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Applied Surface Science xxx (2014) xxxxxx

Contents lists available at ScienceDirect

Applied Surface Science

journal homepage: www.elsevier.com/locate/apsusc

Fabrication and characterization of conductive anodic aluminum

oxide substrates
Sevde Altuntas a , Fatih Buyukserin b,
a
b

Micro and Nanotechnology Graduate Program, TOBB University of Economics and Technology, Ankara 06560, Turkey
Department of Biomedical Engineering, TOBB University of Economics and Technology, Ankara 06560, Turkey

a r t i c l e

i n f o

Article history:
Received 31 October 2013
Received in revised form 2 June 2014
Accepted 3 June 2014
Available online xxx
Keywords:
Biomaterials
Neural tissue engineering
Cues
Anodized aluminum oxide
Conductive surface
PVD

a b s t r a c t
Biomaterials that allow the utilization of electrical, chemical and topographic cues for improved
neuronmaterial interaction and neural regeneration hold great promise for nerve tissue engineering
applications. The nature of anodic aluminum oxide (AAO) membranes intrinsically provides delicate
control over topographic and chemical cues for enhanced cell interaction; however their use in nerve
regeneration is still very limited. Herein, we report the fabrication and characterization of conductive
AAO (CAAO) surfaces for the ultimate goal of integrating electrical cues for improved nerve tissue behavior on the nanoporous substrate material. Paralm was used as a protecting polymer lm, for the rst
time, in order to obtain large area (50 cm2 ) free-standing AAO membranes. Carbon (C) was then deposited
on the AAO surface via sputtering. Morphological characterization of the CAAO surfaces revealed that the
pores remain open after the deposition process. The presence of C on the material surface and inside
the nanopores was conrmed by XPS and EDX studies. Furthermore, IV curves of the surface were used
to extract surface resistance values and conductive AFM demonstrated that current signals can only be
achieved where conductive C layer is present. Finally, novel nanoporous C lms with controllable pore
diameters and one dimensional (1-D) C nanostructures were obtained by the dissolution of the template
AAO substrate.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Development of effective biomaterials for neural tissue engineering requires the optimization of topographic, chemical and
electrical cues that inuence the cellmaterial interactions [14].
Signicant amount of research has been conducted to utilize
combinations of these cues on a rainbow of different substrates
for increased cell adhesion, proliferation and alignment as well
as enhanced neurite outgrowth. For instance, using biomaterials with nanoscale topography that resembles the hierarchical
structure of the extracellular matrix promotes select protein
adsorption/bioactivity and cell interaction [36]. Patterned surfaces with these features create opportunities to align the neurons
which have potential to be used as neuron guidance conduits for
successful neuroregeneration. Regarding chemical cues, a variety of

Corresponding author. Tel.: +90 3122924513.

E-mail address: fbuyukserin@etu.edu.tr (F. Buyukserin).

different strategies were employed for promoting nerve regeneration. These involve the modication of substrate surface with ECM
proteins or neuroactive peptides as well as doping the substrate
with biochemicals such as drugs and growth factors [6,7]. Finally,
studies over the past few decays have demonstrated that electrical
stimulation can accelerate neural tissue regeneration. Here, bulk or
nanober-based conducting polymeric scaffolds [5,7,8] and carbon
nanotubes [9,10] are emerging as novel conductive platforms that
can improve the modulation of neuronal responses.
Nanoporous anodic aluminum oxide (AAO) membranes are a
unique class of biomaterials that can be synthesized by anodization of high purity aluminum [11,12]. Their intrinsic properties
allow one to tune several parameters for obtaining improved tissue
regeneration, and hence these biomaterials are widely used especially for bone tissue engineering applications [13,14]. For instance,
the native porous structure provides a nanoscale topography upon
which improved osteoblast adhesion and matrix formation was
attained when compared with non-porous counterpart that does
not promote osseointegration [15]. Furthermore, the pores of this

http://dx.doi.org/10.1016/j.apsusc.2014.06.007
0169-4332/ 2014 Elsevier B.V. All rights reserved.

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biocompatible material can be lled with chemicals or bioactive

materials to promote osseointegration [11,15]. Despite the ability
to manipulate such topographic and chemical cues, the research for
utilizing AAO membranes for neural tissue engineering is still very
limited and the related studies mainly focus on the role of topography in cellmaterial interaction [1618]. Hence, exploiting the
full potential of AAO as a promising substrate for neuroregeneration by utilizing the topographic, chemical and electrical cues has a
great potential for neural tissue engineering applications, however,
it currently still remains to be a challenge.
In this study, we focus on the fabrication and characterization of
conductive AAO (CAAO) substrates to eventually control nerve tissue behavior via utilizing electrical cues. We rst introduce a new
approach for producing large area free-standing AAO membranes
by the use of paralm as an AAO protecting layer. We then show that
CAAO surfaces with open nanopores can be prepared by carbon (C)
sputtering through morphological, chemical and electrical characterization of the material. Finally, we report the formation of novel
nanoporous C lms and one dimensional (1-D) C nanostructures
after the removal of the AAO templates.

Fig. 1. Schematic representation of free-standing AAO membrane production via

the application of protecting polymer layer.

membranes were obtained by dissolving the paralm in n-hexane

(SigmaAldrich). Nail polish (Plomar), Poly (methyl methacrylate) (PMMA, Aldrich, 350,000 MW) and lacquer (Polisan) were
also used as alternative protecting layers; however, best results
were obtained with paralm as discussed in the following sections.
2.2. Preparation and characterization of CAAO surfaces

2. Experimental details
2.1. Preparation of the free-standing AAO membranes
High purity Al foils (99.999%, Puratronic, 1 mm thickness, Alfa
Aesar) were sanded with 600 grit sand paper, rinsed with deionized
water (18 M, Sartorius) and annealed at 450 C for 4 h. After cooling to room temperature, an electrochemical polishing step at 15 V
was applied to the foils using a Pb cathode for 90 min at 75 C. The
electropolishing solution consisted of 95 wt% H3 PO4 (BDH Prolabo),
5 wt% H2 SO4 (Fluka) and 20 g/ml CrO3 (Fluka). These foils were then
subjected to single or two-step anodization processes depending
on the desired nal nanopore size. Nanopores with 250 nm diameter were obtained using a single step anodization where 160 V
anodization potential was applied to the Al foils in a 0.4 M H3 PO4
electrolyte at 0 C against a stainless steel cathode. The nanopores
were then widened in an aqueous chromic acid solution at room
temperature for 22 min.
In order to obtain monodisperse nanopores with 100 nm pore
diameter, the well-known two-step anodization method [19,20]
was followed. Here, the rst anodization step was carried out at
50 V in a 5 wt% aqueous oxalic acid electrolyte solution for 18 h
at 5 C. A thick non-uniform alumina (Al2 O3 ) lm that forms on
both sides of the Al foil was removed using an aqueous solution
composed of 0.4 M H3 PO4 and 0.2 M CrO3 (Fluka) at 75 C. A second anodization step was then applied at the same conditions of
the rst one for 5 min and the substrate was then immersed into a
5 vol% H3 PO4 solution to widen the nanopores to yield AAO lms
with 100 nm ordered uniform nanopores were attained. Note that
through this report, AAO structures are named as lms if they are
bound to the underlying Al, and membranes when they are liberated.
AAO lms form on both sides of the Al foil and obtaining
free-standing AAO membranes requires the removal of the Al foil
that is sandwiched between these lms (Fig. 1). This is typically
achieved by applying a protecting polymer layer on the surface
of one of the AAO lms. The unprotected AAO lm and the Al
foil were then removed in appropriate solutions and free-standing
AAO membranes were achieved after the protecting polymer layer
was dissolved in an organic solvent (Fig. 1). In our case, paralm
(Bemis) was used, for the rst time, as the protecting layer and the
unprotected AAO lm was dissolved in 1 M NaOH (BDH Prolabo)
to expose the metalic Al surface. Al was then oxidized in 0.1 M
CuCl2 2H2 O (Alfa Aesar) + 6.1 M HCl (Merck) solution, and AAO

The released AAO membranes have two distinct surfaces, the

barrier side and the solution side [21]. Nanopores with 100 or
250 nm pore diameters are located on the solution side of the
membranes. In order to create CAAO surfaces, the solution sides
of the membranes were coated with 20-nm-thick C layer via
sputtering (GATAN Presicion Etching & Coating System) unless
mentioned otherwise. The C thickness value was also veried
by independent ellipsometer studies which conrmed the thickness reading from the sputtering instrument with less than 10%
deviation (data not shown). These CAAO surfaces were then
characterized by using Scanning Electron Microscope (SEM) with
an Energy Dispersive X-ray (EDX) detector (ESEM, Quanta 200
FEG), Atomic Force Microscope (AFM, EZ-AFM, tapping mode, PPP
cantilever, Nanomagnetics), X-Ray photoelectron spectroscopy
(XPS, K-Alpha, Thermo Scientic). The electrical characterization of
the CAAO surfaces was made by measuring the surface resistance
of the structures via a DC Voltage/Current Source (Yokogawa GS
210 sourcemeter). Here, CAAO surfaces were rst fabricated by

Fig. 2. Cross-sectional drawing of the masked C or Au sputtered AAO surface used in

surface resistance calculation (a), and top view optical image of the masked structure
showing the 100 m-gap created by the mask.

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Fig. 3. Photographs showing (a) large area free-standing AAO membrane obtained
by using paralm as a protecting layer, (b) the color difference of naked AAO and
CAAO. SEM image (c) of CAAO surface that was formed by coating 20-nm-thick C on
an AAO membrane grown in H3 PO4 electrolyte.

sputtering AAO membranes with C or Au (as a control) layers. A

mask with 100 m width was then placed on the CAAO surface
and a second 50-nm-thick Au layer was sputtered (Fig. 2).
The mask was removed and ve different potentials were
applied across the top Au pads and the corresponding current values were recorded. The slope of the resultant IV curve was then
used to obtain the surface resistance value corresponding to each
coating condition. Further conrmation of surface conductivity
was characterized by conductive AFM (hp-AFM, Nanomagnetics).
In this setup, part of an AAO substrate was masked and then
the whole AAO surface was exposed to C sputtering. After the
mask was removed a voltage bias (3.5 V) was applied between
a conductive AFM tip (Pt/Ir cantilever) and the substrate. The
current between the conductive tip and the partially C coated
substrate surface was then monitored to distinguish the C coated
areas.
2.3. Liberation and characterization of nanoporous C lms and
1-D C nanostructures
C coated AAO membranes were dissolved in 1 M aqueous NaOH
solution for 4 h. Template synthesized nanoporous C lms and

Fig. 4. (a) AFM image of CAAO surface, and (b) depth prole across a line (red line
in the AFM micrograph) that depicts the open pores.(For interpretation of the references to color in this gure legend, the reader is referred to the web version of the
article.)

1-D C nanostructures were imaged by SEM after this solution

was ltered and extensively washed with water and isopropyl
alcohol. Here, a 5-nm-thick AuPd layer was sputtered prior
to imaging for improved image quality. Further conrmation
of nanoporous C lms and 1-D C nanostructures were carried out by Transmission Electron Microscopy (TEM, FEI Technai
Biotwin). TEM images were obtained after the ltered nanostructures were transferred to a minute volume of ethanol and
this concentrated dispersion was dropped on a 300-mesh TEM
grid.
3. Results and discussion
The general rationale for obtaining free-standing AAO membrane is to protect one of the two AAO faces that form on both
sides of the Al foil (Fig. 1). Popular protecting layers are polymerbased materials including lacquer [22], nail polish [23] or PMMA
[24]. Prior to developing our own methodology, we have tried
these three agents as protecting layers. Despite being successful
to a certain extent, several problems involving surface adherence
or the nal removal of the protecting layer were faced. It should
be noted that the oxide or metal removal steps, occurring in low
pH CuCl2 solutions, are extremely vigorous and proceed at elevated temperatures. These conditions result in surface adherence

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Fig. 5. XPS data from the surface of a CAAO substrate (a), and EDX and SEM results of a conductive AAO membrane cross-section obtained further from the conductive surface
(b) and from the proximity of the surface (c).

problems for the protecting layer in addition to the breaking of the

AAO membrane to small pieces. On the other hand, the difculty
observed in the nal polymer removal step should be due to extensive crosslinking of the polymer layers [25] as the temperature of
the solutions can rise well above 110 C. The application of paralm
as protecting layer proved to be useful during the rst NaOHbased unprotected AAO lm removal as well as the second acidic
Al dissolution step. After the observation of the metal removal, the
AAO-paralm assembly was washed and paralm was dissolved
in hexane to reproducibly yield 50 cm2 -area free-standing AAO
membranes (Fig. 3a).
The conventional approach for coating C on AAO is via chemical vapor deposition (CVD) [26] where carbon nano tubes (CNTs)
[27,28] or CNT membranes [29] can be successfully produced at elevated temperatures. Here, we have used physical vapor deposition
(PVD)-based sputtering as an alternative, practical and versatile
tool to create CAAO surfaces. They can be visually distinguished
from the AAO membranes after 20 nm C coating and as the material turns into a gray color compared to the white AAO (Fig. 3b).
The electron micrograph of a CAAO substrate is illustrated in
Fig. 3c which reveals that the pore diameters are relatively large
(235.28 14.72 nm) as expected from an AAO membrane prepared
in H3 PO4 electrolyte.
AFM was also used to demonstrate the topography of the CAAO
surface, and in particular, the state of the pores (Fig. 4). The dark

circular areas indicate the positions of the pores. A line across this
image was chosen to demonstrate the pore to pore distance of
300 nm which is parallel to the SEM data. More importantly, the
depth prole across this line illustrates that the pores are open
and pore depth is 90 nm. This value is much smaller than the
real pore depth (20 m) and stems from the limited ability of the
AFM tips inside the nanochannels, but nevertheless, it is apparent from Fig. 4b that the periodic pores are not blocked due to C
deposition.
The chemical characterizations of CAAO surface as well as the
nanopores are shown in Fig. 5. High resolution C 1s XPS spectrum
of the CAAO surface (Fig. 5a) illustrates the presence of a broad
peak where the maximum intensity corresponds to the binding
energy of 285 eV, and dictates the dominant presence of amorphous C on the CAAO surface [29]. In order to conrm the presence
of C inside the nanopores, a C coated AAO membrane was broken into half and the membrane cross-section was analyzed by
SEM and EDX. Two different areas, one, close to the conductive
surface, and the other, further down from this surface were investigated.
The EDX data (Fig. 5b and c) displays the abundant presence
of C near the conductive surface and comparably low C content on areas that are placed further deep into the membrane
in addition to the components of the Al2 O3 -based material. This
was expected from a C deposition by a regular sputterer for

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Table 1
The surface resistance values of C or Au coated AAO substrates.
AAO coating material

Thickness (nm)

Resistance (M)

Carbon (C)

0
15
25

195.24 2.97
624.54 10.13

Gold (Au)

0
15
25

22.77 1.10
28.65 2.05

electron microscopy use as there is no control of working pressure

nor substrate temperature that are essential in obtaining conformal coating [30]. It is also worth mentioning that, despite the
improved characteristics for sputtering, PVD methods generally
suffer from conformal coating of substrates with deep features
[30,31].
The electrical properties of the CAAO surfaces were investigated
by calculating their surface resistance values and by imaging a partially C coated AAO substrate with a conductive AFM. In order to
obtain surface resistance values, different DC voltages were applied
on masked substrates (see Section 2) and the resultant IV curves
were used to extract the resistance data. As a comparison, uncoated
and Au coated AAO substrates were also investigated (Table 1).
As expected, the naked AAO substrates were insulators, and Au
coated AAO surfaces had lower resistivity values compared with
C coated ones. In both cases, surface resistance value increased as
the coating got thicker, and similar trend was reported by other
researchers in the literature [32]. It should be noted that, the traditional use of four-point probes for surface resistance calculation
failed in our study due to the fragile nature of the AAO membrane.
In order to conrm the conductive characteristic of the CAAO
surface, part of an AAO substrate was selectively blocked from C
sputtering via the use of a mask. After the mask was removed,
a voltage bias (3.5 V) was applied between a conductive AFM tip
(Pt/Ir cantilever) and the substrate. A large area topographic view of
this selectively blocked CAAO surface as well as its current prole
is shown in Fig. 6. The tiny darker-colored dots in Fig. 6a correspond to the locations of nanopores that are 250 nm in diameter,
and they do not appear clearly in this large area AFM scan. However, when the AFM is switched to the conductive mode, a sharp
distinction between the C coated vs. non-coated area becomes
apparent (Fig. 6b). There is no current ow between the tip and
the masked, non-conductive AAO substrate where as current values up to 0.05 nA can be obtained while the tip scans the conductive
C-sputtered region. The imperfections in the masking should be
responsible for the local current signals observed in the masked
AAO area.
Complete dissolution of a CAAO substrate yields two distinct
group of structures, namely, nanoporous C lms and 1-D C nanostructures as demonstrated in Fig. 7. The diameters of the pores
in the nanoporous C lm (Fig. 7b) as well as the diameter of 1D structures (Fig. 7b and c) are on the order of 240270 nm,
which reects the starting template pore size attained by using
H3 PO4 electrolyte. Utilizing the versatile nature of template synthesis [33], it is also possible to fabricate porous C lms with more
uniform and smaller diameter nanopores. Here, AAO templates
with monodisperse nanopores having 100 nm pore diameters
were rst synthesized via the use of two-step anodization [19] in
oxalic acid electrolyte (Fig. 7d). After C coating and dissolution of
template, nanoporous C lms with ordered and smaller nanopores
(50 nm diameter) were attained (Fig. 7e, curled lm resting on the
lter material). To the best of our knowledge, the template-based

Fig. 6. AFM micrographs showing the topographic view (a) and current prole (b)
of a selectively C coated AAO surface.

formations of such structures are reported for the rst time in the
literature and potential applications involving ltration [34] and
sensing [35] can be foreseen by the control over initial template
pore size.
Careful investigation of the 1D nanostructures and comparison
between the previously published electron micrographs of template synthesized CNTs [2729] dictates that these particles are not
tubular. Note that, after C coating, the pores of the CAAO material were open. One reason behind this observation can be the
collapse of the C-coating inside the nanopores during the template dissolution process. It is also suspected that the non-uniform
conformal C coating by sputtering has a role in the observation of such structures as opposed to the theoretical formation
of CNTs upon dissolution. Note that, by ne tunning the deposition conditions involving substrate temperature and pressure,
CNT fabrication by sputtering is plausible. Systematic control over
such conditions for CNT production via sputtering and detailed
characterization of the 1-D C nanostructures is beyond the scope
of this work, and is currently under investigation for a separate
report.

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Fig. 7. Low (a) and high (b) magnication TEM images of nanoporous C lms and 1-D C nanostructures. SEM images of (c) 1-D C nanostructures after dissolving a AAO
template with large pores (d = 250 nm), (d) AAO template with 100 nm uniform nanopores prior to C deposition, and (e) a curled nanoporous C lm obtained by dissolving
a C deposited AAO template with 100 nm nanopores. Note that, the porous underlying substrate in (c) and (e) is lter membrane and it is not related with the nanoporous C
structures.

4. Conclusion
We have fabricated conductive AAO surfaces to be used as a biomaterial in nerve tissue engineering. Large area free-standing AAO
membranes were rst fabricated by a novel procedure that utilizes
paralm as a protecting lm. These substrates were then coated
with C via sputtering. The morphology of the open nanopores
after C coating was characterized by SEM and AFM studies. Presence of C on the CAAO surface and within the pores that are

close to the sputtered face was characterized by XPS and EDX

analysis. The conductivity of the CAAO surfaces was conrmed
by DC voltagecurrent reading as well as conductive AFM setups
by using masked CAAO substrates. Unique nanoporous C lms
with controllable pore dimensions and 1-D C nanostructures were
obtained by the virtue of template synthesis upon the complete
dissolution of sputtered AAO templates. The potential of AAO and
CAAO substrates for neural adherence and regeneration is currently
being investigated and promising results including cell growth

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stimulation was observed, and these will be the topic of an upcoming report.
Acknowledgment
This research was supported by The Scientic and Technological Research Council of Turkey (Tubitak), grant no: 111M686. We
would like to thank Ms. Hilal Gzler from Nanomagnetics Instruments for her assistance with AFM images.
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Please cite this article in press as: S. Altuntas, F. Buyukserin, Fabrication and characterization of conductive anodic aluminum oxide
substrates, Appl. Surf. Sci. (2014), http://dx.doi.org/10.1016/j.apsusc.2014.06.007