Thin film hydrophilic electroactive polymer coatings for bioelectrodes

Journal ofMaterials Chemistry B

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aGraduate School of Biomedical Engineering

NSW, 2052, Australia. E-mail: r.green@unsbCentre for Advanced Macromolecular Design

NSW, 2052, Australia

† Joint lead authors.

Cite this: J. Mater. Chem. B, 2013, 1,3803

Received 1st February 2013Accepted 10th May 2013

DOI: 10.1039/c3tb20152j

www.rsc.org/MaterialsB

This journal is ª The Royal Society of

Thin film hydrophilic electroactive polymer coatings forbioelectrodes

Sungchul Baek,†a Rylie Green,†*a Anthony Granville,b Penny Martensa

and Laura Poole-Warrena

Hybrids of conducting polymers (CPs) and hydrogels have been explored as soft electroactive coatings for

improving the mechanical and electrical performance of metallic implant electrodes. However, hydrogel

fabrication methods pose a significant challenge to producing thin (sub-micron) coatings, resulting in

bulky implants, which displace a large volume of tissue. To address this issue, polymer brushes of poly(2-

hydroxyethyl methacrylate) (pHEMA) were covalently bound to a gold electrode using surface initiated

atom-transfer radical-polymerization (SI-ATRP). The CP poly(3,4-ethylene dioxythiophene) (PEDOT) was

electropolymersied through the brush layer to form a thin hydrophilic coating. The electrical properties

of the hybrid were shown to be superior to homogenous CPs and the surface chemistry was varied as a

function of PEDOT deposition time to present a graded composition of pHEMA and PEDOT. The

resulting material was shown to support the attachment and differentiation of model neural cells,

signifying the potential of these hybrid coatings for bioelectrode applications.

Introduction

Physical cues presented by implantable materials to the targetcells are critical to the resulting biological response. It is alsoevident that next-generation neural engineering applicationswill demand so, deformable substrates that allow preferentialattachment and differentiation of neurons while suppressingastroglial scar formation.1,2 The demand for so electroactivebiomaterials has encouraged development of composite mate-rials that combine the electrochemical properties of conductivepolymers (CPs) with soer polymers able to mediate mechanicalmismatch between stiff metals and so tissues. These com-posite CPs commonly employ a hydrogel component, which is acrosslinked network of hydrophilic polymers.3,4 It has beenshown that thin CPs and more recently conductive hydrogel(CH) lms present signicant electrochemical and biologicaladvantages over conventional electrode materials such as goldand platinum. However, the formation of thin CHs on metalsubstrates has been limited by the thickness of the hydrogelcomponent and its adhesion to the substrate. As a result, a largeamount of CP is required to form a composite. A new designapproach is required for producing hydrophilic polymer–CPcoatings that will allow ner control of physical and mechanicalproperties.

, University of New South Wales, Sydney,

w.edu.au

, University of New South Wales, Sydney,

Chemistry 2013

Coating thickness can only be minimally controlled byconventional hydrogel application techniques such as dip pro-cessing and gel molding. This results in hybrid coatings ofelectrodes in the micron range. Kim et al.5 described the elec-trodeposition of PPy/PSS through 15–50 mm thick alginate gelformed on silicon microprobes by dip coating and Green et al.6

usedmolding to produce electrode coatings of approximately 10mm thickness. In articial muscle applications, composites havebeen produced at around 600 mm.7,8 As a result these compos-ites, while soer, will signicantly deform the tissue in situ. Toproduce thin hybrids with optimal physico-mechanical prop-erties, it is proposed that a hydrophilic brush structure can beused to impart the mechanical benets of a hydrogel whileallowing a composite to be produced with comparable thick-ness to thin CPs.

Surface initiated atom-transfer radical-polymerization (SI-ATRP) is a method that has been explored for robust, nanoscalesurface modications. Many organic lms are non-adherent tometal surfaces and delamination is a potential challenge indeveloping robust CP–hydrogel composites.3,9,10 SI-ATRP is atwo-step process consisting of immobilization of a thiol-initi-ator followed by ATRP of the monomer graed from the surface.The self-assembly of thiol (SH) compounds, particularly on gold(Au) substrates which induces a S–Au bond,11 provides a stableorganic platform to immobilize brushes on metal electrodes.Metals, especially gold, have strong binding affinity tosulphur.12 The strength of the S–Au bond is �44 kcal mol�1.13

This is stronger than the hydrogen bond,14 1.9–38.6 kcal mol�1,and is roughly one-half that of the C–C covalent bond,15 83–85kcal mol�1. Polymer brushes grown via SI-ATRP are therefore

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Fig. 1 Schematic of the fabrication of the CP–brush hybrid. Step (1) self-assembly of the surface initiator. Step (2) SI-ATRP of pHEMA. Step (3) electrode-position of PEDOT/pTS through pHEMA.

Fig. 2 Synthesis of the surface initiator.

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directly bound to the underlying metal, and hence are likely toimprove the adhesion and mechanical stability of conventionalelectrode coatings.

Poly(2-hydroxyethyl methacrylate) (pHEMA) is a polymerthat can be used to form a hydrogel and has a history of usein biomedical applications, including so contact lenses,nanoparticles for drug delivery and vascular stents. Addi-tionally, it has been shown to be polymerisable by SI-ATRP,making it a useful polymer for hybrid bioelectrode coatings.Yoshikawa et al.16 has used SI-ATRP to gra 15 nm pHEMAbrushes onto a silicon substrate and Huang et al.17 graed�700 nm thick pHEMA brushes on to Au using a wateraccelerated SI-ATRP, previously described by Armes et al.18,19

Polymers end-graed via SI-ATRP are commonly known as“brushes” because the polymer chains are stretched awayfrom the surface when produced at high graing density.20

This property can be used to tune the mechanical propertiesof brushes. As a result it is proposed that thin hybrid elec-trode coatings can be developed consisting of pHEMA poly-mer brushes bound to metallic electrodes, through which theconventional CP, poly(ethylene dioxythiophene) (PEDOT) canbe grown.

A signicant challenge in designing a composite CP–hydrogel coating is developing a method which enables thetwo dissimilar materials to occupy the same volume. In theliterature, CPs have been deposited on substrates bearing self-assembling monolayers (SAMs) and polymer brushes, butthese studies used the monolayer or brush polymer as atemplate,21,22 to block CP deposition in the areas where theywere graed. Gorman et al.21 used micro-patterned thiol-SAMsand Zhou et al.22 used pHEMA brushes as a template for theelectrodeposition of PPy. While most lms were formed on theexposed area of gold, some overgrowth of PPy was alsoobserved. It is hypothesized that as nucleophilic growth of CPsrelies on an initial interaction with the underlying substrate,the susceptibility of SAMs to CP interaction can be utilized toguide CP ingrowth to the graed pHEMA brush. Previousstudies have indicated that SAM covered electrodes are stableduring electrochemical depositions. Willicut and McCarley23

and Sayre and Collard24 created surface bound CPs throughelectrochemical polymerisation of pyrrole-containing alka-nethiol SAMs. Upon the application of electrical charge, thepyrrole pedants polymerized without desorption of the thiolmoiety from the Au surface.23 These studies indicate that CPsand SAMs can be combined to form robust coatings onmetallic substrates. However, electrodeposition of CP throughthe brushes to produce a hybrid biomaterial has not beenreported.

This paper describes a seminal study on formation of ahybrid material using a polymer brush and conducting poly-mer. Integration of the polymer brush and CP will requirecareful control of physical and chemical properties of theconstituent materials. Hence a three step fabrication processhas been devised, as depicted in Fig. 1.

The resulting material has been characterised at each step offabrication across chemical, electrical and biological propertiesconsidered important to bioelectrode applications.

3804 | J. Mater. Chem. B, 2013, 1, 3803–3810

ExperimentalFabrication of PEDOT–pHEMA hybrid

Self-assembly of initiator monolayer. Reactions shown inFig. 2 were performed to produce the initiator, bis[2-(2-bro-moisobutyryloxy) undecyl] disulphide, for the self-assemblyprocess. This was a two-step process which involved synthesis ofthe disulde precursor, followed by a second reaction togenerate the complete initiator. For the rst reaction, ethylacetate (99.5%, Sigma-Aldrich) was degassed with nitrogen gasand cooled on ice. 11-Mercapto-1-undecanol (2.044 g, 10 mmol,97%, Sigma-Aldrich) and sodium iodide (15 mg, 0.1 mmol,99.999%, Sigma-Aldrich) were dissolved in 20 ml of cold ethylacetate (99.5%, Sigma-Aldrich). While stirring, 30% hydrogenperoxide (1.021 ml, 10 mmol, Sigma-Aldrich), was added in adrop-wise manner. The mixture was stirred for 1 h and ethylacetate was rotary evaporated (40 �C, 240 mbar). The aqueousphase was then added to 100 ml of methanol (Ajax Finechem).Aer 20 min stirring, the precipitate was ltered and washedwith 10ml of methanol. The ltrand was then dried in a vacuumoven (0.01 mbar, 40 �C) to yield the disulde.

To complete the surface-initiator, the disulde (1.634 g, 4.02mmol) and pyridine (0.78 ml, 9.64 mmol anhydrous, 99.8%,Sigma-Aldrich) were dissolved in 30 ml of dry dichloromethane(anhydrous, 99.8%, Sigma-Aldrich). a-Bromoisobutyryl bromide(1.2 ml, 9.71 mmol, 98%, Sigma-Aldrich) was slowly added andstirred at 0 �C for 1 h and then at 25 �C for 24 h. The reactionmixture was then diluted with 60 ml of dichloromethaneand extracted twice with 150 ml of 1 N HCl solution, twice with150 ml of saturated sodium carbonate and nally 150 ml ofsaturated brine. The aqueous phase was dried over Na2SO4.Dichloromethane was rotary evaporated at 40 �C. The crudeproduct was puried by column chromatography on silica gel.NMR was used to conrm the product chemistry.

This journal is ª The Royal Society of Chemistry 2013

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Silicon wafers (Universitywafer, MA, USA) were sputteredwith a 100 A layer of titanium (Ti), an adhesive layer, and thenwith a 1000 A layer of gold (Au) using a thermal evaporator(Lesker, Pittsburgh, PA). The substrates were cut into 1.5 � 1.5cm squares using a diamond cutter and washed with water,acetone and methanol. 1 mM of initiator solution was preparedby dissolving in ethanol (anhydrous, 99.8%, Sigma-Aldrich).Self-assembly was carried out by soaking the Au substrates inthe initiator solution at room temperature for 24 h. Thesubstrates were thoroughly rinsed with hexane and methanol,and dried in a nitrogen atmosphere. Layers were characterisedby ellipsometry, sessile drop contact angle measurement, andXPS to yield thickness, surface energy, and chemistry.

Growth of pHEMA brushes. The surface initiated atomtransfer radical polymerisation (SI-ATRP) process of HEMA isillustrated in Fig. 3. Briey, this involved exposing the surfaceinitiator modied substrate to a solution containing thehydrophilic monomer, HEMA, in the presence of an activatorand ligand, to produce the bound brush structure.

Initially, HEMA (97%, Sigma-Aldrich) was puried bycolumn chromatography on silica gel and distillation. In a glovebox, the activator, CuBr (16 mg, 0.11 mmol, 99.999%, Sigma-Aldrich), and the ligand, 2,20-bipyridyl (bpy) (35 mg, 0.22 mmol,99%, Sigma-Aldrich), were dissolved in 2 ml of degassedmethanol and stirred for 1 h. The puried HEMA (3.72 ml, 30.67mmol) and the free initiator, ethyl 2-bromoisobutyrate (15 ml,0.10 mmol, 98%, Sigma-Aldrich), were mixed with 1.5 ml ofmethanol. The resulting solution was degassed on aSchlenk line.

SI-ATRP was started by adding the monomer solution to theactivator–ligandmixture. Aliquots of the resulting solution wereapplied to the SAM coated Au substrates. Polymerisation wascarried out at 25 �C in an incubator shaker for 80 min. A smallportion of the reaction solution was collected to characterisepHEMA molecular weight (Mn) and the polydispersity index(PDI) by gel permeation chromatography (GPC). The goldsubstrates bearing brushes were removed from the reactionsolution and rinsed thoroughly with tetrahydrofuran (THF),ethanol, methanol, and water to eliminate untethered poly-mers. The brushes were dried under nitrogen atmosphere andcharacterized by ellipsometry, contact angle, and XPS.

Electrodeposition of the CP. PEDOT/pTS was electro-deposited on a selective area of the pHEMA brushes bound tothe Au substrate using a silicone gasket system. The gasket(FlexiPERM�, Sigma-Aldrich, Aust), was placed on the brushsubstrate and 150 mL of monomer solution (0.1 M EDOT–0.05 MpTS in 1 part water–1 part acetonitrile) added to the gasket

Fig. 3 SI-ATRP of pHEMA on SAM initiator coated gold.

This journal is ª The Royal Society of Chemistry 2013

dened well area. A platinum (Pt) counter electrode was used tocarry out electrodeposition at 0.5 mA cm�2. Two variants wereproduced by polymerising for either 60 or 100 s, such that thetotal charge applied was 0.03 or 0.05 C cm�2, respectively. Filmswere characterised by XPS, cyclic voltammetry and biologicallyusing neural cell cultures.

Brush characterisation

Brush thickness. Ellipsometry was performed using aGaertner L116A ellipsometer (Gaertner Scientic Corp, IL, USA)with a He–Ne laser (632.8 nm). The angle of incidence was set at70�. Eight measurements were made on each sample. Theellipsometric thickness was determined using the Cauchy layermodel. The refractive index (n) and the extinction coefficient ofthe surface initiator and pHEMA brushes were set to (1.45, 0.01)and (1.46, 0.01), respectively. The graing density (s) of thebrushes was determined from the ellipsometric thickness (L)and eqn (1):16

s ¼ L� r�NA

Mn

(1)

where L is the ellipsometric thickness (nm), r is the bulk densityof pHEMA (1.15 g cm�3), NA is Avogadro's constant (6.022 �1023 mol�1), and Mn is the average molecular weight of thebrush, obtained from GPC.

Hydrophilicity. The static contact angle of substrates wasmeasured by the sessile drop method using a KSV CAM200Contact Angle system (KSV Instruments Ltd., Finland). Deion-ized water was used as probe liquid. A 30 ml drop of water wasplaced on the substrate and images were captured at afrequency of 1 Hz. The static contact angle was analysed usingthe image analysis soware (tting method: Young–Laplace,CAM 200 Soware, KSV). Data is presented as an average �standard deviation of three samples.

Brush structure. Degree of polymerisation (DP) and molec-ular weight (Mn) distributions were estimated by GPC (Shi-madzu Co., Japan) eluting with dimethylacetamide (DMAc)containing 0.05 w/v% LiBr and 0.05% butylated hydroxytolueneat 40 �C. The GPC system consisted of an auto-injector (SIL-10AD), a solvent degasser (DGU-12A), a pump (LC-10AT), acolumn oven (CTO-10A), a refractive index detector (RID-10A)and four Phenomenex columns (100, 103, 104, 106 A pore size, 5mmparticle size). The ow rate was 1 mlmin�1. The column wascalibrated against polystyrene standards with molecularweights ranging from 500 to 106 g mol�1. The theoreticalmolecular weight of the pHEMA was calculated by eqn (2):16

Mn;theo ¼ ½HEMA�0½EBIB�0

� 130:14� %conversion

100(2)

where [HEMA]0 and [EBIB]0 is the concentration of the mono-mer and the free initiator, respectively.

Chemical composition of hybrid. The chemical compositionof material surfaces was measured on an ESCALAB220i-XL X-rayphotoelectron spectrometer using an aluminium Ka radiation(photon energy ¼ 1486.6 eV). The take-off angle was 90�. Surveyscans were obtained using a pass energy of 100 eV and high

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Table 1 Theoretical and empirical atomic composition of the surface initiatormonolayer

ElementsTheoreticalcomposition (%)

Empiricalcomposition (%)

C 83.33 77.28 � 2.52O 10.53 17.64 � 3.15S 5.26 3.04 � 0.47Br 5.26 2.04 � 0.18

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resolution scans were obtained using a pass energy of 20 eV andenergy resolution of 0.1 eV. High resolution C 1s and O 1s scanswere made on gold substrate bearing brushes, 0.05 C PEDOT/pTS and the hybrids formed with 0.05 C cm�2 and 0.03 C cm�2.The energy range for C 1s and O 1s scan was from 280.08 to296.08 eV and from 525.08 to 541.08 eV, respectively. Thegraing of pHEMA brushes on gold substrate was veriedfrom the C 1s scan of brushes laden substrates. Spectra weredeconvoluted using the peak tting soware, Eclipse 2.0. Threedifferent spots on each of three samples from different batchwere analysed.

Electroactivity. The electrical performance of these materialsintended for bioelectronics was assessed through cyclic vol-tammetry (CV). Samples were placed in a 3-electrode cell versusa Pt counter and isolated Ag/AgCl electrode. An eDaq eCorderwith potentiostat was used to ramp the voltage between �700and +700 mV. The area within the curve was calculated to yieldthe charge storage capacity of the hybrid electrode material.

Neural cell response. To investigate the behaviour of neuralcell attachment and antifouling properties of the brushes, cellculture was performed in conventional tissue culture plates. Inthis study thehybridused for testingwas the sample createdwith0.05 C cm�2 of PEDOT. Samples were disinfected by soaking in70%ethanol for 30min. All disinfected substrates were placed inthe laminar ow hood and rinsed with DI water and dried. Tomediate initial cell attachment, sample surfaces were coatedwith 5 mg ml�1 laminin (derived from Engelbreth-Holm-Swarm(EHS) sarcoma, Sigma-Aldrich) in phosphate buffered (Sigma-Aldrich). Fluorescent pheochromocytoma (PC12) cells wereseeded at 2.5 � 104 cells per cm2. Cells were maintained in lowserum medium (1% horse serum in RPMI-1640, Sigma-Aldrich)containing 100 ng ml�1 NGF (N2.5S, Jomar, Aust). Neuriteoutgrowth was assessed using uorescence microscopy at 96 h.For each sample type, 4 images were taken on each of 3 samplesin each experiment which repeated 3 times. The number ofattached PC12 cells and the neurite length was analysed.

Fig. 4 GPC results of SI-ATRP of pHEMA. Data obtained from four samples fromdifferent batches. (a) Low polydispersity index (PDI < 1.2) and (b) the molecularweight close to the theoretical line indicate controlled radical polymerisation viaATRP mechanism.

Results and discussion

A hybrid CP–brush structure was developed, and each step ofthe fabrication process was characterised to dene the resultingmaterial composition and function. The two-step synthesis ofthe surface initiator, bis[2-(2-bromoisobutyryloxy)undecyl]disulde, was successful with both products being obtainedwith high yield. The disulde (compound 1, Fig. 2) and theinitiator (compound 2, Fig. 2) were produced with a yield of 86%and 88%, respectively. Compound 1 was a white solid and thenal product, compound 2, had the appearance of a pale yellowoil. The initiator monolayer was successfully bound to themetallic substrate. This was conrmed by the contact anglemeasurement, ellipsometric thickness and element composi-tion measured by XPS.

Sessile drop analysis of the initiator monolayer determinedthat the static contact angle between DI water and the substratewas 76.6 � 1.9�. Ellipsometry determined that the thickness ofthe SAMwas 1.7� 0.1 nm. Similar results were reported by Shahet al.25 and Rakhmatullina et al.26 The atomic composition was

3806 | J. Mater. Chem. B, 2013, 1, 3803–3810

analysed using XPS. The desired elements such as gold, carbon,oxygen, sulphur and bromine were identied in the monolayer.Gold accounted for 46 � 3% of the spectral area with the otherfour elements comprising the rest. Table 1 compares the theo-retical and empirical atomic composition of the monolayer. Itcan be seen that the empirical results generally correlated withtheoretical values. At the take-off angle of 90�, the calculatedelemental composition tends to understate the proportion ofthe subsurface atoms while overstating the ones on thesurface.27 As a result the sulphur was detected a lower level thanthe theoretical prediction and the oxygen was measured at arelatively higher presence. However, the bromine signal, whichwas obtained from the surface, was weaker than expected. Thiscould be a consequence of beam induced damage. Wassermanet al. have reported that the bromine signal from a SAM could bereduced by up to 66% upon the exposure to X-ray.28

The SI-ATRP growth of pHEMA brushes was rstly charac-terised using GPC as shown in Fig. 4. Aer 80 min of poly-merisation, 87% of themonomer was converted to polymer. Theaverage molecular weight,Mn, measured from GPC was 31� 0.9kDa. The theoretical molecular weight for pHEMA at 87%conversion is 34 kDa.16 The low PDI (<1.2) combined with amolecular weight close to that of the theoretical value conrmthe persistent radical effect of ATRP providing controlledgrowth of polymer chains. The length, L, of the dry pHEMAbrushes was measured by ellipsometry. The average length ofthe brushes was found to be 15.3� 0.5 nm. The graing densitywas determined from the ellipsometry and GPC results (L/Mn) ina similar manner described by Yoshikawa et al.16 For allsamples, the average graing density was 0.35 chains per nm2.Hence, the graed pHEMA falls under the brush regime. Formost neutral polymers, the transition frommushroom to brush

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Fig. 5 XPS analysis of C 1s on pHEMA brushes.

Fig. 6 SEM comparison of (a) hybrid pHEMA–PEDOT/pTS and (b) homogenousPEDOT/pTS. The thickness of the films was �120 nm.36 (Scale bar ¼ 1 mm.)

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regime occurs near 0.1 chains per nm2.29 The density of thesample was typically higher than that of pHEMA brushesprepared via reversible addition–fragmentation chain transfer(RAFT) processes, at 0.3 chains per nm2,30,31 but lower than thatof the brushes prepared on a hard substrate (silicon) using SI-ATRP, at 0.7 chains per nm2.16 The low graing density ispossibly a result of the initiator viability. XPS results showedthat the amount of Br presented by the SAM was half thetheoretically predicted amount. As a result the graing densitywould be comparably reduced, and this would agree withgraing densities reported on Si being reduced by half. It isclear that future investigations will be required to ascertain Brviability of the thiol initiator in comparison to the silane initi-ators used for denser brush constructs. Additionally, surfacedefects and the so nature of gold were considered as variablesthat may have resulted in the lower than expected graingdensity. However, the density of the brushes grown on theannealed gold substrate, which is likely to have less surfacedefects, was also 0.35 chains per nm2.

While this was the maximum graing density obtained in thisstudy, it was considered that the surface shielding effect is likely toincrease with the graing density. Physical changes in the surfaceassociated with graing of the pHEMA brushes was also noted bythe changes in hydrophilicity. The water contact angle of thesurfacewas reduced to 39.9� 1.8� upongraing of thehydrophilicpHEMA brushes. A similar decrease in contact angle was reportedby Yang et al.32whopresenteda 25� decrease in contact angle uponthe graing of �10 nm thick pHEMA brushes on modied stain-less steel. The contact angle of pHEMA brushes has been reportedacross the range of 29 to 61�, depending on the brush length andsubstrate.16,26,32–34XPSdata veries thechemical compositionof thepHEMAbrushes as shown inTable 2. The carbon to oxygen ratio ofthe pHEMA brush was found to very closely align with the theo-retical composition of pHEMA. The XPS spectra of the pHEMAbrushes show that the atomic bonding of the synthesizedcompounds concurs with a previous report by Castner et al.35 forpolymerized pHEMA on glass coverslips. The high resolution XPSscan of C 1s region, shown in Fig. 5, depicts three peaks at 285.03,286.69 and 289.03 eV, which correspond to the C–C, C–O and O–C]Obonds, respectively. The ratio between these three peakswas3.4 : 2 : 1, compared to the theoretical ratio, 3 : 2 : 1, for pHEMA.36

Upon the verication of dense pHEMA brushes on the goldsurface, PEDOT/pTS was electrodeposited through the brushstructure, forming a transparent dark blue lm on the substratefor both deposition parameters. The minimum charge requiredto form a visible lm was 0.03 C cm�2. The SEM micrographrevealed a surface similar to that of homogeneous PEDOTformed on bare gold, shown in Fig. 6. However, the chemicalcomposition of the hybrid formed with 0.03 C cm�2 was

Table 2 Theoretical and empirical atomic composition of the pHEMA brushes

ElementsTheoreticalcomposition (%)

Empiricalcomposition (%)

C 66.67 69.87 � 2.00O 33.33 30.13 � 2.02

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signicantly different from the homogenous PEDOT, as shownby the XPS spectra in Fig. 7. The O 1s and C 1s spectra of the 0.03C cm�2 hybrid contained both PEDOT and pHEMA signals(Fig. 7(a) and (b), respectively). While this result indicated thecoexistence of PEDOT and pHEMA brushes in the hybrid, itdidn't show that the brush structure was maintained over thedeposition of CP. It was considered that unbound pHEMA has alikely chance to present on the surface as well as the inside ofthe CP layer regardless of the thickness. Hence, a thicker CPlayer was deposited on the brushes using a charge of 0.05 Ccm�2. The XPS spectrographs in Fig. 7(c) compare the surfacebound chemistry of the pHEMA and 0.05 C cm�2 PEDOT/pTS tothe hybrids produced from 0.03 C cm�2 and 0.05 C cm�2

PEDOT. The spectrum of the 0.05 C cm�2 hybrid was identicalto that of the pure CP. Since no pHEMA chemistry was elicited,this result suggests that the brushes were not cleaved off duringthe electrodeposition and relatively thick CP was deposited onthe brushes. At the deposition charge of 0.03 C cm�2, the CP wasless likely to have encapsulated the brush structure. Althoughthe SEM images did not show a discernable difference betweenlms formed via the two deposition charges, the maximumproling depth of XPS is less than 10 nm, which suggestsminimal coverage of the upper surface of the brush structure.

The fabrication of pHEMA brushes on gold substrates via SI-ATRP is an established technique.However, the electrodepositionof CP though the brushes to produce a hybrid biomaterial has not

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Fig. 8 CV curves showing electroactivity of hybrids compared to bare gold andhomogenous PEDOT. The brush structure, prior to PEDOT deposition is alsorepresented.

Fig. 7 Chemical composition found by XPS scan of hybrids made with 0.03 Ccm�2 PEDOT compared to 0.05 C cm�2 and homogenous PEDOT and pHEMA. (a)High resolution O 1s scan, (b) high resolution C 1s scan, and (c) a comparison ofsurvey scan graphs. The value shown in the bracket indicates the carbon tooxygen atomic ratio of the sample.

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been reported. This concept may seem counter-intuitive as bothpHEMA brushes and underlying SAM layers have been used fortemplate patterning of electropolymerised CPs. However, bothZhou et al.22 andGorman et al.21 reported electrodeposition ofCPson alkylthiol SAMs patterned on gold electrodes, an affect whichwas considered undesirable. In this study it is thought that the CPpenetration of the SAM was a result of either defects developedduring the fabrication or polymerization of the monomer thatpartitions the SAM. SAMs on gold substrates always carry surfacedefects such as pinholes and domain boundaries that can bepreferred sites for CP nucleation and growth. Previous studiesindicated that about 1% of the current applied is transferredthrough these pinhole defects, and the other 99% of current ispassivated by the SAM.37 Sabatani et al.38,39 has suggested thatpinhole defects can serve as an array of ultra-microelectrodeswithan averagediameter of 5–10nm.Accordingly, it is thought that theelectrodeposition through the pHEMA brush may have alsooccurred through these pinhole defects. The spacing betweenpHEMA brushes is likely to increase during the electrodepositionof the CP as the brushes swell and thermodynamically stretch inthe aqueous monomer solution. The application of electricalcurrent will further elongate the brush through electrostaticinteraction. While the penetration of the CP through the pHEMAbrush remains undetermined, results suggest the co-existence ofthe brush and the CP within the coating material.

Given the potential application of these hybrid materials asbioelectrode coatings, they were further characterised for bothelectroactivity and neural cell interaction. The charge storagecapacity (CSC) of the gold substrate prior to coating was 0.86 �0.18 mC cm�2, as measured from CV curves (Fig. 8). Graing ofpHEMA brushes onto the substrate was found to reduce the CSCby 62.5% to 0.27 � 0.01 mC cm�2. Deposition of the PEDOT to

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form a hybrid then increased the CSC to 11.08 � 0.27 mC cm�2.Interestingly, this was higher than that of homogenous PEDOT/pTS, with a CSC of 9.39 � 0.64 mC cm�2. It is clear that thenanostructure of pHEMA brushes improved the electrochemicalproperties of the hybrid construct. One possibility is that thehybrid provides larger (volumetric) surface area than thehomogenous PEDOT. It is known that the mass of CP poly-merised on a substrate is largely controlled by the depositioncharge.40,41 Hence, a hybrid containing CP deposited with 0.05 Ccm�2 is likely to have a similar mass to a homogenous CP lmdeposited under the same charge. However, due to the presenceof the hydrophilic brushes, charge can access and pass througha greater three-dimensional area across the hybrid electrodethan on the dense hydrophobic CP, where charge can only passthrough the two dimensional surface plane. Physical charac-terisation of the interface between the brushes and the CPremain an important area of future study. Cross-sectional TEMis suggested as a method to assess the polymer phases in thehybrids compared to CPs. However, methods to prepare thincross-sectional samples of these polymers on rigid metallicsubstrates remain an unmet challenge.

The uorescence micrographs of PC12 cells cultured on allsample variants are shown in Fig. 9(a)–(d). The cell density foundon the hybrid, homogenous PEDOT and bare Au samples wereclose to the seeding density of 25 000 cells per cm2. However,neurite data (Fig. 10) clearly indicates that more neurite exten-sion occurred from neural cells cultured on the hybrid and CPthan from the Au. The average number of neurites found on thehybrid was roughly 14 000 neurites per cm2, which is 56%morethan that on the Au, 9000 neurites per cm2. No signicantdifference inneurite lengthwasobservedondifferent substrates.

It was found that the surface graed pHEMA brushes pre-vented the cell adhesion at the border region between thehybrid and the outlying homogenous brush. In Fig. 11 it can beseen that cells selectively adhered to the hybrid CP region. Theprotein and cell repelling properties of pHEMA brushes havebeen reported and the consequent use of the brush layer as an

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Fig. 9 Fluorescence micrographs representative of neural cell growth on (a) Au,(b) pHEMA brushes, (c) PEDOT/pTS and (d) hybrid. (Scale bar ¼ 100 mm.)

Fig. 10 The box plots of (a) the PC12 cell density (b) the number of neurites and(c) the neurite length (N ¼ 4).

Fig. 11 (a) The optical and (b) the fluorescence micrographs of the borderbetween the hybrid and the brush. It can be seen that the brush preventsadhesion of PC12 cells. Red lines indicate the boundary between the hybrid andthe brush. (Scale bar ¼ 400 mm.)

Fig. 12 Schematic illustration of the potential deposition route of CP throughsurface bound brushes.

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antifouling surface has been studied by Yoshikawa et al.16 Thisresult suggests that when the hybrid is produced with thesefabrication parameters, the PEDOT is dominant in determiningmaterial properties. It is also shown that the selective electro-deposition of CP on the brush layer can provide a facile methodof creating an electric contact with selective cell binding. Thedesign of cell specic binding electrodes has been pursued byTourovskaia et al.,42 but the processes are oen complicated,involving multiple masking and etching steps. Hence, it isproposed that with minor manipulation, this hybrid materialcould provide a relatively simple process for cell selective elec-trodes. Additionally, Palanker et al.43 suggested that the fabri-cation of microelectrodes with specic cell binding wouldincrease the spatial resolution of neural stimulation.

The exact structure of the resulting hybrid is yet to bedetermined, but several possible congurations have beenconsidered. These are illustrated in Fig. 12. If the size of the CPnucleate is bigger than that of the pinhole defect, the CP is likelyto nucleate on the brushes and form an overlying lm as shown

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in Fig. 12(a). Depending on the size of the surface defect, thedistance between brushes, the size and percolation of CPnucleates, the alternate structures shown in (b), (c) and (d) canbe achieved. These structures represent the various penetrationoptions for the CP, assuming defects in the SAMs. In Fig. 12(b) itis considered that only defect nucleation occurs and the CPgrows above the brush. In Fig. 12(c) both the defects andspacing between brushes enable CP nucleation and nally inFig. 12(d) the CP forms in intimate integration with the brushstructure, having minimal reliance on defects to polymerise.The scenario depicted in Fig. 12(c) is thought to be the mostpromising for neural interfaces. This structure will enable bothmechanical soening and the passage of electrical charge.

Conclusions

A dense layer of hydrophilic pHEMA brushes was successfullygraed on a SAM carrying gold substrate via a SI-ATRP tech-nique. The electrodeposition of PEDOT/pTS was galvanostati-cally carried out on this brush to form a composite material.The pHEMA nanobrushes fabricated via SI-ATRP techniquesprovide a unique building block to form a new type of CPhybrid. The synergistic incorporation of PEDOT and pHEMAbrushes resulted in a material with high charge storage capacityand promising neural cell interactions. Using the opposing cell/protein adhesion properties of the brush and the hybridprovides selective cell binding for new approaches to high

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density, selective neural interfaces. This approach also haspotential for creation of new hybrids based on functionalbrushes that can act as dopants or as bioactive sites. As anexample, brushes synthesised from amino acids could furtherimprove the biofunctionality of the hybrids. Furthermore,immobilization of dopant species via brushes based on dopingmolecules such as styrene sulfonate could reduce dopantleaching and improve the integration of CP on metal substratesproviding enhanced stability required for implant applications.

Acknowledgements

The authors acknowledge funding fromUniversity of New SouthWales, Silver Star grant scheme, PS24596 and New SouthInnovations Proof of Concept funding, L002317. SB would liketo acknowledge Dr Hisatoshi Kobayashi and Dr Chiaki Yoshi-kawa in the biofunctional materials group at the NationalInstitute for Materials Science (NIMS), Japan for training duringhis internship at NIMS.

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