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Gold Nanostructured Surface for Electrochemical

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Article in Analytical Letters July 2015
DOI: 10.1080/00032719.2015.1043662

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Analytical Letters

ISSN: 0003-2719 (Print) 1532-236X (Online) Journal homepage: http://www.tandfonline.com/loi/lanl20

Gold Nanostructured Surface for Electrochemical

Sensing and Biosensing: Does Shape Matter?
Jana Drbohlavova, Hana Kynclov, Radim Hrd, Kateina Pikrylov, Vojtch
Svato & Jaromr Hublek
To cite this article: Jana Drbohlavova, Hana Kynclov, Radim Hrd, Kateina Pikrylov,
Vojtch Svato & Jaromr Hublek (2016) Gold Nanostructured Surface for Electrochemical
Sensing and Biosensing: Does Shape Matter?, Analytical Letters, 49:1, 135-151, DOI:
10.1080/00032719.2015.1043662
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Date: 11 April 2016, At: 23:31

ANALYTICAL LETTERS
2016, VOL. 49, NO. 1, 135151
http://dx.doi.org/10.1080/00032719.2015.1043662

ELECTROCHEMISTRY

Gold Nanostructured Surface for Electrochemical Sensing

and Biosensing: Does Shape Matter?
Jana Drbohlavovaa,b, Hana Kynclova,b, Radim Hrda,b, Kateina Pikrylova,b, Vojtch
Svatoa,b and Jaromr Hubleka,b
a

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Faculty of Electrical Engineering and Communication, Department of Microelectronics, Brno University of

Technology, Brno, Czech Republic; bCentral European Institute of Technology, Brno University of Technology,
Brno, Czech Republic
ABSTRACT

ARTICLE HISTORY

The construction of an electrochemical sensor based on a gold

working electrode modified with gold nanostructures was achieved by
galvanic deposition into an anodic alumina nanoporous template.
Two approaches were investigated to fabricate the nanostructures of
various sizes and shapes. First, gold deposition was performed
through a thin alumina template resulting in the production
of nanorods. In the second approach, alumina pores were widened
to form cup-shaped nanostructures. The detailed topography of the
nanostructured surfaces was characterized by scanning electron
microscopy. The lengths and the diameters of the nanostructures
were anticipated to have a significant influence on the surface area
and thus on the capacity to promote electron-transfer reactions.
This dependence was investigated by electrochemical impedance
spectroscopy and cyclic voltammetry. The functionalization of
electrode surface with 11-mercaptoundecanoic acid provided a basis
for further bonding with analytes. A modified Randles equivalent
circuit model with a constant phase element was used to interpret the
impedance spectra. The interfacial properties of the modified
electrodes were also evaluated by cyclic voltammetry in the presence
of the Fe(CN)63 /4 redox couple as a probe. The results showed that
the voltammetric behavior of the probe was influenced by surface
modification. The accumulation of 11-mercaptoundecanoic acid on
the nanostructured electrode surface did not result in the expected
increase in charge-transfer resistance and the decreased capacitance,
unlike the cup-shaped nanostructured electrode.

Received 20 November 2014

Accepted 12 April 2015
KEYWORDS

Electrochemistry; electrode;
gold; nanostructures;
sensing

Introduction
Among the metals, gold attracts special attention for sensing due to its excellent optical and
electrical properties and affinity to bind with biomolecules (H-Y. Li et al. 2013; Ravalli et al.
2013; Lv et al. 2014). In the formation of nanostructure arrays, gold is used to modify the
electrodes of electrochemical biosensors which enhances the active surface area and thus
the sensitivity. In addition, gold nanostructures may be functionalized with enzymes or
CONTACT Jana Drbohlavova
jana.drbohlavova@ceitec.vutbr.cz
Faculty of Electrical Engineering and Communication, Department of Microelectronics, Brno University of Technology, Technick 3058/10, 616 00 Brno, Czech Republic.
This paper is part of a special issue of papers presented at the Modern Electrochemical Methods XXXIV (in Jetichovice,
Czech Republic)/47th Heyrovsk Discussion (in Tet, Czech Republic) Conferences.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/lanl.
2016 Taylor & Francis

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J. DRBOHLAVOVA ET AL.

aptamers ensuring biosensor selectivity and specifity. These design settings may substitute
for classic amperometric sensors, which are prone to false positive signals or poisoning
under physiological conditions (Pita et al. 2013).
Instead of rather nanostructured, rough gold electrodes (Garca-Mendiola et al. 2013) or
randomly nanoporous gold surfaces (Patel et al. 2013), anodic alumina oxide templates are
an attractive approach to fabricate various nanostructures with excellent regularity and the
ability to tune their sizes and lengths (Wang, Min, and Zhu 2013; Wu, Zhang, and Du
2013). The construction using anodic alumina oxide is less complicated, less expensive, and
less time-consuming when compared to traditional lithographic approach or epitaxial growth
of highly ordered arrays of nanostructures. Taking into account biosensing applications,
it is well known that surfaces with regularly ordered distribution of nanostructures are more
useful for controllable functionalization, further conjugation, and detection of biomolecules.
The shape of gold nanostructures may include nanowires (Ramulu et al. 2013),
nanorods, and nanodots (Drbohlavova et al. 2014). For example, Cui, Adeloju, and Wu
(2014) fabricated a highly sensitive amperometric glucose biosensor by integration of
glucose oxidase with a gold nanowire array via cross-linking with a mixture of glutaraldehyde and bovine serum albumin.
Comparing electrochemical methods, electrochemical impedance spectroscopy (EIS) is
popular as a sensitive and nondestructive approach to monitor the fabrication of biosensors
(Chen et al. 2011). The technique offers several advantages over cyclic voltammetry and
chronoamperometry because fundamental effects including solution resistance, double layer
charging, and currents due to diffusion may be observed more explicitly (C.-Z. Li, Liu, and
Luong 2005). Thus, EIS may be used for probing the interfacial properties of modified
electrodes and to measure changes in the electron-transfer, resistance, and capacitance of
the biosensor in response to an applied oscillating potential as a function of frequency under
various analyte concentrations (Ding et al. 2005; Gamero et al. 2010). The charge-transfer
kinetics may be modeled from EIS measurements using the equivalent circuit approach using
Randles diagrams showing the elements corresponding to the semicircle part (the Faradaic
impedance) and the nonlinear diffusion part (the Warburg impedance).
Indeed, the interpretation of EIS spectra may be confusing. Therefore, it is useful to
combine EIS with other electrochemical methods such voltammetry to describe the
electrode in more detail. Perusal of the relevant recent literature shows there is a lack of
information related to the characterization of the relationship of gold nanostructure shape
on the electroactive surface area, and thus the sensitivity of the electrode. This paper
reports the comparison of the electrochemical behavior of five gold nanostructured
surfaces with the behavior of flat gold electrodes.

Experimental
Materials
The metallic layers were fabricated using titanium (99.99%, Porexi, Czech Republic), tungsten (99.999%, Porexi, Czech Republic), and aluminium (99.999%, Goodfellow, UK). For
electrochemical fabrication of gold nanostructures, oxalic acid (Penta, Czech Republic),
potassium dicyanoaurate (gold 68.3%, purity 99.9%, Safina, Czech Republic), boric acid
(Penta, Czech Republic), chromium trioxide (Penta, Czech Republic), monobasic

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137

potassium phosphate (Fluka, Japan), and phosphoric acid (98%, Penta, Czech Republic)
were used. The electrochemical characterization of nanostructures was performed using
sulfuric acid (96%, Penta), sodium dihydrogen phosphate dihydrate (99%, Penta,
Czech Republic), sodium hydrogen phosphate dihydrate (98%, Fluka, Germany), 11mercaptoundecanoic acid (95%, Sigma Aldrich, USA), absolute ethanol (per analysis, Penta,
Czech Republic), potassium ferricyanide (99%, Sigma Aldrich, USA), potassium ferrocyanide
(99%, Sigma Aldrich, USA), potassium chloride (99.95%, Penta, Czech Republic), and commercial conductivity standards (100 S cm 1 1%, 706 S cm 1 1%, 1413 S cm 1 1%,
Hamilton, Switzerland). Deionized water (18.2 M) was obtained from Millipore RG system
MilliQ (Millipore Corp., USA). All chemicals were used as purchased without any purification.

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Preparation of thin layers for electrode fabrication

The adhesive titanium layer with a thickness of 20 nm was sputtered on a silicon wafer
covered by SiO2 at a thickness of 500 nm to isolate the nanostructures from n-doped silicon
wafer and thus minimize junction effects at the interface between Ti and Si. The 200 nm
tungsten layer was subsequently deposited by the ion beam sputtering method. Aluminum
films of 100 nm resp. 500 nm were obtained by thermal evaporation. For comparison of the
gold nanostructures on the electrode surface, 250 nm gold layers were prepared by ion
beam sputtering.
Electrochemical fabrication of nanostructured electrodes
Self-assembled gold nanostructured surfaces were produced by pulsed galvanic deposition
of gold from dicyanoaurate and boric acid solutions into the anodic alumina nanoporous
template fabricated by electrochemical anodic oxidation of the thin aluminum layer as
described in our previous papers (Hrdy et al. 2013; Kynclova et al. 2014). The fabrication
procedure is illustrated in Figure 1. In the first step, the upper aluminum layer was
anodically oxidized in 0.3 M oxalic acid at 10C under constant voltage (either 50 or
53 V). During this process, aluminum layer was transformed to nanoporous alumina
(Al2O3) with the hexagonally ordered nanopores perpendicular to the surface. Whereas

Figure 1. Schematic of the electrochemical fabrication of the gold nanostructured electrodes.

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J. DRBOHLAVOVA ET AL.

the anodization was finished, tungsten oxide nanostructures were created on the bottom
as the result of the oxidation tungsten layer below. These WO3 nanostructures were then
etched in pH 7 phosphate buffer at 25C, which allowed the creation of a tungsten nanodimpled surface with the anodic alumina oxide above. The nanodimples strongly anchored
the gold nanostructures slightly below the ambient surface and improved their stability.
Pulsed deposition of gold into the anodic alumina oxide with the nanodimpled bottom
was done using a current of 1 mA with a 400 ms pulse length, 2 s between pulses, and
amplitude of 5 V. The temperature of the solution was maintained at 50C. The number
of pulses changed which affected the length of gold nanocolumns in the anodic alumina
template. The last step of production was the dissolution of the alumina template in the
solution composed of chromium trioxide and phosphoric acid.
The diversity in the shape of gold nanostructures was caused by changes in the thickness
of the aluminum layer, the anodization voltage, the number of pulses, and the etching of
the alumina template in phosphoric acid to enlarge the pores. Five types of nanostructured
surfaces were produced by variation of these parameters.
Characterization of surface properties
The morphology of nanostructures, homogeneity of surface coverage, and the nanostructures size were studied by scanning electron microscopy (SEM, Tescan Mira II, Tescan,
Czech Republic).
Electrochemical characterization
All electrochemical measurements of nanostructured as well as flat gold surfaces were
performed using a AUTOLAB III/FRA2 with the NOVA 1.10.3 software (Metrohm Autolab, NL). A three-electrode cell with a platinum auxiliary electrode and Ag/AgCl/3 M KCl
reference electrode (both Metrohm AG, CH) were used in all experiments. The sample
with gold nanostructures was placed into the non-commercial Teflon electrochemical cell
which defined the working electrode area as a 3 mm diameter circle (i.e., 7 mm2). Prior
to each measurement, the fabricated electrodes were electrochemically cleaned by cyclic
voltammetry in 0.1 M sulfuric acid. The electrochemical cleaning for desorption of impurities from the surface was found to be crucial for the reproducibility of the measurements.
Unfortunately, it was important to be careful to prevent damage to the electrodes. The
impedance spectroscopy (EIS) was measured at zero charge potential from 500 kHz to
0.1 Hz with an amplitude of 20 mV. The same EIS set-up, i.e., zero charge potential, was
used in the following papers (Binh and Thuy 2009; Hrdy et al. 2013). The zero charge
potential enabled the use of constant conditions for all experiments. EIS analysis of electrodes was done in conductivity standards at concentrations of 100, 706, and 1413 S cm 1.
Cyclic voltammetry (CV) was performed from 0.6 V to 0.7 V at a scan rate of 50 mVs 1.
The measurements were performed in 0.1 M potassium chloride with potassium ferricyanide and ferrocyanide at concentrations of 2.5, 5, and 7.5 mM.
Nanostructures biomodification
All nanostructured surfaces and the flat gold electrode were characterized in 5 mM phosphate buffer at pH 7 by EIS and CV. The cyclic voltammetry potential was varied from

ANALYTICAL LETTERS

139

0.2 to 0.7 V. The surfaces were modified by 11-mercaptoundecanoic acid to functionalize

the surface with thiol groups that may have applications for biosensing. The materials were
immersed in 5 mM 11-mercaptoundecanoic acid in absolute ethanol for 24 h, rinsed with
absolute ethanol to remove unadsorbed 11-mercaptoundecanoic acid, and dried in air. The
materials were then characterized by EIS and CV.
Simulation of equivalent circuits

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EIS measurements were analyzed by fitting the experimental impedance data to electrical
equivalent circuit models using NOVA 1.10.3 software (Metrohm, EcoChemie,
Switzerland).

Results and discussion

SEM characterization of gold nanostructured electrodes
The reproducibility of fabrication process of the gold nanostructured surfaces was found to
provide homogeneous surface coverage and uniform nanostructure size regardless of the
fabrication conditions. In order to achieve reproducibility, all preparation parameters
and conditions were investigated and optimized. The adhesion of the metallic films to
the Si wafer with SiO2 layer was used as the substrate. Further uniformity of metallic layer
thickness allowed the production of nanostructures of the same height in the entire
material. The presence of impurities were prevented by using high purity reagents including metallic targets and chemicals employed for the oxidation of Al and W layers and the
galvanic deposition of Au.
The gold nanostructure shape varied according to the fabrication parameters. The anodization voltage strongly affected the diameters of the nanopores in the anodic alumina
template and thus the shape of the deposited gold nanostructures: Generally, higher voltages caused wider nanopores and consequently nanopillars (see Figure 2). The length of
the nanostructures was influenced by the thickness of alumina and the number of pulses.
A higher number of deposition pulses caused higher lengths of nanostructures. After
two-pulse deposition, the nanostructures resembled nanohillocks, while using five and

Figure 2. Schematic of produced gold nanostructures using various deposition pulses and alumina
template sizes: (A) Au nanohillocks, two pulses on the alumina template with 100 nm Al; (B) nanorods,
five pulses on the alumina template with 100 nm Al; (C) nanorods, ten pulses on the alumina template
with 100 nm Al; (D) nanorods, ten pulses on the alumina template with 500 nm Al; and (E) cups, two
pulses in alumina template with 100 nm Al after pore widening.

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J. DRBOHLAVOVA ET AL.

more pulses resulted in nanorods. The increased thickness of the aluminum template
allowed the formation of longer nanostructures for the same number of pulses. An interesting nanostructure shape was achieved by etching the alumina template in 5% phosphoric acid at 50C for three minutes that involved an enlargement of the upper region
of the nanopores. After gold deposition into the template and its dissolution, cup-shaped
nanoparticles were formed. Gold deposition into the etched template was easier because
the pores were wider and hence two deposition pulses were sufficient to produce gold
nanostructures by completely filling the pores of the alumina template. The SEM results
characterizing the shape of the Au nanorods are summarized in Table 1.

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Electrochemical characterization by EIS

The electroactivities of the surface area of the gold nanostructured working electrodes
were characterized by EIS and compared with a flat gold electrode as the thin gold
sputter deposited layer. The EIS measurements revealed the dependence of the active
electrochemical surface on the nanostructure shape, size, and coverage of the electrode.
Nyquist diagrams for selected electrodes (Figures 3, 4, and 5) show typical frequency
regions.
At high frequencies (1000 Hz and higher), semicircular regions corresponded to charge
transfer resistance limiting processes associated with the electrolyte at various concentration (conductivity). The less significant difference due to conductivity was located
on the position of the interface between the diffusion and faradaic parts. The shift of the
interface to the lower real region of impedance shows the electrode had higher conductivity. In this study, the effect of the enlargement of electrode active surface was significant.
It was observed that all nanostructured electrodes had lower values of the real region of the
local maxima in comparison with the flat gold electrode as shown in Figure 6. This fact
demonstrates the influence of the electrode nanostructuring. At lower frequencies,
a diffusion region with non-linear progression and capacitive behavior was observed
for nanostructured electrodes in all cases. All nanostructured surfaces had diffusion areas
of the impedance spectra with curved shapes. The curvature in the diffusion part
of impedance reflected capacitive behavior due to the presence of the nanostructures.
The capacitive character of diffusion part was visible for higher nanostructures. In this case,
the flat gold electrode had higher impedance diffusion values in comparison with other
electrode types. This effect was caused by the lower conductivity of the flat gold electrode.
Also, the curve corresponding to diffusion for the flat gold electrode did not show any
deformation and was nearly linear.
When the electrodes were immersed in the conductivity solutions, a diffusion layer
described by the double layer capacity CDL and charge transfer resistance was created
on the electrode/liquid interface. In other words, the surface potential of electrode pulled
ions from solution and caused the formation of a diffusion layer as well as the charge transfer resistance RCT. The charge transfer resistance characterizes electrochemical properties
and the behavior of the working electrode. Local maximum were provided by the sum
of RS and RCT with minimal contributions from the imaginary region that was dependent
on the concentration of electrolyte. The values of local maxima from the Nyquist diagram
are listed in Table 2. The parameters of the EIS characterization were subsequently simulated using equivalent circuits and are discussed in the following section.

ANALYTICAL LETTERS

Table 1.

141

SEM characterization of the gold nanostructured surfaces.

Fabrication parameter and nanostructure

size

SEM top view

SEM cross-sectional view

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Al thickness: 100 nm
Anodization voltage: 53 V
Au deposition: twopulses
Height: 2030 nm
Diameter: 5060 nm

Al thickness: 100 nm
Anodization voltage: 53 V
Au deposition: 5 pulses
Height: 8090 nm
Diameter: 5565 nm

Al thickness: 100 nm
Anodization voltage: 53 V
Au deposition: ten pulses
Height: 130140 nm
Diameter: 5565 nm

Al thickness: 500 nm
Anodization voltage: 53 V
Au deposition: ten pulses
Height: 190210 nm
Diameter: 6070 nm

(Continued)

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J. DRBOHLAVOVA ET AL.

Table 1. Continued
Fabrication parameter and nanostructure
size

SEM top view

SEM cross-sectional view

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Al thickness: 100 nm
Anodization voltage: 50 V
Au deposition: two pulses
Height: 130150 nm
Diameter: 6070 nm

Figure 3. Nyquist plot of the nanostructured gold electrode prepared with five pulses on the alumina
template from 100 nm Al.

Figure 4. Nyquist plot of the nanostructured gold electrode prepared with ten pulses on the alumina
template from 500 nm Al.

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Figure 5. Nyquists plot of the nanostructured gold electrode prepared with two pulses on the alumina
template from 100 nm Al after pore widening.

Electrochemical characterization by cyclic voltammetry

An important concept involves the electroactive area, which represents the real region participating in the processes between electrode, immersed in the electrolyte, and the bulk of
solution. The electroactive area is influenced by the electrode material, roughness, and
wettability of the surface and may be increased by creating nanostructures on the electrode
surface.
Cyclic voltammetry, which is useful for the characterization of electrochemically active
surfaces of working electrodes, was performed using 2.5, 5, and 7.5 mM potassium ferroferricyanide redox couple in 0.1 M potassium chloride. The electroactive area of electrodes
may vary from the geometrically measured area due to penetration of the electrolyte in the
spaces between the gold nanostructures. A higher current from the voltammogram corresponds to a higher response. The peak height was also dependent on the electrolyte concentration. The results are accurate when the electrochemical system fulfils the
conditions of reversibility, i.e., when the reversible system involves cathodic and anodic

Figure 6. Nyquists plot of the flat gold electrode.

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Table 2.

J. DRBOHLAVOVA ET AL.

Parameters from Nyquist diagrams for nanostructured and flat gold electrodes.

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Electrode
100 nm Al, two
pulses, 53 V
Nanorod height: 2030 nm
Nanorod diameter: 5060 nm
100 nm Al, five pulses, 53 V
Nanorod height: 80 90 nm
Nanorod height: 130140 nm
Nanorod diameter: 5565 nm
100 nm Al, ten pulses, 53 V
Nanorod diameter: 5565 nm
500 nm Al, ten pulses, 53 V
Nanorod height: 190210 nm
Nanorod diameter: 6070 nm
100 nm Al, two pulses, 50 V
Cup height: 130150 nm
Cup diameter: 5060 nm
Flat Au layer

Conductivity
(S cm 1)

Frequency
(Hz)

Impedance
real part
(kX)

Absolute value of
impedance
imaginary part (X)

Impedance
(X)

100

359

47.12

252

47.12

706
1413
100
706
1413
100
706
1413
100
706
1413
100
706
1413
100
706
1413

1263
2371
491
1263
2371
491
1263
2371
359
1263
2371
262
1263
1730
673
2371
3248

10.67
3.51
74.96
15.79
6.11
48.40
16.82
6.37
48.22
9.71
3.54
73.93
10.58
5.33
75.70
13.53
5.86

74
35
297
108
63
300
114
59
247
71
35
255
60
42
512
174
89

10.67
3.51
74.96
15.79
6.11
48.40
16.82
6.37
48.22
9.71
3.54
73.94
10.58
5.34
75.70
13.53
5.86

peaks with potential distance below 0.59 V. In practice, the distance between both peaks,
especially on nanostructured electrodes, is higher.
The electroactive areas were determined according to the Randles-Sevcik equation:
ipa ipc 2; 687:105 n3=2 A:D1=2 v1=2 c

where ipa and ipc are the heights of cyclic voltammograms (A), n is the number of transferred ions (equal 1 for this case), A is the electroactive area (cm2), D is the diffusion coefficient (cm2 s 1), v is the scan rate (V s 1), and c is the concentration of the electrolyte (M
dm3). The average values of the electroactive areas are listed in Table 3.
As shown in Figures 7 and 8, the calculated electroactive area increased with the length
of nanostructures and thus with the number of deposition pulses. Surprisingly, the cupshaped nanostructured surface had an electroactive area value close to the one obtained
Table 3.

Comparison of average electroactive area of nanostructured and flat gold electrodes.

Electrode type
100 nm Al, two pulses, 53 V
Nanorod height: 2030 nm
Nanorod diameter: 5060 nm
100 nm Al, five pulses, 53 V
Nanorod height: 8090 nm
Nanorod diameter: 5565 nm
100 nm Al, ten pulses, 53 V
Nanorod height: 130140 nm
Nanorod diameter: 5565 nm
500 nm Al, ten pulses, 53 V
Nanorod height: 190210 nm
Nanorod diameter: 6070 nm
100 nm Al, two pulses, 50 V
Cup height: 130150 nm
Cups diameter: 5060 nm
Flat Au layer

Electroactive area (cm2)

1.32
1.34
1.45
1.80
1.69
1.85

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Figure 7. Cyclic voltammogram of the nanostructured gold electrode prepared with five pulses on the
alumina template from 100 nm Al.

for the highest nanostructures even though the number of pulses was only two. This
phenomenon is due to the cup-shaped nanostructures with spherical ends that allowed
the deposition of higher masses of gold material with fewer pulses. The highest electroactive area was observed for the flat gold electrode due to the larger amounts of bulk gold
forming this electrode (250 nm layer thickness) compared to the nanostructures. It is
anticipated that only a small increase in the height of nanostructures above 210 nm, i.e.,
the deposition of gold with more than ten pulses, may exceed the active surface of flat gold
electrode.
The other important parameter in these measurements is the stability of the electrode
system, or its reversibility in the presence of redox probe. The flat gold electrode acted
as a standard reversible system (data not shown). The nanostructures prepared with the
anodic alumina template from 100 nm Al layer also behaved reversibly, but the peak
distance was larger in comparison with the flat gold electrode. The curves for cup-like
nanostructures (Figure 8) and for nanostructured electrode made with ten pulses from

Figure 8. Cyclic voltammogram of the nanostructured gold electrode prepared with two pulses on the
alumina template from 100 nm Al after pore widening.

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J. DRBOHLAVOVA ET AL.

Figure 9. Nyquist plot of the nanostructured gold electrode prepared with ten pulses on the alumina
template from 500 nm Al after modification with 11-mercaptoundecanoic acid.

500 nm Al (data not shown) did not show reversible behavior. As a result, the anodic peak
in the voltammograms was much smaller and differently shaped than the cathodic peak.
Electrochemical characterization after modification by 11-mercaptoundecanoic
acid
The 11-Mercaptoundecanoic acid was used to modify the gold nanostructured surfaces and
flat electrodes by providing carboxylic groups for potential biomolecule attachment (not
performed in this work). After modification by 11-mercaptoundecanoic acid, a higher
value of the real region of the impedance and a longer diffusion region were observed
for all electrode types as shown Figures 9 and 10. The most significant increase in the
impedance was observed for nanostructured electrodes prepared with fewer than ten pulses
(data not shown). However, negligible changes in the first semicircle of Nyquist diagram
after modification by 11-mercaptoundecanoic acid were observed with the flat gold

Figure 10. Nyquist plot of the nanostructured gold electrode prepared with two pulses on the alumina
template from 100 nm Al after pore widening and modification with 11-mercaptoundecanoic acid.

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Figure 11. Cyclic voltammogram of the nanostructured gold electrode prepared with ten pulses on
the alumina template from 500 nm Al after modification with 11-mercaptoundecanoic acid.

electrode. However, a significant elongation of the diffusion region of the impedance was
observed.
Similar to previous measurements of the unmodified gold electrodes, cyclic voltammetry
was performed after electrode surface modification with 11-mercaptoundecanoic acid. The
current response in voltammograms after modification by 11-mercaptoundecanoic acid
decreased for all nanostructured electrodes compared to the response obtained in buffer
before modification. The voltammograms for selected types of working electrodes are
shown in Figures 11 and 12.
Simulation of equivalent circuits
In order to calculate the values of electrical equivalent circuits, the semicircle parts of impedance spectra were fitted by an electrochemical circle fit using the instrument software.

Figure 12. Cyclic voltammogram of the nanostructured gold electrode prepared with two pulses
on the alumina template from 100 nm Al after pore widening and modification with 11-mercaptoundecanoic acid.

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Figure 13. Equivalent electrical circuit.

This analytical method uses a simplified Randles circuit (Figure 13) involving the solution
resistance (RS) between the working electrode and the auxiliary electrode, the constant
phase element, and the charge transfer resistance (RCT). The charge transfer parameter,
the capacity of the double layer, was replaced with the constant phase element (CPE)
because of the inhomogeneity of the electrode surface caused by the presence of
nanostructures.
It is very difficult to find the same equivalent circuit for all nanostructured surfaces with
high accuracy. Therefore, interpretation using the common Randles model for an ideal
electrochemical system is inconvenient (Bisquert et al. 1998). In fact, the influence of surface modification in the form of nanostructures causes deformation of the diffusion region
of the Nyquist diagram. There may be contributions from various types of diffusion that
may include spherical, linear, and bounded. Hence, the diffusion impedance was not fitted
in this study.
The formula for calculating the imperfect constant phase element (denoted here as Q) is
given by
Q

1
jx
Q0

where Q0 is the capacitance, j is the imaginary unit, is the angular frequency, and n is a
number between 0 and 1 (H.-Y. Li et al. 2013). When n 1, Q is an ideal capacitor; when
n 0, Q is a pure resistor. When n 0.5, Q corresponds to the Warburg impedance. In
Table 4. Parameters from the simulation of equivalent circuits for nanostructured and flat gold
electrodes.
Electrode
100 nm Al, two pulses, 53 V
Nanorod height: 2030 nm
Nanorod diameter: 5060 nm
100 nm Al, five pulses, 53 V
Nanorod height: 8090 nm
Nanorod diameter: 5565 nm
100 nm Al, ten pulses, 53 V
Nanorod height: 130140 nm
Nanorod diameter: 5565 nm
500 nm Al, ten pulses, 53 V
Nanorod height: 190210 nm
Nanorod diameter: 6070 nm
100 nm Al, two pulses, 50 V
Cup height: 130150 nm
Cup diameter: 5060 nm
Flat Au layer

Conductivity (S cm 1)Solution resistance (k)Charge transfer resistance (k)Capacity (pF)

100
706
1413
100
706
1413
100
706
1413
100
706
1413
100
706
1413
100
706
1413

2.75
0.77
0.40
3.66
1.18
0.70
1.10
0.04
0.55
1.41
0.79
0.54
1.59
0.34
0.77
5.25
0.30
0.72

48.00
9.55
2.98
72.60
14.60
5.25
45.80
15.70
5.69
47.20
8.60
3.02
68.70
10.20
4.48
75.80
13.50
4.96

3.11
10.24
21.94
1.75
7.02
12.66
1.89
4.98
11.17
2.20
10.85
22.51
2.39
10.80
15.93
1.41
6.89
12.92

ANALYTICAL LETTERS

149

general, the constant phase behavior may be attributed to the distributed surface reactivity,
surface inhomogeneity, roughness or fractal geometry, electrode porosity, and to current
and potential distributions associated with electrode geometry (Jorcin et al. 2006). In order
to better evaluate electrode behavior, one can easily compare the values of the charge
transfer resistance from a simulated equivalent circuit (see Figure 13). The value of CDL
was calculated from the semicircular regions of impedance spectra (the lowest value of
imaginary region in the semicircle and relevant frequency) using Equation (2) and the
results are listed in Table 4.

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Conclusions
Five types of nanostructured gold electrodes were fabricated by electrochemical
anodization of aluminum on alumina anodic oxide template and subsequent gold pulse
deposition on nanoporous alumina. Three surfaces included nanorods of various heights,
while a fourth had nanohillocks, and the fifth were cup-shaped. The cup shape was caused
by the etching of nanopore upper parts. The electrical properties of the nanostructured
electrodes were characterized by cyclic voltammetry of a (K3[Fe(CN)6]/K4[Fe(CN)6]) probe
and the charge-transfer resistance from EIS measurements.
The EIS results showed curvature in the low frequency diffusion region of the
impedance spectra. Impedance spectra for the flat gold electrode had straight diffusion
lines. The high frequency charge transfer parts of impedance curves were used to determine
the conductivities of the electrodes. The nanostructured surfaces displayed lower values for
the real impedance which implied higher conductivity. Cyclic voltammetry was employed
to measure the current response and reversibility of the system. The most favorable results
were observed for the flat gold electrode and for the electrode with the longest nanorods
prepared under ten pulses deposited into the alumina template with 500 nm Al. The
nanostructured and flat gold electrodes were subsequently treated by the adsorption of
mercaptoundecanoic acid for characterization of the electrochemical properties before
and after surface modification by thiol groups with application to biosensor development.
As expected, modification of the gold surface by 11-mercaptoundecanoic acid molecules
caused lower cyclic voltammetric currents and an increase in the real values of the local
maxima in most impedance curves.
The interpretation of the EIS and CV results involving the design optimization
of the electrochemical cell involves some speculation, which may result in the parasitic
influence of contacts between gold working electrode and measuring cell. A partial
impediment in these measurements may be due to the complex multilayered electrode
structure. In conclusion, the most suitable shapes for the nanostructures on the electrode
surface were the cup-shaped nanorods or straight nanorods formed by ten pulses.
These surfaces provided larger electroactive areas and are promising for biosensing
applications.

Funding
The research was financially supported by the project number FEKT-S-14-2300, new types of
electronic circuits and sensors for specific applications, and project CEITEC-Central European
Institute of Technology- CZ.1.05/1.1.00/02.0068 from European Regional Development Fund.

150

J. DRBOHLAVOVA ET AL.

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