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Applied Surface Science xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Applied Surface Science

jou rn al h om ep age: www.elsev ier .com/ locate /apsusc

abrication and characterization of conductive anodic aluminumxide substrates

evde Altuntasa, Fatih Buyukserinb,∗

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

r t i c l e i n f o

rticle history:eceived 31 October 2013eceived in revised form 2 June 2014ccepted 3 June 2014vailable online xxx

eywords:iomaterialseural tissue engineeringuesnodized aluminum oxide

a b s t r a c t

Biomaterials that allow the utilization of electrical, chemical and topographic cues for improvedneuron–material interaction and neural regeneration hold great promise for nerve tissue engineeringapplications. The nature of anodic aluminum oxide (AAO) membranes intrinsically provides delicatecontrol over topographic and chemical cues for enhanced cell interaction; however their use in nerveregeneration is still very limited. Herein, we report the fabrication and characterization of conductiveAAO (CAAO) surfaces for the ultimate goal of integrating electrical cues for improved nerve tissue behav-ior on the nanoporous substrate material. Parafilm was used as a protecting polymer film, for the firsttime, in order to obtain large area (50 cm2) free-standing AAO membranes. Carbon (C) was then depositedon the AAO surface via sputtering. Morphological characterization of the CAAO surfaces revealed that thepores remain open after the deposition process. The presence of C on the material surface and inside

onductive surfaceVD

the nanopores was confirmed by XPS and EDX studies. Furthermore, I–V curves of the surface were usedto extract surface resistance values and conductive AFM demonstrated that current signals can only beachieved where conductive C layer is present. Finally, novel nanoporous C films with controllable porediameters and one dimensional (1-D) C nanostructures were obtained by the dissolution of the templateAAO substrate.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Development of effective biomaterials for neural tissue engi-eering requires the optimization of topographic, chemical andlectrical cues that influence the cell–material interactions [1–4].ignificant amount of research has been conducted to utilizeombinations of these cues on a rainbow of different substratesor increased cell adhesion, proliferation and alignment as wells enhanced neurite outgrowth. For instance, using biomateri-ls with nanoscale topography that resembles the hierarchicaltructure of the extracellular matrix promotes select proteindsorption/bioactivity and cell interaction [3–6]. Patterned sur-

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aces with these features create opportunities to align the neuronshich have potential to be used as neuron guidance conduits for

uccessful neuroregeneration. Regarding chemical cues, a variety of

∗ Corresponding author. Tel.: +90 3122924513.E-mail address: fbuyukserin@etu.edu.tr (F. Buyukserin).

ttp://dx.doi.org/10.1016/j.apsusc.2014.06.007169-4332/© 2014 Elsevier B.V. All rights reserved.

different strategies were employed for promoting nerve regenera-tion. These involve the modification of substrate surface with ECMproteins or neuroactive peptides as well as doping the substratewith biochemicals such as drugs and growth factors [6,7]. Finally,studies over the past few decays have demonstrated that electricalstimulation can accelerate neural tissue regeneration. Here, bulk ornanofiber-based conducting polymeric scaffolds [5,7,8] and carbonnanotubes [9,10] are emerging as novel conductive platforms thatcan improve the modulation of neuronal responses.

Nanoporous anodic aluminum oxide (AAO) membranes are aunique class of biomaterials that can be synthesized by anodiza-tion of high purity aluminum [11,12]. Their intrinsic propertiesallow one to tune several parameters for obtaining improved tissueregeneration, and hence these biomaterials are widely used espe-cially for bone tissue engineering applications [13,14]. For instance,

tion and characterization of conductive anodic aluminum oxide014.06.007

the native porous structure provides a nanoscale topography uponwhich improved osteoblast adhesion and matrix formation wasattained when compared with non-porous counterpart that doesnot promote osseointegration [15]. Furthermore, the pores of this

IN PRESSG ModelA

2 ed Surface Science xxx (2014) xxx–xxx

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

the CAAO surfaces was made by measuring the surface resistanceof the structures via a DC Voltage/Current Source (Yokogawa GS210 sourcemeter). Here, CAAO surfaces were first fabricated by

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iocompatible material can be filled with chemicals or bioactiveaterials to promote osseointegration [11,15]. Despite the ability

o manipulate such topographic and chemical cues, the research fortilizing AAO membranes for neural tissue engineering is still very

imited and the related studies mainly focus on the role of topog-aphy in cell–material interaction [16–18]. Hence, exploiting theull potential of AAO as a promising substrate for neuroregenera-ion by utilizing the topographic, chemical and electrical cues has areat potential for neural tissue engineering applications, however,t currently still remains to be a challenge.

In this study, we focus on the fabrication and characterization ofonductive AAO (CAAO) substrates to eventually control nerve tis-ue behavior via utilizing electrical cues. We first introduce a newpproach for producing large area free-standing AAO membranesy the use of parafilm as an AAO protecting layer. We then show thatAAO surfaces with open nanopores can be prepared by carbon (C)puttering through morphological, chemical and electrical charac-erization of the material. Finally, we report the formation of novelanoporous C films and one dimensional (1-D) C nanostructuresfter the removal of the AAO templates.

. Experimental details

.1. Preparation of the free-standing AAO membranes

High purity Al foils (99.999%, Puratronic, 1 mm thickness, Alfaesar) were sanded with 600 grit sand paper, rinsed with deionizedater (18 M�, Sartorius) and annealed at 450 ◦C for 4 h. After cool-

ng to room temperature, an electrochemical polishing step at 15 Vas applied to the foils using a Pb cathode for 90 min at 75 ◦C. The

lectropolishing solution consisted of 95 wt% H3PO4 (BDH Prolabo), wt% H2SO4 (Fluka) and 20 g/ml CrO3 (Fluka). These foils were thenubjected to single or two-step anodization processes dependingn the desired final nanopore size. Nanopores with ∼250 nm diam-ter were obtained using a single step anodization where 160 Vnodization potential was applied to the Al foils in a 0.4 M H3PO4lectrolyte at 0 ◦C against a stainless steel cathode. The nanoporesere then widened in an aqueous chromic acid solution at room

emperature for 22 min.In order to obtain monodisperse nanopores with ∼100 nm pore

iameter, the well-known two-step anodization method [19,20]as followed. Here, the first anodization step was carried out at

0 V in a 5 wt% aqueous oxalic acid electrolyte solution for 18 ht 5 ◦C. A thick non-uniform alumina (Al2O3) film that forms onoth sides of the Al foil was removed using an aqueous solutionomposed of 0.4 M H3PO4 and 0.2 M CrO3 (Fluka) at 75 ◦C. A sec-nd anodization step was then applied at the same conditions ofhe first one for 5 min and the substrate was then immersed into a

vol% H3PO4 solution to widen the nanopores to yield AAO filmsith 100 nm ordered uniform nanopores were attained. Note that

hrough this report, AAO structures are named as films if they areound to the underlying Al, and membranes when they are liber-ted.

AAO films form on both sides of the Al foil and obtainingree-standing AAO membranes requires the removal of the Al foilhat is sandwiched between these films (Fig. 1). This is typicallychieved by applying a protecting polymer layer on the surfacef one of the AAO films. The unprotected AAO film and the Aloil were then removed in appropriate solutions and free-standingAO membranes were achieved after the protecting polymer layeras dissolved in an organic solvent (Fig. 1). In our case, parafilm

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Bemis) was used, for the first time, as the protecting layer and thenprotected AAO film was dissolved in 1 M NaOH (BDH Prolabo)o expose the metalic Al surface. Al was then oxidized in 0.1 MuCl2·2H2O (Alfa Aesar) + 6.1 M HCl (Merck) solution, and AAO

the application of protecting polymer layer.

membranes were obtained by dissolving the parafilm in n-hexane(Sigma–Aldrich). Nail polish (Plomar), Poly (methyl methacry-late) (PMMA, Aldrich, 350,000 MW) and lacquer (Polisan) werealso used as alternative protecting layers; however, best resultswere obtained with parafilm as discussed in the following sec-tions.

2.2. Preparation and characterization of CAAO surfaces

The released AAO membranes have two distinct surfaces, thebarrier side and the solution side [21]. Nanopores with ∼100 or250 nm pore diameters are located on the solution side of themembranes. In order to create CAAO surfaces, the solution sidesof the membranes were coated with 20-nm-thick C layer viasputtering (GATAN Presicion Etching & Coating System) unlessmentioned otherwise. The C thickness value was also verifiedby independent ellipsometer studies which confirmed the thick-ness reading from the sputtering instrument with less than 10%deviation (data not shown). These CAAO surfaces were thencharacterized by using Scanning Electron Microscope (SEM) withan Energy Dispersive X-ray (EDX) detector (ESEM, Quanta 200FEG), Atomic Force Microscope (AFM, EZ-AFM, tapping mode, PPPcantilever, Nanomagnetics), X-Ray photoelectron spectroscopy(XPS, K-Alpha, Thermo Scientific). The electrical characterization of

tion and characterization of conductive anodic aluminum oxide014.06.007

Fig. 2. Cross-sectional drawing of the masked C or Au sputtered AAO surface used insurface resistance calculation (a), and top view optical image of the masked structureshowing the 100 �m-gap created by the mask.

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Fig. 3. Photographs showing (a) large area free-standing AAO membrane obtainedby using parafilm as a protecting layer, (b) the color difference of naked AAO andCAAO. SEM image (c) of CAAO surface that was formed by coating 20-nm-thick C ona

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Fig. 4. (a) AFM image of CAAO surface, and (b) depth profile across a line (red linein the AFM micrograph) that depicts the open pores.(For interpretation of the refer-ences to color in this figure legend, the reader is referred to the web version of the

n AAO membrane grown in H3PO4 electrolyte.

puttering AAO membranes with C or Au (as a control) layers. Aask with 100 �m width was then placed on the CAAO surface

nd a second 50-nm-thick Au layer was sputtered (Fig. 2).The mask was removed and five different potentials were

pplied across the top Au pads and the corresponding current val-es were recorded. The slope of the resultant I–V curve was thensed to obtain the surface resistance value corresponding to eachoating condition. Further confirmation of surface conductivityas characterized by conductive AFM (hp-AFM, Nanomagnetics).

n this setup, part of an AAO substrate was masked and thenhe whole AAO surface was exposed to C sputtering. After the

ask was removed a voltage bias (3.5 V) was applied between conductive AFM tip (Pt/Ir cantilever) and the substrate. Theurrent between the conductive tip and the partially C coatedubstrate surface was then monitored to distinguish the C coatedreas.

.3. Liberation and characterization of nanoporous C films and

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-D C nanostructures

C coated AAO membranes were dissolved in 1 M aqueous NaOHolution for 4 h. Template synthesized nanoporous C films and

article.)

1-D C nanostructures were imaged by SEM after this solutionwas filtered and extensively washed with water and isopropylalcohol. Here, a 5-nm-thick Au–Pd layer was sputtered priorto imaging for improved image quality. Further confirmationof nanoporous C films and 1-D C nanostructures were car-ried out by Transmission Electron Microscopy (TEM, FEI TechnaiBiotwin). TEM images were obtained after the filtered nano-structures were transferred to a minute volume of ethanol andthis concentrated dispersion was dropped on a 300-mesh TEMgrid.

3. Results and discussion

The general rationale for obtaining free-standing AAO mem-brane is to protect one of the two AAO faces that form on bothsides of the Al foil (Fig. 1). Popular protecting layers are polymer-based materials including lacquer [22], nail polish [23] or PMMA[24]. Prior to developing our own methodology, we have triedthese three agents as protecting layers. Despite being successfulto a certain extent, several problems involving surface adherence

tion and characterization of conductive anodic aluminum oxide014.06.007

or the final removal of the protecting layer were faced. It shouldbe noted that the oxide or metal removal steps, occurring in lowpH CuCl2 solutions, are extremely vigorous and proceed at ele-vated 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(

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b) and from the proximity of the surface (c).

roblems for the protecting layer in addition to the breaking of theAO membrane to small pieces. On the other hand, the difficultybserved in the final polymer removal step should be due to exten-ive crosslinking of the polymer layers [25] as the temperature ofhe solutions can rise well above 110 ◦C. The application of parafilms protecting layer proved to be useful during the first NaOH-ased unprotected AAO film removal as well as the second acidicl dissolution step. After the observation of the metal removal, theAO-parafilm assembly was washed and parafilm was dissolved

n hexane to reproducibly yield ∼50 cm2-area free-standing AAOembranes (Fig. 3a).The conventional approach for coating C on AAO is via chem-

cal vapor deposition (CVD) [26] where carbon nano tubes (CNTs)27,28] or CNT membranes [29] can be successfully produced at ele-ated temperatures. Here, we have used physical vapor depositionPVD)-based sputtering as an alternative, practical and versatileool to create CAAO surfaces. They can be visually distinguishedrom the AAO membranes after ∼20 nm C coating and as the mate-ial turns into a gray color compared to the white AAO (Fig. 3b).he electron micrograph of a CAAO substrate is illustrated inig. 3c which reveals that the pore diameters are relatively large

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235.28 ± 14.72 nm) as expected from an AAO membrane preparedn H3PO4 electrolyte.

AFM was also used to demonstrate the topography of the CAAOurface, and in particular, the state of the pores (Fig. 4). The dark

circular areas indicate the positions of the pores. A line across thisimage was chosen to demonstrate the pore to pore distance of∼300 nm which is parallel to the SEM data. More importantly, thedepth profile across this line illustrates that the pores are openand pore depth is ∼90 nm. This value is much smaller than thereal pore depth (20 �m) and stems from the limited ability of theAFM tips inside the nanochannels, but nevertheless, it is appar-ent from Fig. 4b that the periodic pores are not blocked due to Cdeposition.

The chemical characterizations of CAAO surface as well as thenanopores are shown in Fig. 5. High resolution C 1s XPS spectrumof the CAAO surface (Fig. 5a) illustrates the presence of a broadpeak where the maximum intensity corresponds to the bindingenergy of 285 eV, and dictates the dominant presence of amor-phous C on the CAAO surface [29]. In order to confirm the presenceof C inside the nanopores, a C coated AAO membrane was bro-ken into half and the membrane cross-section was analyzed bySEM and EDX. Two different areas, one, close to the conductivesurface, and the other, further down from this surface were inves-tigated.

The EDX data (Fig. 5b and c) displays the abundant presence

tion and characterization of conductive anodic aluminum oxide014.06.007

of C near the conductive surface and comparably low C con-tent on areas that are placed further deep into the membranein addition to the components of the Al2O3-based material. Thiswas expected from a C deposition by a regular sputterer for

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

AAO coating material Thickness (nm) Resistance (M�)

Carbon (C) 0 ∞15 195.24 ± 2.9725 624.54 ± 10.13

Gold (Au) 0 ∞15 22.77 ± 1.1025 28.65 ± 2.05

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

lectron microscopy use as there is no control of working pressureor substrate temperature that are essential in obtaining confor-al coating [30]. It is also worth mentioning that, despite the

mproved characteristics for sputtering, PVD methods generallyuffer from conformal coating of substrates with deep features30,31].

The electrical properties of the CAAO surfaces were investigatedy calculating their surface resistance values and by imaging a par-ially C coated AAO substrate with a conductive AFM. In order tobtain surface resistance values, different DC voltages were appliedn masked substrates (see Section 2) and the resultant I–V curvesere used to extract the resistance data. As a comparison, uncoated

nd Au coated AAO substrates were also investigated (Table 1).s expected, the naked AAO substrates were insulators, and Auoated AAO surfaces had lower resistivity values compared with

coated ones. In both cases, surface resistance value increased ashe coating got thicker, and similar trend was reported by otheresearchers in the literature [32]. It should be noted that, the tradi-ional use of four-point probes for surface resistance calculationailed in our study due to the fragile nature of the AAO mem-rane.

In order to confirm the conductive characteristic of the CAAOurface, part of an AAO substrate was selectively blocked from Cputtering via the use of a mask. After the mask was removed,

voltage bias (3.5 V) was applied between a conductive AFM tipPt/Ir cantilever) and the substrate. A large area topographic view ofhis selectively blocked CAAO surface as well as its current profiles shown in Fig. 6. The tiny darker-colored dots in Fig. 6a corre-pond to the locations of nanopores that are ∼250 nm in diameter,nd they do not appear clearly in this large area AFM scan. How-ver, when the AFM is switched to the conductive mode, a sharpistinction between the C coated vs. non-coated area becomespparent (Fig. 6b). There is no current flow between the tip andhe masked, non-conductive AAO substrate where as current val-es up to 0.05 nA can be obtained while the tip scans the conductive-sputtered region. The imperfections in the masking should beesponsible for the local current signals observed in the maskedAO area.

Complete dissolution of a CAAO substrate yields two distinctroup of structures, namely, nanoporous C films and 1-D C nano-tructures as demonstrated in Fig. 7. The diameters of the poresn the nanoporous C film (Fig. 7b) as well as the diameter of 1-

structures (Fig. 7b and c) are on the order of ∼240–270 nm,hich reflects the starting template pore size attained by using3PO4 electrolyte. Utilizing the versatile nature of template syn-

hesis [33], it is also possible to fabricate porous C films with moreniform and smaller diameter nanopores. Here, AAO templatesith monodisperse nanopores having ∼100 nm pore diametersere first synthesized via the use of two-step anodization [19] in

xalic acid electrolyte (Fig. 7d). After C coating and dissolution of

Please cite this article in press as: S. Altuntas, F. Buyukserin, Fabricasubstrates, Appl. Surf. Sci. (2014), http://dx.doi.org/10.1016/j.apsusc.2

emplate, nanoporous C films with ordered and smaller nanopores∼50 nm diameter) were attained (Fig. 7e, curled film resting on thelter material). To the best of our knowledge, the template-based

formations of such structures are reported for the first time in theliterature and potential applications involving filtration [34] andsensing [35] can be foreseen by the control over initial templatepore size.

Careful investigation of the 1D nanostructures and comparisonbetween the previously published electron micrographs of tem-plate synthesized CNTs [27–29] dictates that these particles are nottubular. Note that, after C coating, the pores of the CAAO mate-rial were open. One reason behind this observation can be thecollapse of the C-coating inside the nanopores during the tem-plate dissolution process. It is also suspected that the non-uniformconformal C coating by sputtering has a role in the observa-tion of such structures as opposed to the theoretical formationof CNTs upon dissolution. Note that, by fine tunning the depo-sition conditions involving substrate temperature and pressure,CNT fabrication by sputtering is plausible. Systematic control oversuch conditions for CNT production via sputtering and detailedcharacterization of the 1-D C nanostructures is beyond the scopeof this work, and is currently under investigation for a separate

tion and characterization of conductive anodic aluminum oxide014.06.007

report.

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Fig. 7. Low (a) and high (b) magnification TEM images of nanoporous C films and 1-D C nanostructures. SEM images of (c) 1-D C nanostructures after dissolving a AAOt nopoa ing sus

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emplate with large pores (d = ∼250 nm), (d) AAO template with 100 nm uniform na C deposited AAO template with 100 nm nanopores. Note that, the porous underlytructures.

. Conclusion

We have fabricated conductive AAO surfaces to be used as a bio-aterial in nerve tissue engineering. Large area free-standing AAOembranes were first fabricated by a novel procedure that utilizes

Please cite this article in press as: S. Altuntas, F. Buyukserin, Fabricasubstrates, Appl. Surf. Sci. (2014), http://dx.doi.org/10.1016/j.apsusc.2

arafilm as a protecting film. These substrates were then coatedith C via sputtering. The morphology of the open nanopores

fter C coating was characterized by SEM and AFM studies. Pres-nce of C on the CAAO surface and within the pores that are

res prior to C deposition, and (e) a curled nanoporous C film obtained by dissolvingbstrate in (c) and (e) is filter membrane and it is not related with the nanoporous C

close to the sputtered face was characterized by XPS and EDXanalysis. The conductivity of the CAAO surfaces was confirmedby DC voltage–current reading as well as conductive AFM setupsby using masked CAAO substrates. Unique nanoporous C filmswith controllable pore dimensions and 1-D C nanostructures were

tion and characterization of conductive anodic aluminum oxide014.06.007

obtained by the virtue of template synthesis upon the completedissolution of sputtered AAO templates. The potential of AAO andCAAO substrates for neural adherence and regeneration is currentlybeing investigated and promising results including cell growth

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timulation was observed, and these will be the topic of an upcom-ng report.

cknowledgment

This research was supported by The Scientific and Technologi-al Research Council of Turkey (Tubitak), grant no: 111M686. Weould like to thank Ms. Hilal Gözler from Nanomagnetics Instru-ents for her assistance with AFM images.

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