Bioelectroanalysis with nanoelectrode ensembles and arrays

TL;DR: This review deals with recent advances in bioelectroanalytical applications of nanostructured electrodes, in particular nanoelectrode ensembles (NEEs) and arrays (NEAs), and nanofabrication techniques, principles of function, and specific advantages and limits of NEEs and NEAs.

Abstract: This review deals with recent advances in bioelectroanalytical applications of nanostructured electrodes, in particular nanoelectrode ensembles (NEEs) and arrays (NEAs). First, nanofabrication techniques, principles of function, and specific advantages and limits of NEEs and NEAs are critically discussed. In the second part, some recent examples of bioelectroanalytical applications are presented. These include use of nanoelectrode arrays and/or ensembles for direct electrochemical analysis of pharmacologically active organic compounds or redox proteins, and the development of functionalized nanoelectrode systems and their use as catalytic or affinity electrochemical biosensors.

Summary

Introduction

• In the last decade there has been growing interest in the development of innovative electrochemical sensors and devices for bioanalytical purposes.
• Moreover, by separating biorecognition and transduction on the nanoscale it is possible to engineer the sensor surface so that one can protect, by use of self-assembled monolayers (SAMs) of thiols, the nanoelectrodes from undesired nonspecific adsorption yet confine biorecognition to the proximity of (but not on) the nanoelectrode [11, 12].
• In contrast with electrochemical template deposition, in the electroless method the metal layer grows from the catalytic nuclei, which are located on the pore walls, toward the centre of the pores.
• These holes can be used as recessed nanoelectrodes, and by further electrochemical deposition of gold, it is possible to fill the holes partially or totally to obtain arrays of inlaid nanodisc electrodes (Fig. 6).
• As the electrode decreases in size, the diffusion layer thickness approaches the electrode dimensions.

I t ! 1ð Þ ¼ nFAC =d t ! 1ð Þ ð2Þ

• Dividing Eq. (2) by A reveals that smaller (nano)electrodes will furnish higher 3718 M. Ongaro, P. Ugo current densities as a consequence of this enhanced mass transport.
• From perspective of diffusion, the voltammetric responses of NEEs/NEAs can vary, depending on the scan rate or the reciprocal distance among the nanoelectrodes [60–62].
• Typical values of the geometric area range from 0.008 to 0.580 cm2 [47]; this property is defined at the moment of fabrication of the NEE from the dimension of the hole punched into the insulator.
• The effect of the distance between and radius of the nanoelectrodes, and of their number (with regard to negligible border effects) has been explained above.
• NEE and NEA-based biosensors Direct detection strategies are not always feasible, especially for more complex or non-electrochemically active biomolecules.

Conclusion

• Nanoelectrode ensembles and arrays can be obtained both by “bottom-up” and “top-down” nanotechnology.
• A typical “bottom-up” method is membrane templated deposition of ensembles of nanoelectrodes in self-standing track-etched polymer membranes.
• Control of the geometry of the composite enables one to obtain functional materials with unique electroanalytical characteristics.
• Moreover, the small active area can limit the amount of biorecognition molecules which can be immobilized with the purpose of obtaining suitable biosensors.
• The possibility of moving from current NEEs/ NEAs (inwhich all nanoelectrodes are interconnected) tomore sophisticated nanoelectrode systems, in which multiple analyte determination is achieved, and the extrememiniaturization of such devices, would be particularly suitable for sensors to be used in bioanalysis, both for “in vitro” and “in vivo” analysis.

Figures

Fig. 4 Schematic representation of an NEE section, prepared by using a track-etched polycarbonate membrane as template: (a) track-etched golden membrane; (b) copper adhesive tape with conductive glue to connect to instrumentation; (c) aluminium adhesive foil with nonconductive glue; (d) insulating tape. Note: the dimensions of the pores (nanofibres) are only indicative and not to scale (reprinted, with permission, from Ref. [46])

Fig. 5 a SEM micrograph of a nanohole matrix on a PC membrane, obtained by e-beam lithography, development at 70 °C for 60 s and subsequent electrochemical gold deposition. b Top view of 75 nm radius dots in a hexagonal array on PC film; inset: higher magnification detail (reprinted, with permission, from Ref. [55])

Fig. 6 SEM images of NEAs with holes 500 nm in diameter with gold electrochemically deposited inside for 0 s (a), 10 s (b), 20 s (c), and 30 s (d). Estimated recession depths: (a) 450 nm; (b) 300 nm; (c) 150 nm; (d) 0 nm (reprinted, with permission, from Ref. [55])

Fig. 10 CVs recorded in 10−4molL−1 ferrocene methanol and 0.5 mol L−1 NaNO3. Scan rates: 5 (full line), 10 (dashed line), 20 (dotted line), and 50 mVs−1 (dash–dot line). Geometrical characteristics: nanodisc radius=75 nm, distance centre to centre=3 μm, estimated number of nanoelectrodes in the array=1.1×10−4 (reprinted, with permission, from Ref. [55])

Fig. 16 Schematic illustration of two DNA biorecognition systems The probe DNA strand is first attached to the polymer membrane; the target GOx-conjugated strand is then hybridized. The mediator reacts with the reduced enzyme and gives an electrochemical signal at the nanoelectrodes

Fig. 15 Design of DNA hybridization sensor based on NEE assembly: (a) activation of –COOH groups of the PC surface and immobilization of the capture amino-end DNA probe on to the activated carboxylic functionalities; (b) hybridization of DNA-GOx conjugate on to modified PC surface (reprinted, with permission, from Ref. [88])

Fig. 14 Schematic illustration of the HER2 detection mechanism. A specific antibody is first attached to the polycarbonate to capture the target protein (blue square). A primary antibody binds to the protein and, subsequently, a secondary antibody tethered with the enzyme label (EL)

Fig. 13 Schematic illustration of the target ssDNA detection mechanism by a so called AuNPs-NEE: (a) modification of NEE with cysteamine; (b) immobilization of gold nanoparticles; (c) functionalization with probe sequences (SHD1) and subsequent hybridization with complementary target conjugated with GOx (D2-GOx). Note: the dimensions are not to scale (reprinted, with permission, from Ref. [88])

Fig. 8 Zone diagram of cyclic voltammetric behaviour at microelectrode arrays. d is the centre-to-centre distance of individual electrodes in the array (measured in units of a), V is the dimensionless scan rate, and θ is the fraction of electrochemically active area in the array (reprinted, with permission, from Ref. [63])

Fig. 7 Simulated concentration profiles, with isoconcentration contour lines, over a microelectrode array representing the five main categories of diffusion modes (forms I to V). In the scale bar next to the figure, the red colour represents the bulk concentration and the blue colour represents zero concentration. The second scale bar represents a relative concentration scale for the contour lines. Typical CVs of the each category are shown at the right (reprinted, with permission, from Ref. [63])

Fig. 1 Schematic diagram of a nanoelectrode ensemble in a template membrane: (a) overall view; (b) lateral section

Fig. 2 Schematic representation of the cell setup. On raising the elevator the membrane lying over the sponge, soaked with the electrolyte, is pressed on the surface of the electrode (reprinted, with permission, from Ref. [25])

Fig. 11 Schematic illustration of Ru(III)/Fe(III) electrocatalysis at a DNA-modified Au NEE (reprinted, with permission, from Ref. [85])

Fig. 12 Sketch of the electrochemical MUC-16 detection method. Step 1: αMUC16 immobilized on the 3D NEE exposed wires. Step 2: MUC-16 immunoconjugated with the antibody on the surface of the nanowires. Step 3: sandwich immunocomplex with immunoliposomes. Step 4: disruption of immunoliposomes and release of the redox species, whose concentration is determined by SWv (Step 5) (reprinted, with permission, from Ref. [1])

Fig. 9 Cyclic voltammograms, recorded at different scan rates, at an NEE (geometric area 0.07 cm2; active area 0.004 cm2), 50 mmolL−1 ferrocene in [tris(n-hexyl)tetradecylphosphonium][bis(trifluoromethylsulfonyl)amide]. Scan rates: full line 5 mVs−1; dashed line 50 mVs−1; dotted line 500 mVs−1 (reprinted, with permission, from Ref. [67])

Fig. 3 Time transient current for electrochemical deposition using a track-etch membrane as templating material (reprinted, with permission, from Ref. [21])

Full text

1 23
Analytical and Bioanalytical
Chemistry
ISSN 1618-2642
Volume 405
Number 11
Anal Bioanal Chem (2013)
405:3715-3729
DOI 10.1007/s00216-012-6552-z
Bioelectroanalysis with nanoelectrode
ensembles and arrays
Michael Ongaro & Paolo Ugo

1 23
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REVIEW
Bioelectroanalysis with nanoelectrode ensembles and arrays
Michael Ongaro & Paolo Ugo
Received: 14 September 2012 /Revised: 31 October 2012 /Accepted: 5 November 2012 /Published online: 28 November 2012
#
Springer-Verlag Berlin Heidelberg 2012
Abstract This review deals with recent advances in bioelec-
troanalytical applications of nanostructured electrodes, in par-
ticular nanoelectrode ensembles (NEEs) and arrays (NEAs).
First, nanofabrication techniques, principles of function, and
specific advantages and limits of NEEs and NEAs are criti-
cally discussed. In the second part, some recent examples of
bioelectroanalytical applications are presented. These include
use of nanoelectrode arrays and/or ensembles for direct elec-
trochemical analysis of pharmacologically active organic
compounds or redox proteins, and the development of func-
tionalized nanoelectrode systems and their use as catalytic or
affinity electrochemical biosensors.
Keywords Nanoelectrode
.
Ensemble
.
Array
.
Voltammetry
.
Biosensor
.
Mediated electrochemistry
Introduction
In the last decade there has been growing interest in the devel-
opment of innovative electrochemical sensors and devices for
bioanalytical purposes. The final applications include biomed-
ical diagnostics [1–3], environmental [4] and food control [5,
6], and safety and biohazard assessment [7, 8]. In practice,
unique characteristics distinguish (bio)electrochemical sensors
from classical instrum ental meth ods, for example spectroscopy,
chromatography, and mass spectrometry; these include low
cost, mini aturizability, ease of use, no interference from
coloured or turbid samples, and applicability to raw samples
for “in situ” and decentralized. However, some problems and
limits must still be overcome. One crucial aspect is optimization
of the surface modification procedure to maximize biorecogni-
tion capabilities and reduce sensitivity to non-specific adsorp-
tion and fouling. In principle, use of sensor surfaces with
appropriate nanostructure can contribute to solving some of
these problems, for example by increasing the specific area
available for immobilization of large amounts of the biomole-
cules involved in the recognition while, at the same time,
keeping the overall size of the sensor very small [9, 10].
Moreover, by separating biorecognition and transduction on
the nanoscale it is possible to engineer the sensor surface so
that one can protect, by use of self-assembled monolayers
(SAMs) of thiols, the nanoelectrodes from undesired non-
specific adsorption yet confine biorecognition to the proximity
of (but not on) the nanoelectrode [11, 12]. Use of an array of
nanostructured electrodes also enables extreme miniaturization
of the sensor, keeping the overall size to dimensions 1–2orders
of magnitude lower than with micrometre-sized electrodes [13].
Taking this approach to the extreme, the possibility of devel-
oping multiplexed arrays is particularly attractive [14, 15]. For
instance, Zoski et al. [14] built and tested complex arrays
composed of groups of nanoelectrode ensembles, each group
being individually addressable via a separate current collector.
In the following text we will discuss some relevant exam-
ples of the state of the art of preparation of ensembles and
arrays of nanoelectrodes (NEEs and NEAs respectively), and
models and theories explaining their electrochemical behav-
iour, before discussing some significant ap plications in
bioanalysis.
Templated ensembles of nanoelectrodes
Nanoelectrode ensembles are useful electroanalytical tools
which are applied in many fields ranging from sensors to
Published in the topical collection Bioelectroanalysis with guest
editors Nicolas Plumeré, Magdalena Gebala, and Wolfgang
Schuhmann
M. Ongaro
:
P. Ugo (*)
Department of Molecular Sciences and Nanosystems,
University Ca’ Foscari of Venice, S. Marta 2137,
30123, Venice, Italy
e-mail: ugo@unive.it
Anal Bioanal Chem (2013) 405:3715–3729
DOI 10.1007/s00216-012-6552-z
Author's personal copy

electronics, from energy storage to magnetic materials [16].
The first template synthesis of NEEs for electrochemical use
was described by Menon and Martin [17] who deposited gold
nanofibres with a diameter as small as 10 nm within the pores
of track-etched polycarbonate (PC) membranes by a chemical
(electroless) method and obtained a random ensemble of metal
nanodisc electrodes surrounded by the insulating polymer. All
the nanoelectrodes were interconnected, so they all experi-
enced the same electrochemical potential. A schematic dia-
gram of the structure of an NEE is shown in Fig. 1.
Membrane-templated synthesis is based on the idea that the
pores of a host material can be used as a template to direct the
growth of new materials. Historically, template synthesis in
track-etched materials was introduced by Possin [18] and
Williams and Giordano [19], who prepared different metallic
wires with diameters as small as 10 nm within the pores of
etched nuclear damage tracks in mica. This method was
designed to image the shape of the pores rather than to obtain
a functional composite with electrochemical sensing capabil-
ities, as achieved later by Menon and Martin [17]. Avariety of
examples of membrane templated electrochemical deposition
of nanowires of semiconductors [20], metals (e.g. Ni and Co)
[21], oxides, and conducting polymers [16] have subsequently
appeared in the literature.
In the template synthesis of nanoelectrodes, each pore of
the membrane is filled with a metal nanowire or nanofibre.
Growth of the metal fibres can be achieved by use of both
electrochemical [21, 22]orelectroless[17, 23, 24]methodsof
deposition.
In both methods of deposition, the pore density of the
template determines the number of metal nanoelectrode
elements on the NEE surface and, correspondingly, the
average distance between them, whereas the diameter of
the pores in the template determines the diameter of the
individual nanoelectrodes. Track-etched membranes with
pore diameters ranging from 10 nm to 10 μm are com-
mercially available.
Template electrochemical deposition of metals
Electrochemical deposition inside the pores of a nanoporous
membrane requires that one side of the membrane be made
conductive. This can be achieved by plasma or vacuum de-
position of a thin (typically 100–200 nm) layer of metal on
one side of the membrane. The metal layer can be the same or
different from the metal which will be electrodeposited inside
the pores and the membrane should be sufficiently robust to
tolerate this kind of treatment. As an alternative, it is possible
to place the membrane directly in contact with a solid elec-
trode. Figure 2 shows the interesting cell setup recently pro-
posed by Gambirasi et al. [25], in which the membrane is
placed between a solid electrode and a sponge drenched in the
electrolyte; the pressure of the electrode on the sponge keeps
the membrane fixed tightly to the electrode for the deposition
time. In electrochemical template deposition, the coated film
is placed in an electrochemical cell, and acts as the cathode
whereas the counterelectrode is the anode.
Deposition can be performed under potentiostatic or gal-
vanostatic conditions. In the former, it is possible to monitor
the time course of the deposition and the progressive filling
of the pores by analysing the time transient current. As
Fig. 1 Schematic diagram of a nanoelectrode ensemble in a template
membrane: (a) overall view; (b) lateral section
Fig. 2 Schematic representation of the cell setup. On raising the
elevator the membrane lying over the sponge, soaked with the electro-
lyte, is pressed on the surface of the electrode (reprinted, with permis-
sion, from Ref. [25])
3716 M. Ongaro, P. Ugo
Author's personal copy

shown in Fig. 3, the deposition curve can be divided in three
parts [21, 26, 27](I–III in Fig. 3) associated with the three
steps of deposition sketched below. Immediately after clos-
ing the circuit (phase I) an intense peak then rapid decay of
the current is observed, because of depletion of metal ions
after the rapid initial deposition and the increased resistance
inside the pores of the membrane. Subsequently, the current
slowly decreases, reaching a plat eau (phase II) which corre-
sponds to progressive filling of the pores. At the beginning
of phase III, the current increases again because of the
increase of the electrode area caused by growth of the metal
outside the pores. In this phase it is possible to observe caps
on the tips of the nanowires with a typical mushroom shape
[21]. Finally, the overgrown caps merge together producing
an almost flat surface; this leads to a second plateau in the
current transient. If the objective is preparation of ensembles
of nanodisc electrodes, it is essential to stop the electrode-
position at the end of stage II, i.e. before the “mushroom
caps” start to grow.
Because the process is based on progressive growth and
filling of the pores from the bottom metallic layer toward the
open end of the pores, final products are nanowires and not
hollow structures (e.g. nanotubes).
Electrodeposition of metals has been used to obtain nano-
wires not only of gold, but also of other materials, for
example, other metals (Co [21, 28, 29]Ni[21, 26, 30]Cu
[21, 26], Pt and Pd [31]), alloys (NiFe [29], FeSiB [30]), or
salts (Bi
2
Te
3
[32], CdS [ 20]).
Template electroless deposition
Electroless deposition involves chemical reduction of a met-
al salt from solution to metal on a surface. Non-catalytic
surfaces, for example insulating polymers, must be activated
(made catalytic) before the electroless deposition. Usually,
this is achieved by g enerating metal nuclei on the surface of
the non-catalytic material. By this way, the metal ion is
preferentially reduced at the sensitized surface so that only
this surface is finally plated with the desired metal [33].
The principles of electroless deposition on nanoporous
membranes are exemplified by the Au deposition method
developed in Charles Martin’s laboratory [16 , 17] for tem-
plate fabrication of gold nanowires, nanotubes, and other
shaped gold materials. The process involved in electroless
deposition of gold can be divided in four steps:
1. “sensitization” of the membrane, during which Sn
2+
ions are adsorbed by the substrate;
2. deposition of Ag nanoparticles by reduction of an Ag
+
solution by the adsorbed Sn
2+
ions;
3. galvanic displacement of the Ag particles by reduction
of a Au(I) solution; and
4. catalytic reduction of more gold on the deposited Au
nuclei, by addition of a reducing agent (formaldehyde).
A detailed description of the gold electroless deposition
process may be found in the original papers [17, 34].
In contrast with electrochemical template deposition, in the
electroless method the metal layer grows from the catalytic
nuclei, which are located on the pore walls, toward the centre
of the pores. When step 4 is stopped after a short time (e.g. 40–
60 min at pH 10 [23]) one can obtain hollow tubes instead of
nanowires. This procedure enables the preparation of micro-
filtration membranes with gold pores [35, 36] which can be
further functionalized, for instance by use of well known thiol
chemistry [37], and have interesting applications as molecular
sieves. A sensitive detection approach based on such modified
membranes involves application of a constant potential across
the membrane and measuring the drop in the trans-membrane
current on the addition of the analyte. Detection limits as low
as 10
−11
molL
−1
have been obtained [38].
Other metals, for example Cu [39], Pd [40], and Ni–P
[41] can also be deposited in polycarbonate templates by
electroless deposition. In this case the procedure must be
suitable for the desired metal.
When the purpose of deposition is to obtain freestanding
metallic structures it is possible to completely etch the
template. Polycarbonate can be dissolved by use of organic
solvents, for example CH
2
Cl
2
–C
2
H
5
OH mixtures [9, 42], or,
as an alternative, by etching with oxygen plasma [43].
For fabrication from a metalized membrane, an easily
handled electrode system, the following procedure is typical
[9, 17, 23, 35, 44–46].
1. Remove the outer gold layer from the smooth side of the
membrane by peeling it off with adhesive tape (3 M
Magic). In this way the tips of the nanowires remain
exposed, under t he shape of an ensemble of gold
nanodiscs.
2. Attach a piece of copper adhesive tape (5 mm×60 mm)
with conductive glue (Ted Pella) on a small adhesive
Fig. 3 Time transient current for electrochemical deposition using a
track-etch membrane as templating material (reprinted, with permis-
sion, from Ref. [21])
Bioelectroanalysis with nanoelectrode arrays 3717
Author's personal copy

Frequently asked questions

Q1. What have the authors contributed in "Bioelectroanalysis with nanoelectrode ensembles and arrays" ?

This review deals with recent advances in bioelectroanalytical applications of nanostructured electrodes, in particular nanoelectrode ensembles ( NEEs ) and arrays ( NEAs ). 

Q2. What are the future works in "Bioelectroanalysis with nanoelectrode ensembles and arrays" ?

An alternative approach is the possibility of immobilizing the biorecognition layer on the insulating polymer which surrounds the nanoelectrodes, rather than on the nanoelectrodes themselves. Future research effort should be devoted to the development of singly addressable electrodes or of groups of nanoelectrodes. The possibility of moving from current NEEs/ NEAs ( inwhich all nanoelectrodes are interconnected ) tomore sophisticated nanoelectrode systems, in which multiple analyte determination is achieved, and the extrememiniaturization of such devices, would be particularly suitable for sensors to be used in bioanalysis, both for “ in vitro ” and “ in vivo ” analysis. 

Q3. What is the main idea behind the use of a templated synthesis?

Membrane-templated synthesis is based on the idea that the pores of a host material can be used as a template to direct the growth of new materials. 

Q4. How can the authors achieve growth of the metal fibres?

Growth of the metal fibres can be achieved by use of both electrochemical [21, 22] or electroless [17, 23, 24] methods of deposition. 

Q5. Why is the IF proportional to the area of the ensemble exposed to the sample?

This is because, for NEEs, operating under total overlap diffusion conditions, the Faradaic current (IF) is proportional to the total geometric area of the ensemble exposed to the sample solution (Ageom, area of the nanodiscs plus insulator area) whereas the double layer capacitive current (IC), which is the maincomponent of the noise in electroanalytical chemistry, is proportional to the nanodisc area only (active area, Aact) [17]. 

Q6. What is the advantage of NEAs for bioanalytical applications?

The improved S/N ratio typical of NEEs makes them particularly suitable for direct determination of electroactive species at low concentrations. 

Q7. Why is the NEAs used for bioanalytical applications so often recessed?

it is worth stressing that, because of the nanolithographic process itself, quite often the nanoelectrodes obtained are slightly recessed, so that theoretical model for such geometry must be taken into account [50, 55]. 

Q8. What are some recent examples of bioelectroanalytical applications of nanostructure?

These include use of nanoelectrode arrays and/or ensembles for direct electrochemical analysis of pharmacologically active organic compounds or redox proteins, and the development of functionalized nanoelectrode systems and their use as catalytic or affinity electrochemical biosensors. 

Q9. What is the process of forming a PC-based nanoelectrode?

These PC-based nanoelectrodes are fabricated by patterning arrays of holes in a thin film of PC spin-coated on a gold layer on Si–Si3N4 substrate. 

Q10. What is the simplest way to make a PC-based nanoelectrode?

These holes can be used as recessed nanoelectrodes, and by further electrochemical deposition of gold, it is possible to fill the holes partially or totally to obtain arrays of inlaid nanodisc electrodes (Fig. 6). 

Q11. What is the main idea behind the use of a templated ensemble of nanoelectrod?

The first template synthesis of NEEs for electrochemical use was described by Menon and Martin [17] who deposited gold nanofibres with a diameter as small as 10 nm within the pores of track-etched polycarbonate (PC) membranes by a chemical (electroless) method and obtained a random ensemble ofmetal nanodisc electrodes surrounded by the insulating polymer. 

Q12. How is the analyte deposited on the surface of the gold nanowires?

In this approach the analyte is adsorbed on the surface of the gold nanowires and analysed directly by SWV, resulting in an LOD as low as 8.9×10−8molL−1 (S/N=3) [84]. 

Q13. What is the way to measure the amount of biomolecules on nanowires?

Alternative designs: gold nanoparticles on NEEsUse of etched 3D NEEs to increase the amounts of biomolecules adsorbed on gold nanowire surfaces proved to be a viable process, although with the drawback of an increase of the capacitive current and, consequently, an increase of the S/N ratio [9]. 

Q14. What is the difference between NEEs and conventional electrodes?

Because the main advantage of NEEs over conventional macro (mm-sized) or even ultramicro (μm-sized) electrodes is a dramatic lowering of double-layer capacitive currents [17, 69], if it is not possible to directly characterize the morphology of the electrodes, the lack of this characteristic should be taken into account to discriminate well-prepared from defective NEEs. 

Q15. What is the drawback of etching the polymer?

One way of reducing this drawback has recently been proposed [88]—increasing the nanoelectrode area not by etching the templating polymer but depositing gold nanoparticles on the gold nanodisc electrodes. 

Q16. What is the simplest way to make a PC-based array of nanoelectrod?

As shown in Fig. 5, because the properties of PC enable its use as a high-resolution e-beam resist, it is possible to obtain a perfectly ordered array of nano-holes, of controlled diameter, as small as 50 nm [55].