Micropatterning of Proteins and Mammalian Cells on Indium Tin Oxide

Micropatterning of Proteins and Mammalian Cells on Indium Tin Oxide
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  Micropatterning of Proteins and MammalianCells on Indium Tin Oxide Sunny S. Shah, † Michael C. Howland, ‡ Li-Jung Chen, #  Jaime Silangcruz, † Stanislav V. Verkhoturov, # Emile A. Schweikert, # Atul N. Parikh, §, ⊥ and Alexander Revzin* ,† Department of Biomedical Engineering, Department of Chemical Engineering and Materials Science, Department of Biophysics, and Applied Science Graduate Group, University of California, Davis, California 95616, and Departmentof Chemistry, Texas A&M University, College Station, Texas 77843 ABSTRACT  Thispaperdescribesanovelsurfaceengineeringapproachthatcombinesoxygenplasmatreatmentandelectrochemicalactivationtocreatemicropatternedcoculturesonindiumtinoxide(ITO)substrates.Inthisapproach,photoresistwaspatternedontoan ITO substrate modified with poly(ethylene) glycol (PEG) silane. The photoresist served as a stencil during exposure of the surfaceto oxygen plasma. Upon incubation with collagen (I) solution and removal of the photoresist, the ITO substrate contained collagenregions surrounded by nonfouling PEG silane. Chemical analysis carried out with time-of-flight secondary ion mass spectrometry(ToF-SIMS) at different stages in micropatterned construction verified removal of PEG-silane during oxygen plasma and presence of collagenandPEGmoleculesonthesamesurface.Imagingellipsometryandatomicforcemicroscopy(AFM)wereemployedtofurtherinvestigate micropatterned ITO surfaces. Biological application of this micropatterning strategy was demonstrated through selectiveattachmentofmammaliancellsontheITOsubstrate.Importantly,afterseedingthefirstcelltype,theITOsurfacescouldbeactivatedby applying negative voltage ( - 1.4 V vs Ag/AgCl). This resulted in removal of nonfouling PEG layer and allowed to attach another celltypeontothesamesurfaceandtocreatemicropatternedcocultures.Micropatternedcoculturesofprimaryhepatocytesandfibroblastscreated by this strategy remained functional after 9 days as verified by analysis of hepatic albumin. The novel surface engineeringstrategydescribedheremaybeusedtopatternmultiplecelltypesonanopticallytransparentandconductivesubstrateandisenvisionedto have applications in tissue engineering and biosensing. KEYWORDS:  indium tin oxide • photolithography • switchable surfaces • protein micropatterning • cell micropatterning •imaging ellipsometry • microfabrication INTRODUCTION T he ability to design cellular interactions is importantfor creating in vitro models that mimic complexityof native tissue (1–3). One approach employed fre-quently in biology is cultivating two cell types as a way toensure that cell function is better maintained in a culturedish.Cultivationoftwocelltypes,orcoculture,isparticularlyimportant in liver tissue engineering where hepatocytescoculturedwithsupporting(nonparenchymal)cellsmaintainhepatic function longer and at a much higher level thanhepatocytes cultured alone (4, 5).Traditionally,cocultureswerecreatedbyrandomseedingof the two cell types in a culture. In a series of seminalpapers, Bhatia and colleagues proposed to employ surfacemicropatterning (photolithography) to define sites for at-tachment of hepatocytes and supporting cells, and showedimprovedhepaticfunctionofsuchcellculturesystem(6–8).In this approach, photoresist was patterned on the glasssurface and used as a stencil for adsorption of cell-adhesiveproteinmolecules(collagenI).Constructionofthecoculturewasbasedonpreferenceofhepatocytestoattachtocollagenand the ability of fibroblasts to adhere elsewhere on thesurface (8). While offering outstanding insight into interac-tionsbetweentwocelltypes,thiscoculturemicropatterningapproach relied on cell type-specific adhesion preferencesand therefore could not be easily translated to other celltypes.Anumberofalternativemicropatterningapproacheshave been developed to better control cell-surface interac-tionsandtoorganizemultiplecelltypesonthesamesurface.Theseapproacheshaveutilizedmicrofluidicchannels(9,10),polymerstencils(11,12),layer-by-layerelectrostaticinterac-tions (13), stimuli-responsive polymers (14), and electro-chemical activation (15, 16) to define the time and place of cell attachment.Electricalstimulationisparticularlyappealingasamethodfor controlling composition of biointerface (17) and forguiding cell-surface interactions (15, 16, 18–23). This strat-egy requires minimal handling of the culture substrate;electrical stimulus can be applied in cell culture media sothat the location and time of stimulation may be controlledprecisely through the use of electrode arrays (21, 22, 24).The majority of electrochemical switching strategies havefocused on altering properties of gold substrates modified * To whom correspondence should be addressed. Mailing address: Departmentof Biomedical Engineering, University of California, Davis, 451 East HealthSciences St. #2619, Davis, CA, 95616. E-mail: arevzin@ucdavis.edu. Phone:530-752-2383. Fax: 530-754-5739.Received for review July 30, 2009 and accepted October 14, 2009 † Department of Biomedical Engineering, University of California. ‡ Department of Chemical Engineering and Materials Science, University of California. # Department of Chemistry, Texas A&M University. § Department of Biophysics, University of California. ⊥ Applied Science Graduate Group, University of California.DOI: 10.1021/am900508m© 2009 American Chemical Society        A       R       T       I       C       L       E 2592  VOL. 1  •  NO. 11  •  2592–2601  •  2009 www.acsami.org Published on Web 10/30/2009  with self-assembled monolayers (15, 17, 18). Gold is anexcellent electrode material; however, it is not opticallytransparent and therefore is not optimal for cell cultivation.Whilegoldcanbesemitransparentwhendepositedasathinlayer and may serve as a cell culture substrate (25), it willno longer function properly as an electrode because of increased resistivity. ITO, on the other hand, combinesexcellent conductivity with optical transparency and hasbeenusedinelectrophysiology(26),cellcultivation(27),andcell-basedbiosensing(28).ElectrochemicalswitchingofITOsurface properties has been demonstrated in a few recentpublications (21, 24, 29, 30).Functionalizationwithpoly(ethyleneglycol)(PEG)renderssurfaces resistive to adhesion of cells and proteins (31, 32).Previously,wedemonstratedelectrochemicaldesorptionof a nonfouling PEG silane layer from ITO substrate as a waytoexercisespatialandtemporalcontrolovercellattachment(21). Building on this prior work, the present paper soughtto develop and characterize a simple and effective methodfor micropatterning cells in mono- and co-cultures on ITO.A novel micropatterning approach developed for this pur-pose involved patterning photoresist on nonfouling (PEG-silane modified) ITO substrate and then treating this sub-strate in oxygen plasma to selectively remove PEG silanefrom regions not protected by photoresist (see Figure 1).Immersion of this surface in a solution of cell-adhesiveprotein (e.g., collagen I) followed by photoresist lift-off resulted in a surface comprised of cell-adhesive collagen (I)islands within a layer of nonfouling PEG. Chemical andtopographicanalysisofthesemicropatternedsubstrateswasperformed at different stages in surface preparation usingtime-of-flightsecondaryionmassspectrometry(ToF-SIMS),imaging ellipsometry (IE) and atomic force microscopy(AFM). To further develop biological application of thismicropatterningstrategywedemonstratedthatprimaryrathepatocytes attached selectively on regions of the ITOsubstrate containing matrix proteins and did not attach onPEG-modified regions. Applying reductive voltage ( - 1.4 Vvs Ag/AgCl) to the ITO substrate containing hepatocytes ledtodesorptionofthesurroundingPEG-silanelayer.Asecondcell type (fibroblasts) could now be added to the surface tocomplete the coculture. Importantly, hepatocytes were notaffected by the electrical stimulation of the surface andremained functional on ITO. MATERIALS AND METHODS Chemicals and Materials.  Indium Tin Oxide (ITO) coatedglass slides (75 × 25 mm) were obtained from Delta Technolo-gies (Stillwater, MN). The ITO coated glass slides had sheetresistance of 4 - 8 Ω with nominal transmittance of   > 82% andan ITO thickness of 150 - 200 nm. 2-[Methoxy(polyethylenox-y)propyl]trichlorosilane(MWrange470 - 610)(PEGsilane)waspurchased from Gelest, Inc. Ethanol, acetone, anhydrous tolu- FIGURE 1. Novel surface engineering strategy for micropatterning one or two cell types on ITO. Step 1: ITO coated glass slides are modifiedwith a nonfouling PEG silane. Photoresist is then patterned on top of PEG-modified ITO. Step 2: The substrate is exposed to oxygen plasmawhich removes PEG from regions of ITO not protected by photoresist. Step 3: The substrate is then incubated with cell-adhesive ligands(collagen I in our case) followed by photoresist liftoff. Upon incubation with the patterned ITO substrate, cells of type I selectively attach tothecell-adhesivedomainstoformmicropatterns.Step4:Applyingreductivepotential( - 1.4vsAg/AgClreferencefor60s)resultsindesorptionoftheremainingPEGsilane.ElectricalstimulationswitchesITOsurfacepropertiesandallowsforattachmentofcelltypeIItoformacoculture. A  R  T  I     C  L  E   www.acsami.org VOL. 1  •  NO. 11  •  2592–2601  •  2009  2593  ene, and collagen from rat tail (type I) were purchased fromSigma-Aldrich (St. Louis, MO). Phosphate-buffered saline (PBS)10XwaspurchasedfromCambrex.Dulbecco’smodifiedEagles’medium (DMEM), Minimal Essential Medium (MEM), sodiumpyruvate, nonessential amino acids, fetal bovine serum (FBS),FITC-labeled collagen type I were purchased from InvitrogenLife Technologies (Carlsbad, CA). The 384-well polypropylenemicroarray plates were obtained from Genetix (New Milton,NH). Goat antirat albumin antibody, goat antirat albuminantibody-HRP conjugate, reference serum, and goat IgG ELISAquantitation kit were obtained from Bethyl Laboratories (Mont-gomery, TX). PEG Silane Modification and Photoresist Patterning onITO Coated Glass Substrates.  ITO coated glass slides weretreated in an oxygen plasma chamber (YES-R3, San Jose, CA)at300Wfor5min.Afterward,theslideswereincubatedin2%v/v 2-[methoxy(polyethylenoxy)propyl] trichlorosilane (PEG si-lane) dissolved in anhydrous toluene for 2 h. This reaction wasperformed in a glovebag under nitrogen purge to avoid atmo-sphericmoisture.AfterPEGsilanemodification,theslideswererinsed in fresh toluene, dried under nitrogen, and cured at 100°Cfor2h.Modifiedslideswerethenstoredinadesiccatoruntilfurther use.Positive photoresist (AZ 5214-E) was spin-coated on PEGsilane modified ITO substrate at 800 rpm for 10 s, followed by4000 rpm for 30 s. The photoresist-coated slide was then soft-baked on a hot plate at 100 °C for 105 s. After baking, thephotoresist layer (PR) was exposed to UV light (10 mW/cm 2 )through a photomask for 45 s using a Canon PLA-501F MaskAligner. Exposed photoresist was then developed for 5 min inAZ 300 MIF developer solution, briefly washed with DI waterto remove residual developer solution, and then dried usingnitrogen.Theresultantphotopatternedsubstratewasthenhardbaked for 30 min at 120 °C. Characterization of PEG Silane Removal and ProteinDeposition. Aphotopatternedsubstratewasexposedtooxygenplasmaat300Wfor10mintoremovePEGsilanefromregionsunprotected by photoresist. This resulted in formation of holesinthePEGsilanemonolayer.Todepositproteinsintheseholes,the substrate was incubated with 0.1 mg/mL collagen I in 1 × PBS solution for 60 min, followed by a rinsing with DI waterand drying under nitrogen. The substrate was then sonicatedinacetonefor30mintoremove(lift-off)remainingphotoresistfrom the surface leading to the formation of collagen domainssurrounded by PEG silane on ITO.Secondaryionmassspectrometry(SIMS)coupledwithtime-of-flight (ToF) spectrometer was used to verify the removal of PEGsilaneusingoxygenplasmaandthedepositionofcollagenon ITO. Effusion C 60 + ions were accelerated to 16 keV andsteeredtowardanegativelybiasedtarget( - 10keV)tocreateatotal impact energy of 26 keV. The feature of this technique isrunning SIMS in the event-by-event bombardment/detectionmode. The secondary ions generated from each single impactwere detected by an eight-anode detector. Each single impactwas detected and recorded as an individual event. As reportedby us previously, the secondary ion emission hemisphericvolumeofasingleprojectileimpactwasdeterminedtobe5 - 10nmindiameter(33,34).Theaccumulationof  ∼ 2 × 10 6 eventsconstituted a conventional secondary ion mass spectrum. Thesecondary ion yield is the number of secondary ions emittedperprojectileimpact.TheyieldofadetectedionAiscalculatedusing eq 1 Y  A  )  ∑ x A x A N  ( x A ) N  total )  ∑ x A x A  P ( x A )  )  I  A N  total (1) where  x A  is the number of detected ions A in a single event (0 e  x A  <  8),  N  ( x A ) is total number of events when ions A weredetected, N  total isthetotalnumberofprojectileimpacts,  P ( x A )istheprobabilitydistributionofdetectingionsAinasingleevent,and  I  A isthemeasurednumberofeventswhenionAisdetected.Imaging ellipsometry (IE) was also used to characterize thetopologyofthesubstrateandfurtherverifytheremovalofPEGsilane and the deposition of collagen. For these experiments,collagen I patterns were created on PEG silane-modified ITOusing photoresist lithography protocols detailed above. Forcomparison and ease of characterization, identical procedureswere followed to produce patterns on silicon wafers. Themeasurements were taken using an iElli2000 imaging nullellipsometer(Nanofilm,Gottingen,Germany)witha20mWNd:YAG frequency-doubled laser operating at 1% power. Spatialmaps of the ellipsometric parameter,  δ , were acquired using a10 ×  objective, giving a field of view of 645  ×  430  µ m and alateral resolution of 2  µ m.  δ  maps were acquired using a seriesof 70 images collected over 4° of polarizer rotation. The nullconditionsofeachpixelwerethendeterminedfromthisseriesof images and used to calculate  δ  values. Topographical mapsofthicknessweregeneratedfromthese δ valuesusinganopticalmodel that assumed isotropic parallel slabs and a refractiveindex of 1.47 for both the PEG silane and collagen whereas arefractive index of 2.0  +  0.0075i was used for ITO. Averagevalues were calculated on four separate 25 × 25 pixel regionson collagen and silane to characterize the roughness of theorganic layer.Inaddition,atomicforcemicroscopy(AFM)wasusedtoverifythe thickness changes during PEG silane removal and collagenadsorption. All surface scans employed a Dimension 3100ScanningProbeMicroscopewithaHybridclosed-loopXYZheadand Nanoscope IVa controller (Vecco, Santa Barbara, CA). Allsamples were imaged in air or under Millipore water with adirect drive cantilever holder for fluids (Vecco, Santa Barbara,CA).Sampleswerescannedat0.5Hzincontactmodewith512points collected in each of 512 scan lines. A silicon nitridecantileverwithaspringconstantof0.05N/mwasused.Athirdorder flattening routine was used to correct for bowing of thepiezo during sample scanning. Micropatterning of Proteins and Cells on ITO Substrates. To demonstrate the ability to define protein attachment sites,the micropatterned ITO surfaces were incubated in collagen-FITC solution (0.1 mg/mL) for 30 min, washed in 1X PBS threetimes, and rinsed in DI water. The samples were dried undernitrogen and imaged using LSM 5 Pascal confocal microscope(Carl Zeiss).ITO substrates containing protein micropatterns were alsoincubated with cells to demonstrate that cell attachment couldbe controlled in a spatially resolved fashion (see Figure 1, step3). The cell types used in our studies were human hepatic cellline (HepG2), primary rat hepatocytes, and murine 3T3 fibro-blasts.HepG2cellsweremaintainedinMEMsupplementedwith10% FBS, 200 U/ml penicillin, 200  µ g/mL streptomycin, 1 mMsodiumpyruvate,1mMnonessentialaminoacidsat37°Cinahumidified5%CO 2 atmosphere.Primaryrathepatocyteswereisolated from adult female Lewis rats (Charles River Laborato-ries, Boston, MA) weighing 125 - 200 g, using a two-step colla-genase perfusion procedure as described previously (35). Typi-cally,100 - 200millionhepatocyteswereobtainedwithviabilityof   > 90% as determined by trypan blue exclusion. Primaryhepatocytes were maintained in DMEM supplemented withepidermal growth factor, glucagon, hydrocortisone sodiumsuccinate,recombinanthumaninsulin,200units/mLpenicillin,200  µ g/mLstreptomycin,and10%FBS.Murine3T3fibroblastswere maintained in DMEM supplemented with 10% FBS, 200U/ml penicillin, 200  µ g/mL streptomycin at 37 °C in a humidi-fied5%CO 2 atmosphere.Priortocellseeding,anITOsubstratewith collagen micropatterns was sterilized with 70% ethanol,washed twice with 1 ×  PBS and placed into a well of a 6-wellplate before being incubated with a 3 mL of cell suspension of hepatocytes in culture medium with 10% FBS at a concentra-        A       R       T       I       C       L       E 2594  VOL. 1  •  NO. 11  •  2592–2601  •  2009 Shah et al. www.acsami.org  tionof1 × 10 6 cells/ml.After1hofincubation,unattachedcellswere aspirated and the medium replaced to leave behindpatterned cellular arrays visualized using Zeiss Axiovert 40microscope. Electrochemical Desorption of PEG Silane Layer andFormation of Cocultures.  The surface modification and cellseeding procedure described in the previous section resultedin cells attaching onto microdomains defined within a PEGsilane layer (see Figure 1, step 3). To form cocultures, electro-chemical desorption of the PEG layer was employed to makepreviously nonfouling regions of the ITO substrate conduciveto cell adhesion. Electrochemical desorption of PEG silane self-assembled on ITO substrates was described by us previously(21).Briefly,anITOsubstratecontainingislandsofhepatocytes(50 to 300  µ m diameter) was placed into a custom-madePlexiglas electrochemical cell and immersed in 500  µ L of cellculture media acting as an electrolyte solution. Ag/AgCl refer-ence and Pt counter electrodes were positioned in the sameelectrochemical cell with ITO region serving as a workingelectrode and a steel wire providing electrical contact with the FIGURE 2. Characterization of PEG silane removal and protein micropatterns using ToF-SIMS. (A) The negative ion mass spectra of before andafteroxygenplasmatreatmentshowsdisappearanceofPEGpeaks.(B)Thenegativeionmassspectraof100  µ mcollagenmicropatternsformedonPEG-modifiedITOafterremovalofthephotoresist(PR)layer.(C)CoincidenceionmassspectrumshowsthatemissionofCNO - ions(peptidebond components) is not associated with PEG or PR ion signatures. This suggests successful micropatterning of collagen. A  R  T  I     C  L  E   www.acsami.org VOL. 1  •  NO. 11  •  2592–2601  •  2009  2595  substrate. Reductive potential of  - 1.4 V was applied for 60 s,followed by washing with fresh media. This procedure led toremoval of the PEG silane layer surrounding the islands of hepatocytes and allowed to add another cell type, 3T3 fibro-blasts, onto the same surface. Fibroblasts were incubated at aconcentration of 0.5  ×  10 6 cells/mL to create hepatocyte-fibroblastcocultures.Productionofalbuminbythehepatocyteswas assessed using standard ELISA protocols to evaluate func-tion of hepatocytes on ITO substrates (35). RESULTS AND DISCUSSION Inthisstudy,wepresentanovelapproachthatcombinesphotolithography, oxygen plasma treatment and electro-chemical switching of the biointerface to create micropat-terned cocultures on ITO substrates (Figure 1). Differentsteps in this surface micropatterning procedure were char-acterized by ToF-SIMS, imaging ellipsometry and AFM.Micropatterning of cells and creation of functional hepato-cyte-fibroblast cocultures on ITO substrates was also dem-onstratedpointingtofutureapplicationsofthisapproachincell cultivation, tissue engineering and biosensing. Chemical Analysis of Micropatterned SurfacesUsing ToF-SIMS.  The goal of this study was to develop asimple and effective strategy for patterning two cell typeson the same surface. A first step in creating this complexmicropatterned substrate was to define cell-adhesive micro-domains on a nonfouling, PEG-modified ITO substrate. Wechose to employ a strategy described by Folch and co-workers whereby nonfouling substrates are covered with astencil and then treated with oxygen plasma to removenonfouling molecules from specific regions (36). In ourapproach, described in Figure 1, a photoresist patternfabricated on PEG silane-modified ITO substrate served asa protective layer during the oxygen plasma exposure.Plasma treatment removed PEG molecules from regions of ITOsubstratesnotprotectedbyphotoresist.Thisallowedforprotein (collagen I) adsorption to occur within etched-outregions of the surface. Removal of the photoresist createdan array of protein islands within a layer of PEG-silane (seeFigure 1).ToF-SIMS analysis was employed to characterize chemi-cal composition at different stages in the construction of micropatterned surfaces. Unlike imaging ToF-SIMS that isused more commonly for micropattern characterization(37,38),oursurfaceswerecharacterizedby26keVC 60 + ToF-SIMSrunningintheevent-by-eventbombardment-detectionmode (34, 39). In this approach, surface is bombarded byprojectiles(e.g.,C 60 + particles)andasingleprojectileimpactcreates a hemispherical “crater” of 5 - 10 nm in diameter(40).Massspectraofeachimpactaredetectedone-at-a-timeandareresolvedintimeandspace.Forexample,usingthismethod we could investigate ion masses coinciding withphotoresist or PEG-silane signature peaks and could inferabout contaminating chemical species present on the sur-face. Modification of ITO with PEG silane was confirmed bythe presence of a silane related peak (CH 3 SiO 2 - ) at  m /  z   ) 75(datanotshown).Themassspectraofnegativesecondaryions emitted from surfaces of PEG silane-modified ITObeforeandafterplasmaashingareshowninFigure2A.Thecharacteristic ion of PEG molecules is at  m /  z   )  223 whilethe indium oxide ion (InO - ) is at  m /  z  ) 131. The yields of 131and223secondaryionscorrespondingtoITOandPEGsilane-modified ITO were calculated based on mass spectraobtained before and after oxygen plasma treatment. Thesedata,presentedinTable1,pointtocompleteremovalofPEGmolecules after exposure to oxygen plasma. In addition,absence of the silane peak ( m /  z  ) 75) after oxygen plasmatreatment also indicated removal of silane molecules. Itshould also be noted that in addition to disappearance of PEG mass (223) oxygen plasma treatment also caused adecrease in the yield of   m /  z  ) 131 associated with InO - ionofITOandanincreaseintheyieldsofglasscorrelatedpeaksat  m /  z  ) 137 (SiO 2 ) 2 OH - and  m /  z  ) 257 (SiO 2 ) 4 OH - (Figure2A and Table 1). This suggested etching or chemical modi-fication of the outer layer of ITO. This did not impact ourability to culture cells on the surface; however, the oxygenplasma exposure may need to be optimized in the future toeliminate overetching. Given that the depth of penetrationof C 60 + ions used in surface bombardment was estimatedto be ∼ 10 nm (40), appearance of secondary InO - ions inmassspectraofPEG-modifiedITOindicatesthatPEGsilanethicknessislessthan10nm.ImagingellipsometryandAFMdatapresentedinthefollowingsectionvalidatethisobserva-tion.ToF-SIMS analysis was also used to characterize deposi-tionoftheproteininregionsexposedtooxygenplasma.Fortheseexperiments,photolithographywasusedtomake100  µ m photoresist arrays on PEG silane-modified ITO sub-strates.OxygenplasmawasusedtoremovePEGsilanefromtheexposedregionsasdescribedearlier.Followingthis,thesurface was incubated with collagen and sonicated in ac-etone to dissolve remaining photoresist. After the lift-off process the collagen molecules adsorbed on ITO wereexpected to remain on the surface while protein moleculesdeposited on photoresist were expected to be removed.After photoresist removal, the surface was expected tocontaincollagenislandssurroundedbythePEG-silanelayer(see Figure 1 for description). SIMS analysis of this micro-patterned surface (shown in Figure 2B) pointed to thepresence of several ion fragments associated with peptidebonds (CN - , CNO - , and C 3 N - ). The lift-off process led to adecrease in intensity of protein segments confirming theremoval of collagen adsorbed on top of the resist. Impor-tantly, protein fragments were colocalized in the samespectrum with fragments characteristic of PEG silane at Table 1. Yields of Negative Ions InO - at   m /  z  ) 131and PEG Fragments at   m /  z  ) 223 from Bare ITO,PEG Silane Modified ITO and PEG Silane Coated ITOTreated with Oxygen Plasma to Remove PEG fromthe Substrates samples  Y  InO - ( m /  z  ) 131)  Y  PEG  ( m /  z  ) 223)1. Bare ITO 1.11 × 10 - 3 a 2. PEG coating 2.60 × 10 - 4 2.70 × 10  - 3 3. after O 2  clean 1.95 × 10  - 4 a a Indicates absence of a peak.        A       R       T       I       C       L       E 2596  VOL. 1  •  NO. 11  •  2592–2601  •  2009 Shah et al. www.acsami.org
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