 | Lab Chip, 2009, 9, 3574–3579

We find a linear red-shift of the minima and maxima in

transmission and reflectivity as function of the increasing effec-

tive refractive index (see Fig. 3) which allows us to calculate the

bulk sensitivity in transmission (338 nm/RIU) and reflectivity

(301 nm/RIU). The two values are slightly different, which can be

related to different numerical apertures of optical fibres for light

collection in transmission and reflection experiments. The plas-

monic sensitivity is similar with previously reported data for

a nanohole array (333 nm/RIU) and gold nanoshells (328 nm/

RIU) but it is higher that of regular arrays of metallic nano-

particles prepared by standard nanosphere lithography (191 nm/

RIU)11 or gold nanorods (195–288 nm/RIU) and colloids

(66.5 nm/RIU) deposited on a solid substrate.29

In the next step we explore the limit of detection when

a monolayer of probe molecules (p-ATP) was absorbed onto the

film. A shift of 16 nm was measured between the transmission

maximum recorded before and after the adsorption of p-ATP

(see Fig. 4).

From the shift value and the liner dependence of bulk sensi-

tivity in Fig. 3, an effective refractive index of 1.188 for a mono-

layer of p-ATP is determined.

This journal is ª The Royal Society of Chemistry 2009

Fig. 4 Normalized transmission spectra through a metallic array of

nanoholes without (—) and with an adsorbed p-ATP monolayer (----).

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The result can be compared with the value given by a theo-

retical approach from literature for determining the effective

refractive index.30 Accordingly, the effective refractive index of

a thin molecular layer can be calculated using eqn (3):

neff ¼2

ld

ðN0

nðzÞ�expð�z=ldÞ

�2dz (3)

where

nðzÞ ¼ncap; 0 # z # dcap

nair; dcap # z\N

((4)

Here ncap is the bulk refractive index of p-ATP (ncap¼ 1.665), nair

is the refractive index of air (nair ¼ 1.000), dcap represents the

thickness of the capping molecular monolayer of p-ATP (dcap ¼0.667 nm) and ld represents the characteristic decay length (ld �5 nm) of the electromagnetic field. From the above equation we

Fig. 5 (a) Normal Raman spectrum of solid p-ATP (----) and SERS spect

(b) Simulation of the electric field |Ez| (normalized by the Fourier transform

This journal is ª The Royal Society of Chemistry 2009

found an effective refractive index of 1.155, which is close to the

experimental determination.

Although the plasmon resonance shift provides high sensi-

tivity, this sensing mechanism lacks molecular specificity. On the

contrary, SERS offers enormous molecular information about

the analyte, as the SERS signal results directly from the molec-

ular vibration when molecules are in close proximity to nano-

meter-sized metallic structures. Fig. 5(a) presents the recorded

SERS spectrum of p-ATP together with its corresponding ordi-

nary Raman spectrum collected from a bulk sample. We are

aware about the fact that when excited by laser, the small amount

of ‘‘defects’’ existing in the patterned film are able to sustain and

concentrate a much higher electric field in such localized surface

plasmon resonances and increase the Raman signal. Therefore,

in order to check the reproducibility of the recorded signal, many

SERS spectra were collected from different spots on the nano-

structured film. The recorded spectra exhibit only small varia-

tions (<10%) of the relative intensities of the bands from spot to

spot, while the spectral position and width of the Raman bands

show no noticeable differences. The result clearly demonstrates

two aspects: (a) the main Raman signal is generated and

controlled by periodic plasmonic interactions with little contri-

bution from the randomly distributed, local defects mentioned

previously, and (b) the molecular surface coverage is consistent

with a uniform monolayer of self-assembled target analyte.

Relative to the ordinary Raman spectrum, the most noticeable

differences in the SERS spectrum are the frequency shifts and

changes in relative intensity for most of the bands. The C–S

stretching vibrations mode shifts from 1093 to 1080 cm�1,

whereas the C–C stretching vibrations band shifts from 1596 to

1581 cm�1. It was shown that the enhancement of the p-ATP

Raman bands located at 1080 and 1581 cm�1 assigned to the C–S

stretching vibration has a pure electromagnetic origin.31 The

absence of the S–H stretching band at 2580 cm�1, corroborated

with the enhancement of the C–S stretching mode at 1080 cm�1,

demonstrates that the p-ATP molecules are adsorbed onto the

gold film through their sulfur atoms. There is also an enhance-

ment of the bands around 1147, 1391 and 1440 cm�1 which are

rum of p-ATP molecules adsorbed on a metallic nanohole array (—).

of the source pulse) at the metal/air interface (0xy plane) at l ¼ 633 nm.

Lab Chip, 2009, 9, 3574–3579 | 3577

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conventionally ascribed to the charge transfer from the metal to

the adsorbed molecules. It should be noted that the UV-Vis

absorption spectrum of the p-ATP molecule presents two bands

around 256 and 297 nm and thus no resonant Raman contri-

bution would be involved in the overall SERS enhancement at

633 nm excitation.

However, for a molecule adsorbed onto a metal, vibronic

mixing between the electronic and vibrational wave functions of

adsorbed molecules and those of the metal leads to electron

transfer, strongly increasing Raman scattering associated with

mixed modes by inducing an effect analogous to resonant

Raman. The apparent enhancement of the bands around

1147 cm�1 (ascribed to C–H bending) and 1391 and 1440 cm�1

(ascribed to a combination of the phenyl group C–C stretching

and NH2 rocking) together with the decrease of the intensity of

the band around 1190 cm�1 (ascribed to C–N stretching)

demonstrates that there is at least a subset of molecules which

adopt a tilted orientation relative to the metal surface. Normally,

p-ATP molecules should orient perpendicular to the metal

surface in a close-packed molecular layer, making it difficult for

a molecule to tilt toward the metal surface. However, it is

conceivable that at rims of hole cavities, the close-packing could

be interrupted, allowing molecules to lie flat on the surface and

undergo charge transfer. In order to get a better insight into the

amplification and localization of the electromagnetic field inside

and near the rims of metallic holes, numerical simulations by

employing the FDTD method were conducted, using FDTD-

Solutions� software from Lumerical Inc.32 The simulated

structure consisted of a thin gold film of 40 nm thickness,

perforated with a periodic array of cylindrical holes with diam-

eters of 320 nm sandwiched between two infinite half-spaces of

air and glass. The rectangular computational cell used had

dimensions of 152 � 262 � 224 grid points spaced at 3 nm

distance from each other. Perfectly matched layer (PML)

boundary conditions were applied on the boundaries normal to

the incident light to prevent reflections (z direction) and periodic

boundary conditions were applied in the x and y directions. A

frequency domain profile monitor at the air/metal interface was

used to record the fields. Numerical calculations in Fig. 5(b)

showed that the plasmonic electric field at the metal/air interface,

excited by laser line at 633 nm is located near the contour of

circular holes and exhibits a multipolar like configuration.

Indeed, this theoretical result implies that the majority of the

SERS signal measured from our sample is due to the excitation of

a very small percentage of the adsorbed molecules adsorbed near

the edges of the holes.

We estimate the SERS enhancement factor (EF) per adsorbed

molecule using eqn (5):33

EF ¼ ASERS

Nsurf

Nbulk

Anorm

(5)

where ASERS, Anorm, Nbulk and Nsurf are the SERS and normal

Raman areas of the n(CS) band and the number of the probe

molecules under laser illumination in the bulk sample and

adsorbed on the substrate, respectively. For the normal Raman

experiment, the probe volume was considered a focal ‘‘tube’’ with

a waist diameter of �5 mm and a depth of �19 mm. By using the

density of the bulk sample, Nbulk was calculated, yielding 2.13� 1012

molecules. In order to estimate Nsurf we considered that only the

3578 | Lab Chip, 2009, 9, 3574–3579

molecules adsorbed on the inner surface of the holes and those

adsorbed near the edge of the holes contribute to the SERS

enhancement. Given the diameter of the holes (320 nm), the

height of the holes (40 nm), the diameter of the corona (consid-

ered as 2 nm), the diameter of the spot (5 mm) and the area

occupied by each p-ATP molecule (�0.39 nm2)34 we calculated

Nsurf�1.33� 107 molecules. Thus we calculated an enhancement

factor of 2 � 104, which is in good agreement with similar results

obtained previously on regular arrays of nanoparticles.26

Future work will be focused on increasing the sensitivity of the

fabricated structures upon changes in the refractive index of the

local environment and SERS activity by optimizing the size of

the holes, the period of the hole array, as well as the thickness of

the metallic film. As regard to considering the above fabricated

substrate as a potent biosensing platform for SERS, the issue of

the reproducibility of the spectral response from the target

biomolecule can arise in some cases. There are two standard

SERS configurations that have to be addressed. First, for

detecting small biomolecules (nucleic acid bases, amino acids and

pharmaceuticals) the bio-analyte can be directly linked to the

nanostructured surface, as in the case of the p-ATP probe

molecule, and the analyze should work well.35 As for proteins,

a typical kind of biomacromolecule, most of them have three-

dimensional structures of large diameters (2–20 nm) which can

lead to different orientations and structural changes on the

surface and, consequently, to differences in selective enhance-

ments of certain molecular groups. This obstacle may be over-

come by employing analyte capture methods, e.g. using specific

molecular linkers such as antibodies and taking into account the

statistical reliability of the method. Solutions for these critical

issues are currently addressable in SERS literature36 and the

extension of this work to detection of proteins is now in progress

in our laboratory.

Conclusions

We have reported a study on the optical and spectroscopic

properties of regular arrays of subwavelength holes in a thin gold

film with the aim of exploiting the entire sensing ability of such

a substrate by combining LSPR and SERS. The binding of the

p-ATP monolayer was quantified by SPR shift measurements

and SERS was used to verify the identity and orientation of the

adsorbed molecules. Apart from LSPR sensitivity of the

substrate, which can detect the presence of a thin layer of p-ATP

molecules, the SERS has the ability to detect the p-ATP mole-

cules at much lower concentration and provide more molecular

specific information.

We have successfully demonstrated the integration of a LSPR

transducer with SERS activity on low-cost subwavelength metallic

nanohole arrays. The feasibility of such an approach can contribute

to the development of new multifunctional sensing platforms, not

only for signalling molecular binding events, but also elucidating the

orientation of molecular species at a metal surface.

Acknowledgements

This work was supported by The National University Research

Council (CNCSIS) from Romania in the frame of the PN-II

program (Project No. 477/2007 and Project TD-28/2007).

This journal is ª The Royal Society of Chemistry 2009

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