Nanoporous anodic aluminum oxide for chemical sensing and biosensors

Trends in Analytical Chemistry, Vol. 44, 2013
Trends
Nanoporous anodic aluminum
oxide for chemical sensing and
biosensors
Abel Santos, Tushar Kumeria, Dusan Losic
Nanoporous anodic aluminum oxide (AAO) has become one of the most popular materials with potential applications in
numerous areas, including molecular separation, catalysis, energy generation and storage, electronics, photonics, sensing, drug
delivery, and template synthesis.
The fabrication of AAO is based on simple, cost-effective, self-ordering anodization of aluminum, which yields verticallyaligned, highly-ordered nanoporous structures. Due to its unique optical and electrochemical properties, nanoporous AAO has
been extensively explored as a platform for developing inexpensive, portable sensing and biosensing devices.
This article reviews AAO-based sensing and biosensing technologies, highlighting key examples of different detection concepts
and device performance. We conclude with a perspective on the exciting opportunities for further developments in this research
field.
Crown Copyright ª 2012 Published by Elsevier Ltd. All rights reserved.
Keywords: Anodic aluminum oxide (AAO); Biosensor; Chemical sensor; Detection; Device performance; Electrochemical sensor; Nanopore;
Nanoporous alumina; Optical biosensor; Self-ordering anodization
1. Introduction
Abel Santos,
Tushar Kumeria,
Dusan Losic*
School of Chemical
Engineering, University of
Adelaide, Adelaide
SA 5005, Australia
*
Corresponding author.
Tel.: +61 8 8313 4648;
E-mail: [email protected]
au
The development of ultra-sensitive sensing
and biosensing devices using nanomaterials has received a great deal of attention
in recent years, due to their unique physical and chemical properties. In particular,
nanoporous materials prepared by selfordering synthesis based on electrochemical anodization have enormous potential
for the development of such devices [1].
This process has successfully generated
highly-ordered, vertically-aligned nanoporous and nanotubular structures with
well-defined, controllable geometry, anodic aluminum (Al) oxide (AAO) and titania nanotubes (TNTs) being two
outstanding examples [2,3]. Compared to
conventional time consuming and expensive lithographic techniques, self-ordering
anodization offers numerous advantages,
including simple, cost-competitive fabrication, controllable pore structure with
nanometric precision, structure with high
aspect ratio and fast, industrially-scalable
production [4,5]. In addition, AAO has a
unique set of chemical, optical, mechanical, transport and electrical properties,
which include chemical resistance, thermal stability, hardness, biocompatibility
and large surface area.
Nanopore structures demonstrate a
dramatic increase in surface-to-volume
ratio that enhances the signals corresponding to interaction between analyte
and surfaces, including biomolecular
reaction. The potential of nanopores to
mimic protein nanochannels in cell
membranes, which have single molecule
sensitivity and selectivity is critical toward
the development of novel bioinspired biosensors [6]. The size and the surface
chemistry of nanopores can provide
selective molecular transport and attachment, which enables the integration of
separation and sensing functions into one
device.
All these properties make AAO an
excellent platform with exciting opportunities for development of advanced, smart,
simple, cost-effective sensing devices for
numerous analytical applications. Over
the past decade, significant research efforts
have been made to explore the properties
and emerging applications of AAO,
resulting in more than 2000 publications
0165-9936/$ - see front matter Crown Copyright ª 2012 Published by Elsevier Ltd. All rights reserved. doi:http://dx.doi.org/10.1016/j.trac.2012.11.007
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Trends in Analytical Chemistry, Vol. 44, 2013
in the past five years. However, this is the first literature
review to highlight their sensing and biosensing applications. All this has led to the use of AAO for more
sophisticated and relevant applications as selective
molecular separators, chemical/biological sensing devices, catalysis, cell adhesion and culture, data storage,
energy generation and storage, drug delivery and template synthesis [3,5–7]. These applications require AAO
to have well-defined structural and chemical properties,
and levels of complexity that can be achieved through
structural engineering during fabrication and suitable
functionalization in subsequent post-treatments. The use
of AAO for sensing and biosensing is a very fast developing research area focused on different detection principles and broad applications, with potential in the near
future to translate this research into commercial devices.
Herein, we review the major advances and developments of chemical sensing and biosensing systems based
on nanoporous AAO. First, we briefly describe the
nanoporous structures of AAO prepared by self-ordered
anodization, showing their key optical and electrochemical properties, which make AAO ideal for specific
sensing applications. Second, we present the most outstanding examples of recent advances in optical and
electrochemical sensors and biosensors, reporting their
detection principles, performance and practical applications. Finally, we conclude with a prospective outlook
over the future trends in this research field and future
developments of AAO-based sensing and biosensing devices.
2. Fabrication, structure and properties of
nanoporous AAO
2.1. Fabrication and structure
Self-ordered AAO can be described as a nanoporous
alumina (Al2O3) matrix with close-packed arrays of
hexagonally-arranged cells containing a cylindrical
central pore, which grows perpendicularly to the surface
of the underlying aluminum substrate [5]. During the
anodization process, an electrochemical equilibrium between the formation of Al2O3 and its dissolution takes
place through a thin oxide-barrier layer located at the
pore bottom tip (i.e. closed pores). After anodization, this
oxide barrier layer therefore has to be removed, together
with the remaining Al substrate to obtain free-standing
AAO membranes (i.e. open pores). The pore structure of
AAO can be defined by structural parameters [e.g., pore
diameter (dp), interpore distance (dint), pore length (Lp)
and oxide-barrier layer thickness (sobl)].
Fig. 1 shows a typical AAO structure (top and crosssection). These geometric pore features can be accurately tuned by the anodization conditions in the range
10–400 nm for dp, 50–600 nm for dint, from several nm
to hundreds of lm for Lp and 30–250 nm for sobl. Other
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characteristic structural parameters of AAO are the
pore density (qp) and its porosity (P), which can be
modified between 109–1011/cm2 and 5–50%, respectively [5].
Over several decades, the influence of the anodization
conditions on the AAO-pore structures was studied
extensively in order to achieve highly-ordered pores with
controllable dimensions [2,4,5,8,9]. The anodization
voltage, electrolyte type, concentration and temperature
have been recognized as the most critical parameters to
control the self-ordering process and the geometry of the
resulting porous structures. Generally, aqueous solutions
of sulfuric acid (H2SO4) at 25 V, oxalic acid (H2C2O4) at
40 V and phosphoric acid (H3PO4) at 195 V are the most
commonly used electrolytes for preparation of AAO by
conventional anodization process, called ‘‘mild’’ anodization (MA). [4,5]. Several studies have reported on the
self-ordering mechanism of AAO in other electrolytes
(e.g., aqueous solutions of citric, maleic, malonic, tartaric and sulfamic acids), but they achieved poor pore
ordering [10].
The ground-breaking milestone in the fabrication of
AAO with highly-ordered pore structures was first reported by Masuda and Fukuda, who introduced a twostep approach to anodization [11]. In this method, after
the first anodization step, the resulting porous oxide
layer is removed to pre-structure the Al surface and
make the propagation of well-defined pores from the top
to bottom possible during the second anodization step.
The main disadvantage of this conventional MA process
is the slow pore-growth rate (i.e. 2–7 lm/h), which requires around two days to prepare well-ordered AAO
structures.
To address this problem, a new approach, so-called
‘‘hard’’ anodization (HA), was introduced by the Göseles
group. Under these conditions, the pore-growth rate is
considerably higher (50–100 lm/h) [12]. Furthermore,
this method spreads the fabrication of AAO with interpore distances not attainable by MA.
These two anodization strategies, using different
electrolytes and anodization conditions combined with
chemical etching, provide broad scope to engineer
AAO with desired pore dimensions and shapes. To
fabricate AAO with complex pore geometries, several
electrochemical approaches have been successfully
implemented, including periodic changing of anodization conditions (voltage or current) with or without
replacement of the acid electrolyte [5,13–15]. This has
enabled the generation of AAO with many different
morphologies, including funnel-type, branched pores,
periodically-shaped pore structures, and hierarchical
and multi-structured pores [14–18]. The structural
engineering of AAO pores, combined with other
modification methods, provides enormous scope to
improve the properties of AAO for specific sensing
applications.
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Figure 1. Anodic aluminum oxide (AAO) structure. (a) Pore structure in AAO and definition of the main structural parameters: pore diameter (dp), interpore distance (dint), pore length (Lp), pore
wall thickness (sW) and oxide barrier thickness (sobl). (b) SEM images showing the top and cross-section views of AAO structure.
Trends in Analytical Chemistry, Vol. 44, 2013
2.2. Properties for sensing and biosensing applications
The unique set of physical and chemical properties of
porous structures makes AAO an excellent platform to
develop sensing devices. The most characteristic optical
and electrochemical properties of AAO are photoluminescence (PL), transmittance, reflectivity, absorbance,
electron transfer, impedance, electric resistance, and
conductance, which can be used as detection principles
to fabricate highly sensitive, selective chemical and biological sensors. The miniaturization and easy integration
of AAO films into microchips with current microfabrication methods used in silicon technology is another
important feature of AAO. Nanoporous AAO can act not
only as an active sensing layer, but also as a container to
accommodate biological events inside its nanopores. It is
worth noting that these properties of AAO, relevant to
sensing applications, can be optimized by designing the
pore geometry and its surface chemistry. Numerous soft
and hard surface-modification techniques of nanoporous
AAO (e.g., molecular self-assembly, layer-by-layer
deposition, plasma polymerization, atomic layer deposition, dip coating, chemical-vapor deposition, sol-gel,
physical and electrochemical metal deposition) have
been demonstrated [19–22]. The capability of these
modifications is not only to improve or to introduce new
properties, but also to endow AAO with multifunctional
properties (e.g., optical and electrochemical activity)
provides flexibility to develop advanced sensing and
biosensing devices for multiple-analyte detection.
In terms of chemical composition, two main regions
can be distinguished in the pore structure of AAO [4].
The first region is an inner layer close to the aluminumalumina interface, which is mainly composed of pure
alumina. The second one is an outer layer located between the inner layer and the alumina-electrolyte
interface. This layer is contaminated during the anodization process by anionic species incorporated into the
alumina structure from the acid electrolyte (e.g., sulfate,
oxalate, and phosphate) [5]. The important function of
these layers is that they provide AAO with specific
optical properties (e.g., PL), which depend fundamentally
on the acid electrolyte used during the anodization
process (i.e. on the anionic species incorporated into the
AAO structure).
As stated previously, another outstanding characteristic of AAO is its capability to adjust the surface
chemistry inside the pores in a controlled manner by
surface functionalization with specific molecules. This
process provides AAO with high selectivity toward target
biomolecules (e.g., lipids, antibodies, DNA, proteins, and
enzymes). In this way, the large specific surface area of
AAO can be activated for interacting or capturing target
molecules, which can be subsequently analyzed by
optical or electrochemical techniques. These modifications can also improve the properties of AAO (e.g.,
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Table 1. Optical sensors and biosensors based on nanoporous anodic alumina oxide (AAO): detection principle, application and performance
Optical technique
Detection limit/concentration
Ref.
6
DNA
Oxazine 170
Glucose
5Æ10 M
40 lg/mL
0.1 mg/mL
100 mM
6.5Æ103 M
0.1 M
[32]
[32]
[31]
[33]
[34]
[34]
SPR-LSPR
Avidin
Anti-5-Fluorouracil
Ru[BPhen3]2+
Fe[Phen3]2+
BSA
Invertase
Melittin
10 lg/mL
100 mg/mL
2 lM
1 lM
60 nM
10 nM
100 ng/mL
[37]
[37]
[40]
[40]
[39]
[38]
[37]
SERS
p-aminothiophenol
4-mercaptopyridine
3-mercaptobenzoic
0.5 M
1Æ106 M
3 mM
[50]
[51]
[53]
RIfS
H2S
DNA
Circulating tumor cells
Immunoglobulin
0.5% v
2 nmol cm2
1000 cells/mL
0.1 mg/mL
[63]
[61]
[62]
[58]
PL
Analyte
Morin
Trypsin
reflectivity, hydrophobicity or hydrophilicity, antifouling, and accommodation and maintenance of biomolecules for specific and high-throughput assays) [23,24].
This review presents the classification and a brief
description of nanoporous AAO sensors and biosensors
on the basis of their optical and electrochemical-detection principles.
3. Optical biosensors
Due to their dimensions, geometry and chemical composition, AAO structures show characteristic responses
when interacting with light. This makes them an
attractive material to develop optically-active devices.
Many studies have demonstrated the applications of
AAO for optical filters, waveguides, anti-reflective surfaces, resonators or microcavities [25,26]. AAO has been
shown to be a particularly outstanding platform for the
development of sensing devices with an exclusive set of
optical properties including reflectance, transmittance,
absorbance, PL, chemiluminescence, and wave-guiding.
A new generation of optical sensing and biosensing devices based on the AAO recently emerged, and their
performance was successfully explored for many analytical applications, including environmental and clinical
analysis, industrial and food control, defense and
homeland security. The most representative advances in
development and applications of optical AAO sensors
and biosensors are described in the following sections
and summarized in Table 1 with their analytical performances.
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3.1. Photoluminescence (PL) spectroscopy
The PL properties of AAO were reported several decades
ago, followed by extensive studies to understand and to
control PL behavior of AAO, but its origin is still in doubt
[10,27,28]. It is generally accepted that this phenomena
is related to two types of PL field centers:
(1) F centers, which rely on the amount of carboxylate
impurities incorporated into the AAO structure
from the acid electrolyte in the course of the anodization process [60]; and,
(2) F+ centers, which are related to ionized oxygen
vacancies in the AAO structure [27,28].
Previous studies have demonstrated that PL of AAO
depends on the acid electrolyte, anodization voltage,
pore diameters, thermal treatment and anodization regime [10,29,30]. As an example, it has been observed
that AAO fabricated in oxalic acid has a higher PL
intensity than AAO produced in sulfuric or phosphoric
acids [29]. Pore widening or thermal treatment also increases the PL intensity in AAO [30]. Another advantage
of using AAO for developing PL-sensing devices is that,
unlike other materials (e.g., porous silicon), AAO has a
stable PL spectrum and it is not necessary to passivate
AAO to prevent changes over the PL spectrum over the
course of time.
The unique fingerprint of PL in AAO is clearly useful
for developing optical sensors, in which a high degree of
resolution, sensibility and biocompatibility are required
[31]. In a basic configuration, AAO nanopores can be
used as a nanocontainer to accommodate targeted
molecules and record their PL response compared with
the spectrum of unmodified AAO. It has been observed
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Figure 2. Photoluminescent (PL) optical biosensors based on anodic aluminum oxide (AAO). (a) Experimental set-up used to perform PL measurements. (b) PL spectrum. (c) Resulting barcode
generated from the PL spectrum.
Trends in Analytical Chemistry, Vol. 44, 2013
that the presence of adsorbed molecules in the AAO can
be detected by a shift in its PL spectrum. The adsorption
of large biomolecules (e.g., morin, trypsin and human
serum albumin) on AAO was studied by Jia et al.
showing significant enhancement of PL signal [32].
Feng et al. studied a more complex process of DNA
hybridization by this method using quantum dots as
biological markers [33]. In this study, AAO nanopores
were functionalized with mercapto-undecanoic acid followed by layer-by-layer deposition of positively or negatively charged dendrimers and ZnCdSe quantum dots.
A different approach was taken by Santos et al., who
developed an optical barcode system for sensing based on
the PL of AAO in the UV-visible region [34]. The origin
of these PL oscillations is the Fabry-Pérot effect, which
amplifies the PL oscillations by enhancing the PL at
wavelengths corresponding to the optical modes of the
cavity formed by the system air–AAO–Al. In this way,
the PL spectrum of AAO shows an abundance of narrow,
well-resolved oscillations, which are very useful for biosensing. It is worth mentioning that the number, the
intensity and the position of these oscillations can be
tuned by modifying the pore length and diameters (i.e.
by changing the effective medium).
Fig. 2 shows this type of PL sensor, which has been
tested with success for detecting biological substances
(e.g., organic dyes, enzymes and glucose) [31,33]. This
sensing method enables the generation of a wide range
of PL barcodes, which are suitable for developing smart
optical biosensors for a broad range of analytes.
3.2. Surface-plasmon resonance (SPR), waveguiding
spectroscopy (WS) and localized surface-plasmon resonance (LSPR)
SPR is recognized as the most popular analytical method
to probe biological interactions. SPR is capable of realtime and in-situ measurements of a wide range of surface
interactions, including ligand-binding affinity, association/dissociation kinetics, affinity constant, and highly
sensitive surface-concentration measurements [35]. SPR
and WS systems are based on a Kretschmann configuration, where the excitation of surface plasmons is generated by an evanescent electromagnetic wave produced
by the incidence of light on the surface of a prism coated
with metal. SPR-AAO biosensors are integrated into this
configuration, based on a prism on which a thin metallic
film with AAO layer is grown. The plasmonic properties
of this surface rely strongly on the refractive index of the
adjacent medium within distances of 200 nm beyond
the metal film surface. This makes it possible to use SPR
with a thin layer of AAO for detecting biological binding
events [35]. Fig. 3(a) shows a typical SPR configuration
used in SPR-AAO biosensing systems. These devices
have been successfully used to study the adsorption and
the desorption of bovine serum albumin (BSA) at different values of pH and bioaffinity interactions between
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Figure 3. Surface-plasmon resonance (SPR) and waveguiding anodic aluminum oxide (AAO) sensors. (a) SPR set-up in a Kretschmann
configuration for SPR-AAO sensors (left). Reflectance spectrum showing the different modes of the reflected light (right). (b) Waveguiding
spectroscopy (WS) based on AAO (left). SEM image of AAO/Al layer and reflection spectra measured from this layer in contact with different
concentration of bovine serum albumin (BSA) (right) (Reprinted from [40] with permission).
biotin and avidin [36,37]. More recently, a SPR-AAO
biosensor was used to measure the activity of immobilized enzyme invertase along the pores of AAO, which
demonstrates that these devices are useful not only to
perform qualitative analysis but also to quantify biological molecules or even to study biological events as
enzymatic kinetics [38].
Nanoporous AAO with a well-defined cylindrical pore
geometry and pore diameters 0.1 times smaller than the
wavelength of the incident light can confine light optical
modes. This makes AAO an attractive sensing material
for WS. Furthermore, AAO porous structures minimize
scattering losses at visible and longer wavelengths,
which is important to achieve sensitive probing of the
binding of biomolecules inside nanopores [39]. Fig. 3(b)
shows a typical configuration of a WS-AAO biosensor
and its signal response [40].
Hotta et al. demonstrated that surface plasmons of
WS-AAO biosensors can be exquisitely tuned and optimized by changing the AAO geometry via the anodiza30
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tion process or by subsequent post-treatments (e.g.,
chemical etching) [40]. Several new approaches to
designing and optimizing SW-AAO sensors for detection
of biomolecules using surface modification of AAO pores
and their replication into polymer-nanorod structures
were reported by the Knolls group [41,42].
Another configuration for developing optical biosensors is the so-called localized SPR (LSPR) or localized
plasmon resonance (LPR), where surface plasmons are
generated from metallic nanoparticles (NPs) when they
are irradiated with light [43]. The advantages of LSPR
systems compared with SPR are that this is a simpler,
inexpensive, portable system, which does not require a
Kretschmann configuration (i.e. the prism element is
not necessary). Highly ordered arrays of metallic NPs of
gold or silver (i.e. nanocaps) can be easily fabricated by
thermal deposition or other deposition and assembly
methods on the top surface of the AAO. This makes
AAO a very popular substrate for designing LSPR
devices.
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Figure 4. Local surface-plasmon resonance (SPR) anodic aluminum oxide (LSPR-AAO) biosensor with metal-film (Au)-capped AAO substrate
modified sensing biomolecules (antibody) (Reprinted from [45] with permission).
Fig. 4 shows a typical LSPR-AAO label-free biosensor
fabricated on a gold-capped AAO substrate for detection
of antigen-antibody binding [44]. The observed shift in
the LSPR signal linearly depends on the adsorbed
amount of biomolecules on the outer and inner surfaces
of the AAO porous layer. Development of simple, costcompetitive and highly-sensitive LSPR-AAO biosensors
for specific detections of proteins (e.g., BSA, avidin, DNA
and thrombin) or their binding events with bioaffinity
couples (e.g., biotin-avidin and 5-fluorouracil-anti-5fluorouracil) have been reported [36,44,45].
The combination of LSPR-AAO with an electrochemical system was also reported for sensitive detection of
toxic peptide (i.e. melittin, the venom from the honey
bees) with a limit of detection (LOD) of 10 ng/mL. This
validates the flexibility of AAO to develop multi-detection
sensing devices [46].
3.3. Surface-enhanced Raman scattering (SERS)
spectroscopy
SERS spectroscopy is based on an increase in the local
optical field, which excites the target molecules, multiplying and amplifying the radiated Raman scattered
light [47]. This effect generates a huge enhancement (i.e.
of the order of 106) of the Raman signal from molecules
adsorbed onto a metal surface with nanometric surface
roughness (i.e. so-called ‘‘hot spots’’ or ‘‘hot junctions’’).
For that reason, SERS is an extremely sensitive, espe-
cially attractive system for detecting, identifying and
quantifying trace amounts of molecules [47]. The use of
AAO for developing SERS-AAO active substrates has
received significant attention in recent years because of
its cost-competitive fabrication, reliable reproducibility,
well-defined nanostructure and applicability over large
surface areas. SERS-AAO devices are commonly fabricated by evaporating or sputtering a metallic layer of
silver or gold on the top or bottom side of an AAO
substrate. In this way, periodic metallic nm structures
with well-controlled geometry can be produced. The size
and the inter-distance of these structures can be tuned at
will by changing the geometry of AAO pores and the
conditions of the metal deposition, which enable optimization of these SERS-AAO sensors for specific sensing
applications [48].
Strong Raman signal enhancements (105–106) have
been reported in AAO substrates with gold and silver NPs
grafted onto or synthesized inside AAO nanopores and
used for biosensing applications [49,50]. Ko et al. decorated AAO with gold NPs to detect trace amounts of 2,4dinitrotoluene, which were not detectable by conventional Raman spectroscopy [49]. In another study, Lu
et al. used silver NPs to decorate AAO and the performance of the resulting SERS-AAO sensor was studied by
detecting p-aminothiophenol [50]. A similar strategy was
used by Ji et al. to detect other mercapto-based molecules
(i.e. 4-mercaptopyridine). The metal-decoration process
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was performed by Lu et al. using electrodeposition of
silver NPs along the AAO pores [51].
Other studies have shown that other metal nanostructures combined with AAO as nanowires and
nanotubes can be used to develop SERS-AAO sensors
[52,53]. Lee et al. prepared silver nanowires inside AAO
templates by electrodeposition and showed their application to SERS detection to study the adsorption of
4-aminobenzenethiol [52]. Valleman et al. applied
electroless deposition of gold inside an AAO template in
order to fabricate a metallic AAO/gold composite membrane, which was subsequently used as a SERS substrate
[53]. Furthermore, Kondo et al. used AAO templates as a
deposition mask to grow 3D multi-layered gold-AAO
cone-like arrays by vacuum evaporation deposition. The
performance of this SERS biosensor was tested in
analyzing pyridine, which is a precursor used in pharmaceutical and agrochemical applications and an
important solvent and reagent [54].
3.4. Reflectometric interference spectroscopy (RIfS)
RIfS is another highly sensitive optical sensing technique, which is based on the interaction of white light
with thin films [55]. In the past decade, nanoporous thin
films based on porous silicon have emerged as more
efficient, attractive substitutes for planar thin films used
in earlier studies to develop RIfS biosensors [56,57].
Following this success, AAO was recently considered as
an outstanding substrate, offering several advantages
(e.g., better chemical stability and more controllable and
defined pore structures) [58]. This makes RIfS devices
based on AAO more stable and reproducible than porous
silicon RIfS systems [58]. Likewise in PL, the RIfS spectrum of AAO presents fringes with well-resolved peaks
generated by the Fabry–Pérot effect [58,59]. Fig. 5
shows a RIfS set-up used to detect binding molecules in
AAO nanopores [59]. The wavelength of each peak
maxima in the RIfS spectrum follows the Fabry–Pérot
relationship (i.e. 2neffLp = mk), where neff is the effective
refractive index of AAO, Lp is the pore length and m is
the order of the RIfS fringe, the maximum of which is
located at wavelength k. These peaks in the RIfS spectrum are useful for sensing, as the binding molecules on
the surfaces inside pores can be detected through shifts
in the peak positions [56,58]. The RIfS spectrum of AAO
(i.e. number, intensity and position of the fringes) can be
tuned by modifying the AAO structures (i.e. pore length
and its diameter). This enables the generation of multiple
RIfS spectra, which are envisaged to develop accurate
sensing devices with capability for qualitative, highly
sensitive quantitative measurements within the UVvisible region.
In the past few years, RIfS-AAO sensing and biosensing technology was rapidly progressed and successfully
used for numerous sensing applications, including gases,
organic molecules, label-free biological molecules (e.g.,
DNA, proteins, antibodies, and aptamers), and study of
the binding of biomolecules (e.g., DNA and antibodyantigen) [58,60].
The pioneering work by Pan et al. showed the
capability of the RIfS-AAO for label-free detection of
complementary DNA with sensitivity to 1 nmol with a
0.5-cm2 probe area [61]. Alvarez et al. developed an
RIfS-AAO immunosensor to detect binding events
Figure 5. Schematic diagram of a reflectometric interference spectroscopy anodic aluminum oxide (RIfS-AAO) set-up for biosensing applications
showing reflection of light from the AAO structure, RIfS set-up, typical raw signal and processed interference signal.
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Table 2. Electrochemical sensors and biosensors based on nanoporous anodic alumina oxide (AAO): detection principle, application and
performance
Electrochemical technique
Analyte
Voltammetry/amperometry
Glucose Oxidase
Detection limit/concentration
Ref.
DENV-2
WNV-DIII
Glucose
Cholesterol
Na+/K+
DNA
Immunoglobulin
Thrombin
Marker CA 15-3
Formaldehyde
H2O2
100 ng/L
0.1 lM
1 pfU/mL
4 pg/mL
1 lM
0.5 mg/mL
5 mM
9.55Æ1012 M
50 ng/mL
1.8 ng/mL
52 U/mL
0.5 lM
0.1 lM
[77]
[73]
[74]
[75]
[80]
[68]
[81]
[78]
[71]
[78]
[71]
[65]
[67]
Impedance
Escherichia coli
DENV-2
1Æ102 CFU/mL
1 pfU/mL
[84]
[81]
Capacitance
PCB77
H2O
8Æ108 M
15 pf/RH%
[88]
[87]
Resistance/conductance
NO2
DNA
1 ppm
10 lM
[89]
[85]
between antibodies (i.e. rabbit anti-sheep IgG) and their
corresponding antigens (i.e. sheep IgG) [58]. These results confirmed that only selective antibody-antigen
binding events were detected by a significant change in
the optical thickness, demonstrating the capability of this
system to design selective and sensitive, label-free
immunosensors for broad applications.
A similar approach using an RIfS-AAO microchip
biosensor was demonstrated by Kumeria et al. fabricated
by modification of gold-coated AAO with biotinylated
anti EpCAM antibodies [62]. This device was used to
capture and to detect circulating tumour cells (CTCs) in
one step. This group also demonstrated gas sensing of
volatile sulfur compounds in oral malodor and hydrogen
sulfide for biomedical applications, by using RIfS-AAO
modified with a metal or other chemical-sensitive layer
with affinity to gas adsorption [63].
The combination of two techniques – RIfS and LSPR
detection using AAO – was recently demonstrated using
gold film on AAO [44]. The results obtained showed
exciting opportunities to develop smart, multi-functional
optical devices with microchip design.
4. Electrochemical sensors and biosensors
Electrochemical sensors based on AAO, depending on
their detection principles, can be divided into several
different types, including voltammetric, amperometric,
impedometric, conductometric, capacitative, and resistive. The most relevant examples of AAO electrochemical
sensors and their performances are presented in the
following sub-sections and summarized in Table 2.
4.1. Voltammetric and amperometric
Nanoporous AAO membranes are an excellent platform
for the development of membrane-type nanosensors with
amperometric or voltammetric detection, considering
their large surface area for effective immobilization of
sensing and biosensing elements inside pores, which
allows optimal interaction with analyte molecules flowing through these pores. As was commented upon previously, AAO is an electrical insulating material and its
pores have to be modified with a conventional electrode
conductive layer to turn them into a transducer able to
measure the changes in electrochemical response. To this
end, several methods using metal or metal-oxide deposition (Au, Pt, SnO2), growth of carbon nanotubes (CNTs),
conductive polymers (polypyrrole) or other electrochemically active materials (Prussian blue) were reported
[3,22,64]. These AAO-based electrochemical devices
were applied to a wide range of sensing applications,
including gas detection (vinyl chloride, ammonia, and
formaldehyde) [65], glucose [66], hydrogen peroxide
[67], cholesterol [68], DNA, nucleotides, blood proteins,
antibodies, cancer biomarkers [9,69–71], study of enzymatic activity [72], electron transfer [73] and detection of
cells (viruses, bacteria and cancer cell) [74,75]. The
electrochemical signals from AAO-modified electrodes
were derived indirectly via redox mediator or redox
reaction between the AAO substrate and the immobilized
molecules or directly by electrical-conducting linkers
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Trends in Analytical Chemistry, Vol. 44, 2013
Figure 6. Electrochemical anodic aluminum oxide (AAO) sensors. (a) AAO biosensor for electrochemical (voltammetric) sensing of DNA hybridization (Reprinted from [70] with permission). (b)
AAO protein imunobiosensor showing the blood cells outside the nanopores, and the protein molecules entering inside to bind specific antibodies generating a blockage in the diffusion of
electroactive species (left). The determination of cancer biomarker CA 16-3 in blood and buffer showing two functions of sensor, filtering (blood cells) and detection (right) (Reprinted from
[71] with permission).
Trends in Analytical Chemistry, Vol. 44, 2013
using amperometric and voltammetric methods (i.e. differential pulse voltammetry (DPV) and cyclic voltammetry (CV)).
The first AAO-nanopore probe to detect DNA hybridization by voltammetric measurements of changing
conductance through nanopores was demonstrated by
Smirnovs group [76]. The hybridization 21-mer singlestranded DNA (ssDNA) on internal pore structures
modified by covalent immobilization of ssDNA was
detected as result of the blocking effect in the diffusion of
electroactive species [Fe(CN)6]4/[Fe(CN)6]3 measured by
the decrease of the cyclic voltammetric peaks [76]. The
significant contribution in improving this voltammetric
electrochemical biosensing concept based on AAO
membranes came from Merkocis and Tohs groups, who
developed AAO immunobiosensors [6,71,74,75,77,78].
The binding of antigen inside pores was successfully
demonstrated and an limit of detection (LOD) of 100 ng/L
was reached by measuring the change of the voltammetric signal caused by impeded diffusion of the redox
probe (i.e. ferrocenemethanol) [74,75]. A similar
biosensing device prepared by modifying AAO with
5 0 -aminated DNA probe showed an ultra-sensitive LOD of
3.1 · 1013 M for quantification of single-stranded DNA
sequences [70]. Fig. 6 shows this DNA biosensor sensor.
This sensing concept was further extended to develop
ultra-sensitive nanobiosensors for the detection of viruses and pathogen bacteria (e.g., dengue type 2 mosquito
virus, West Nile viral particles and Legionella pneumophila), using specific antibodies immobilized inside AAO
pores [74,75,78]. DPV and redox probe were used to
improve sensitivity, achieving the ultra-low LOD of
1 pfU/mL (dengue type 2) and 4 pg/mL (West Nile viral)
in blood with an insignificant cross-reaction with nonspecific viruses.
This concept of AAO immunobiosensor was further
improved by the Merkocis group, by introducing gold
NPs inside nanopores in order to enhance the blockage
of the pores [71,78,79]. The reproducible detection of
target ssDNA was achieved with linear correlation in the
range 50–250 ng/mL and LOD of 42 ng/mL [78]. Furthermore, it was shown that the nanopore blockage
could be further enhanced by silver deposition to decrease the diffusion of the redox probe through the
nanopores, and increase signal amplification and biosensor sensitivity [71]. This concept was successfully
applied [Fig. 6(b)] to develop more advanced AAO
nanobiosensors with a filtration function and used for
analysis of complex samples (e.g., whole blood) [71,79].
Efficient immunoassay of thrombin, immunoglobulin
and cancer-biomarker CA 15-3 in blood samples with
LODs of 1.8 ng/mL, 50 ng/mL and 52 U/mL, respectively, was demonstrated [71,79]. This separation and
detection integrated on the same platform is particularly
interesting for analysis of composite samples for biomedical, environmental and food analysis.
Trends
The development of electrochemical-based AAO sensors by modifying pores with Prussian blue (PB), ironhexacyanoferrate polymer, have gained tremendous
attention in recent years due to the unique ion-exchange
and electrocatalytic properties of PB [64,80]. PB was
electrodeposited inside AAO membranes coated with
gold on the bottom to form arrays of PB nanoelectrodes
for amperometric measurements of glucose concentration [80]. The resulting glucose biosensor showed
excellent performance with a broad linear concentration
range over three orders of magnitude, and a low LOD of
1 lM glucose, comparable with commercial enzymebased glucose biosensors.
Furthermore, PB-AAO sensors were also successfully
utilized for ion-selective detection of Na+ and K+ ions
[81].
Wong et al. recently reported development of an advanced PB-AAO amperometric nanotube sensor for the
detection of hydrogen peroxide and glucose with a unique self-powered function [67].
4.2. Impedance spectroscopy (IS)
IS measures the dielectric properties of a material, as a
result of the interaction between the applied electric field
(frequency) and the electric dipole moment of this
material. The electrical behavior of AAO can be expressed by an equivalent circuit, the components of
which depend on the model used. If the oxide-barrier
layer of AAO is removed from the pore bottom tips, the
electrical resistance of the system is significantly reduced
and the current can flow exclusively through the pores.
In this way, AAO pores can be used not only as containers to accommodate biological events, but also as an
impedometric sensor by monitoring changes of conductance or impedance produced by binding reactions inside
AAO pores. The most important applications of impedance AAO biosensors include DNA biosensing [82],
study of protein and lipid membrane interactions [83],
and detection of cancer cells and bacteria [84].
IS using modified AAO to study DNA hybridization
was reported by Smirnov et al. [82]. First, the pore
diameter at the pore mouth of produced AAO was reduced by a hydrothermal treatment with boiling water,
followed by modification with 3-aminopropyltrimethoxysilane (APTES) and covalent attachment of complementary ssDNA inside AAO pores. DNA hybridization
was studied by IS showing an impedance increment of
50% when that hybridization process took place inside
the pores. The importance of surface-charge effect in
controlling the ionic conductance through AAO
nanopores is highlighted by the same group and applied
as a convenient detection method for unlabeled DNA
[85]. Wang et al. used a more advanced approach by
combining voltammetry and IS to characterize DNA
hybridization through AAO pores [86]. AAO pores were
functionalized in a similar way with 5 0 -aminated ss-DNA
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Trends in Analytical Chemistry, Vol. 44, 2013
and, subsequently, a complementary target bacterium
(i.e. Escherichia coli O157:H7) was detected by CV and IS,
confirming significant blockage of pores as result of this
hybridization process.
Miniaturization and integration of AAO into microchips for the development of microchip impedance sensors to detect pathogens (e.g., E. coli O157:H7 and
Staphylococcus aureus) were reported by Tan et al. [84].
The AAO membrane was functionalized with 3-glycidoxypropyl)trimethoxysilane (GPMS) and embedded in a
polydimethylsiloxane matrix. Nguyen et al. developed an
impedance-based AAO sensor for dengue-virus particles
by anodizing aluminum sputtered on platinum electrodes [81]. The impedance change was recorded in response to the binding of dengue-virus particles serotype
2 (DENV2) with its serotype 2 immunoglobulin G antibody.
4.3. Capacitive, conductometric and resistive sensors
Porous metal oxides, including AAO, are attractive
materials to develop capacitance-based gas-sensing devices because of their stability at large temperature, high
surface area and affinity to adsorb gases and vapors.
Relative humidity (RH) capacitance sensors based on
AAO have been explored by several groups, but showed
limitations caused by non-linearity of response at higher
RH levels. This problem was successfully addressed by
Juhász et al. by fabricating an AAO sensor for RH using a
CMOS-MEMS process with AAO films grown on silicon
wafers [87]. The porous structure was used as the
sensing layer, while two types of electrodes were grown
on the AAO surface (i.e. a vapor-permeable palladium
layer and a gold grid). The sensitivity of this sensor (i.e.
15 pF/RH%) was found to be much higher than that of
many commercial humidity sensors.
Jin et al. developed an AAO-based capacitance gas
sensor to detect and quantify polychlorinated biphenyls
(PCBs), which are important environmental contaminants, with an LOD of 8 · 108 M at room temperature
[88]. The behavior of capacitance in the AAO sensor was
modeled on a parallel-plate capacitor and indicated an
exponential increase of capacitance with the PCB concentration. The integration of AAO with other materials
(e.g., tungsten-trioxide layers) was introduced by Khatko
et al. to improve the sensitivity of a resistance-based
sensor for toxic gas detection (i.e. nitrogen dioxide,
ammonia and ethanol) [89]. The resistance increased
with the concentration of these gases in air with an LOD
of 1 ppm.
Apart from gas sensing, Wang et al. developed an
AAO-conductometric biosensor for the label-free detection of DNA molecules based on changes of ionic conductance of nanopores as result of DNA binding [87].
In another study, Yang et al. used a similar approach
to fabricate AAO-conductometric sensors for establishing
the activity of enzyme immobilized inside AAO pores
36
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[72]. The urease enzyme was used as a model and
immobilized onto AAO by four different strategies (i.e.
physical absorption, absorption and reticulation,
absorption and chitosan covering and reticulation and
chitosan covering) [72].
5. Miscellaneous principles for chemical sensing
and biosensing
In addition to optical and electrochemical methods, AAO
has been used as a substrate for sensing applications
based on other detection principles, of which surface
acoustic wave (SAW), matrix assisted laser desorption
ionisation (MALDI) and quartz crystal microbalance
(QCM) are the most noteworthy examples. Several
developments of SAW-AAO and QCM-AAO sensing devices for humidity, gas, organic vapor detection and
enzyme activity have been reported, demonstrating high
sensitivity and outstanding performance [90,91].
6. Conclusions
In this review, we summarized recent advances in the
application of nanoporous AAO as a platform to develop
chemical-sensing devices and biosensors. We presented
relevant examples of detection concepts and developed
devices based on optical and electrochemical detection
principles. The key features of AAO include simple,
inexpensive self-ordering fabrication, large surface area,
well-defined and controllable porous structures at the
nanometric scale, biocompatibility, easy functionalization of inner pore surfaces, and stable optical, thermal
and chemical properties. The combination of these
characteristics makes nanoporous AAO a highly
attractive material for the development of a broad range
of sensing devices for many applications. Furthermore,
AAO can be structurally engineered with complex pore
structure and chemically modified with desired functionalities, which can considerably improve its performance in multi-functional and smart sensing devices.
We expect that further progress on structural and
chemical modifications of AAO will facilitate the development of AAO-based devices with superior performances. This research field is broad and
multidisciplinary, involving several disciplines from
materials science, nanotechnology, optics, electrochemistry, cell biology and medicine.
AAO chemical sensors and biosensors have been used
in a broad range of applications from gases, vapors, organic molecules, biomolecules (DNA, proteins, antibodies) and cells (viruses, bacteria, cancer cell) in air, water
and biological environments. In general, devices based
on optical detection (SERS, SPR, LSPR or RIfS) showed
lower LODs than those based on electrochemical
Trends in Analytical Chemistry, Vol. 44, 2013
analytical systems. In particular, the development of
small optical spectrometers (i.e. mobile spectrometers)
and their cost-competitive price make them an attractive
tool to develop portable point-of-care biosensing systems.
Additional advantages of optical systems are their easy
fabrication and implementation into more complex systems (e.g., microfluidics). Worth noting is that the speed
of optical measurements is faster than that of electrochemical systems, although the choice of the biosensing
system will depend on many factors (e.g., speed of
measurements, price of equipment, analytes, measurement conditions, stability and accuracy).
Finally, we conclude that there is a bright opportunity
for further advances and developments of sensing and
biosensing devices based on AAO, especially through
further miniaturization and integration into lab-on-chip
systems. The design of implantable biosensors with the
ability to monitor biological systems in vivo and real time
is promising for the application of AAO immunosensors,
even though it is yet to be explored. Biomedical applications for point-of-care biodiagnostics and environmental detection of toxic agents are two areas of focus
for future applications.
Acknowledgments
The authors acknowledge the financial support of the
Australian Research Council (FT 110100711) and the
University of Adelaide for this work.
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