Biointerfaces on IndiumâTin Oxide Prepared from

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Biointerfaces on Indium Tin Oxide Prepared From Organophosphonic Acid Self-Assembled Monolayers Muthukumar Chockalingam, Astrid Magenau, Stephen G. Parker, Maryam Parviz, SRC Vivekchand, Katharina Gaus, and J. Justin Gooding Langmuir, Just Accepted Manuscript • Publication Date (Web): 24 Jun 2014 Downloaded from http://pubs.acs.org on June 27, 2014

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Biointerfaces on Indium Tin Oxide Prepared From Organophosphonic Acid Self-Assembled Monolayers Muthukumar Chockalingam1,2, Astrid Magenau1,3, Stephen G. Parker1,2, Maryam Parviz1,2, S. R. C. Vivekchand1,2, Katharina Gaus1,3, J. Justin Gooding1,2,4*. 1

Australian Centre for NanoMedicine, 2School of Chemistry, 3Centre for Vascular Research,

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ARC Centre of Excellence in Coherent Bio-Nano Science and Technology, The University of

New South Wales, Sydney 2052, Australia KEYWORDS Self-assembled monolayers, organophosphonic acids, biointerfaces, cell-surface interactions.

ABSTRACT

Herein we show the development of biointerfaces on indium tin oxide surfaces prepared from organophosphonate self-assembled monolayers. The interfaces were prepared in a stepwise fabrication procedure containing a base monolayer modified with oligo(ethylene oxide) species to which biological recognition ligands were attached. The density of ligands was controlled by varying the ratio of two oligo(ethylene oxide) species such that only one is compatible with

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further coupling. The final biointerface on ITO was assessed using cell adhesion studies which showed that the biointerfaces prepared on ITO performed similar to equivalent monolayers on gold or silicon.

Introduction

Indium tin oxide (ITO) is both a conducting and transparent surface that is widely used in optoelectronic applications.1 These twin features of conductivity and transparency also give ITO the potential to be used in dual detection systems where electrochemistry and optical microscopy are performed simultaneously. Nowhere is this potential better exemplified than in the elegant work of Amatore and co-workers where fluorescence microscopy and amperometry of single cells was performed in real time.2 In these studies, stimulation of surface immobilized cells resulted in neurotransmitters being released from cells via secretary vesicles. Total internal reflectance fluorescence (TIRF) microscopy showed that amperometric spikes correlated with vesicles docking to the basal membrane of the cell. This study revealed the power of the dual electrochemical/fluorescence microscopy output. However, in these cases there was no control over the surface chemistry presented by the ITO to the cells. Control over cell adhesion has however been shown using organophosphonic acid-based SAMs on ITO by Yousof and coworkers where an electroactive hydroquinone moiety is oxidized to the benzoquinone to allow coupling of amines via an oxyamide tether.3 With adherent cells, we have shown that the number of cell adhesive ligands presented to the cell by a model surface impacts on cell signaling pathways and the migratory ability when induced by a soluble growth factor.4, 5 Many other studies of cell-surface interactions also illustrate that the cell shape, size and cytoskeletal organization is exceedingly sensitive to surface design.6 In


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fact cells can sense changes in adhesive ligand spacing of only 1 nm.7 The implication of this surface sensitivity on cell adhesion is that exploring the influence of surfaces of cell response to stimuli requires surface modification with the molecular level control afforded by self-assembled monolayers.6 On ITO self-assembled monolayers (SAMs) with good control over the presentation of adhesive ligands to cells can potentially be formed using organophosphonic acid-based self-assembled monolayers.8-11 To form such SAMs, the organophosphonic acid moieties bind to the surface via a heterocondensation reaction where loss of water from the hydroxylated ITO surface forms covalent bonds between the surface and the oxygen atoms of the phosphonate. This heterocondensation reaction is frequently facilitated by annealing with the resultant SAMs being quite stable on the ITO surfaces. We have studied parameters such as the impact of the surface structure12 and the solvent from which the SAMs are assembled13 on the quality of the resultant monolayer. Importantly, an organophosphonic acid with a carboxylic acid at the other end from the phosphonic acid, will assemble on a surface exclusively by the phosphonic acid moiety giving a monolayer with a distal carboxylic acid. The importance of the distal carboxylic acid is it is then readily amenable to further coupling reactions as is typically important in forming biointerfaces. The purpose of the present paper is to demonstrate the ability to fabricate biointerfaces on ITO surfaces for cell adhesion studies using the interface as in scheme 1. The two important features of this interface are as follows. Firstly, after formation of the base SAM, carbodiimide coupling is used to attach aminated tetra(ethylene oxide) (EO4) species to the surface. The purpose of the EO4 species is to limit nonspecific adsorption of cells or proteins to the ITO surface as has been extensively demonstrated for gold14,

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surfaces. Secondly, once attached to the


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surface, the EO4 molecules possess two different distal moieties, either a hydroxyl or a methoxy. Further coupling of cell adhesive ligands can be achieved with the hydroxyl16 using disuccinimidyl carbonate, but not to the methoxy terminated species. Thus, by adjusting the ratio of these two molecules during their attachment to the surface, control over the density of cell adhesive ligands presented to the cells is achieved.4, 5 Experimental Methods Indium tin oxide surfaces were from SPI (USA) where smooth amorphous surfaces of ITO on microscope slides gave the highest density SAMs as described previously.12 16Phosphohexadecanoic acid of 99.5% purity (PHDA), dimethylaminopropyl carbodiimide (EDC), N-hydroxysuccinimide, fetal bovine serum, dichloromethane, K2CO3, N,N’-disuccinimidyl carbonate (DSC), 4-dimethyl aminopyridine (DMAP) dimethylformamide (DMF) and methanol were from Sigma-Aldrich (Sydney, Australia). The antifouling molecules 1-amino tetra (ethylene oxide) (H2N-EO4-OH) and 1-amino tetra(ethylene oxide) monomethyl ether (H2N-EO4-OCH3) were obtained from Tim Tec (USA). The fluorescently-labeled pentapeptide GRGDC-Alexa fluor 647 was from Genscript (Sydney). The Alexa Fluor 555 phalloidin, cell culture medium, Dulbecco modified eagle medium (DMEM) and L-glutamine were purchased from Invitrogen. Paraformaldehyde (PFA) 16% w/v, used to fix cells was purchased from Thermo Scientific, and Triton X-100 was obtained from Sigma Ultra. Surface preparation. ITO surfaces were first cleaned in an ultrasonicator with dichloromethane and then methanol for 10 minutes each, followed by sonication in 0.5 M K2CO3 in a 3:1 methanol:MilliQ water mixture for 30 min to remove any residual organic contaminants. The surfaces were then rinsed in copious amounts of MilliQ water. PHDA SAMs were assembled


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from a 5 mM PHDA in methanol solution for 12 h followed by rinsing with methanol and dried under nitrogen gas. The substrates were then annealed at 150˚C for 48 h under nitrogen. To further modify the SAMs, the carboxylic acid terminal of PHDA SAM was activated with 50 mM EDC and 50 mM NHS in an aqueous solution for 2 h. The activated surfaces were then rinsed with MilliQ water and ethyl acetate and incubated for 12 h in 10 mM EO4 in DMF solution containing various ratios of 1-amino tetra (ethylene oxide) to 1-amino tetra(ethylene oxide) monomethyl ether. The modified surfaces were rinsed with ethyl acetate several times and dried under a stream of nitrogen. The hydroxyl terminated-tetra(ethylene oxide) molecules were activated with 0.1 M solution of dry dimethylformamide containing N,N-disuccinimidyl carbonate (DSC) and 4-dimethyl aminopyridine (DMAP) for 12 h. The activated surfaces were then washed with copious amount of ethyl acetate and dried under a stream of N2. The samples were then immersed in 20 mM phosphate buffer containing 15 ug/mL of Alexa fluor 647 tagged GRGDC for 30 min at room temperature. After bioconjugation, the samples were washed with MilliQ water and in PBS containing 0.3% Triton X for 5 min in order to remove any nonspecifically adsorbed peptides from the modified samples. Finally, the samples were rinsed several times with MilliQ water, dried under a stream of N2 and stored in a dry and dark condition in a glass sample tube filled with N2 at 4°C. The samples were once again rinsed with 70% ethanol just before use. Scheme 1 shows the reaction steps of the modification procedure. XPS Characterisation. XPS measurements of unmodified and modified ITO surfaces were taken using an ESCALAB 220iXL spectrometer with Al Kα monochromatic source (1486.6 eV), hemispherical analyzer and multichannel detector. Spectra were recorded in normal emission with the analyzing chamber operating below 10-10 mbar and selecting a spot size of approximately 1 mm2. The angle of incidence was set to 58° with respect to the analyzer lens


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with maximum sensitivity. Wide scans were obtained using pass energy of 100 eV and highresolution spectra were obtained using pass energy of 20 eV. All the binding energies are referenced to the main hydrocarbon peak binding energy of 285.0 eV. The fitting of the spectra was performed using a nonlinear least square procedure that involved a simple LorentzianGaussian function, prior to peak fitting using a background subtraction (Shirley method). Cell Culture. HeLa cells were cultured to confluence in DMEM culture media supplemented with 5% fetal bovine serum, 0.1% glutamine at 37 ˚C in 5% CO2. Every 3-4 days, the cells were detached from the culture flask using trypsin. After washing with 0.9% sodium chloride, the cells were resuspended in fresh media. The resuspended cells were counted using a hemocytometer. From that, 50,000 cells per mL in culture media was added to each modified ITO surface. The surfaces were incubated for 3 h. The adhered cells on the modified ITO surfaces were fixed in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) for 20 min. The fixed cells were washed with PBS twice and permeabilized in 0.1% Triton X in PBS for 5 min. The permeabilized cells on surfaces were then washed twice with PBS and stained with Alexa Fluor 555 phalloidin (1:200 dilution in PBS) for 20 min. The stained cells and surfaces were washed with PBS twice and mounted on a glass slide with mowiol gel. Epi-fluorescence microscopy was used to investigate cells with a relatively good resolution at 20X magnifications. Attached HeLa cells on different ITO surfaces were imaged for cell numbers and cell spreading. Alexa Fluor 555 phalloidin with excitation at 510-560 nm and emission above 610 nm was used to investigate changes in cytoskeleton of cells on different ITO


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surfaces. The obtained images were processed using ImageJ analysis software with appropriate plugins for distance, area and cell counting analysis.17 Results and Discussion Surface characterisation of the biointerface. The basic fabrication of the biointerface is shown in Scheme 1. Initially, a cleaned ITO surface is modified with PDHA using the T-Bag method9 followed by activating the distal carboxylic acid with EDC/NHS to create an active succinimide ester-terminated SAM which is then susceptible to nucleophilic attack from amines such as from the H2N-EO4-OH and H2N-EO4-OCH3. The distal hydroxyl on the H2N-EO4-OH is then activated with DSC/DMAP in DMF16 to give another succinimidyl ester-terminated SAM to which the cell adhesive peptide GRGDC can attach via formation of an amide bond.18 The RGD peptide motif is well known to bind to integrins on cell surfaces as the first stage of the formation of cell focal adhesions to which the cell cytoskeleton is linked.6 Therefore, by varying the ratio of H2N-EO4-OH to H2N-EO4-OCH3, the density of RGD ligands on the modified ITO surface can be controlled.


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Scheme 1. The stepwise fabrication of biointerfaces on ITO surface using organophosphonate SAMs. Carboxyl terminated PHDA base layer SAMs were activated with EDC/NHS, followed by coupling with 100% hydroxyl terminated 1-amino tetra(ethylene oxide) molecules for XPS surfaces or a mixture of hydroxyl terminated and methoxy terminated 1-amino tetra(ethylene oxide) molecules for surfaces used for cell-surface interactions studies. In next step only hydroxyl terminal groups of tetra(ethylene oxide) was activated and coupled with GRGDCAlexa fluor 647.


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We have previously used a very similar strategy to fabricate model surfaces for investigating cell adhesion on silicon surfaces.5 In that study, the RGD ligand spacing was estimated to vary from 1.4 nm for 100% H2N-EO4-OH, 44 nm for a ratio of 1:103 H2N-EO4-OH:H2N-EO4-OCH3, 1,380 nm for 1:106 and 43,700 nm for 1:109. Furthermore, that study showed the greatest number of cells on the 1:106 surface but the greatest cell spreading and greatest outside-in signaling on the 1:103 surface. The key difference between the monolayers formed on silicon in the previous study5 and the new biointerfaces presented herein for ITO is the base monolayer to form a carboxylic acid-terminated surface. Thereafter the fabrication is identical. The steps involved in fabricating the biointerface were characterized using XPS. Figure 1 shows the C1s, N1s, P2p and S2p narrow scans for each of the steps in forming the interface, A) modification of ITO with PHDA, B) attachment of H2N-EO4-OH and H2N-EO4-OCH3 and C) attachment of the resultant peptide. After step A, The high-resolution C1s scan (Figure 1A) was deconvoluted with fitting to three functions: a) a main peak centred at 285 eV attributed to aliphatic carbon-bonded carbons (C-C)20-22; b) a signal at 287 eV corresponding to oxygenbonded carbon (C-O)20, 22 originating from the ITO surface; and c) a high-binding energy signal at 289.5 eV assigned to the electron-deficient carbon atom within the carboxylic group (OC=O)20,

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. The N1s XPS data showed the absence of nitrogen in PHDA modified surfaces.

However, the N1s region after attachment of the 1-amino tetra(ethylene oxide) (Figure 1B) revealed nitrogen species present on the surface in three distinct environments. A peak at 399 eV, attributed to the electron-rich nitrogen atoms within the acylurea intermediates formed during the activation of the acid groups,23 a peak at 400.2 eV which correlates to the formation of an amide bond20 and provides evidence for the successful nucleophilic attachment of the 1-amino tetra(ethylene oxide) and a peak at 402 eV, assigned to unreacted NHS groups still present on the


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surface.24 The strong presence of this peak in an NHS-activated PHDA surface gives further confidence in the assignment of this peak [figure S1 in the supporting information (SI)]. The C1s region of this surface revealed the same peaks present on the acid-terminated surface; however an increase in the size of the peak at 287 eV provides further evidence for the C-O rich ethylene oxide molecule. As can be seen in scheme 1, for the modification of the PHDA with the ethylene oxide, there is a linear relationship between the amide:phosphonate stoichiometric ratio and the coupling efficiency where a 1:1 stoichiometric ratio is expected for a 100% coupling efficiency. Therefore, the observed ratio of 0.72 (calculated by comparing the area under the amide peak in the N 1s region with the phosphonate peak in the P 2p region for the H2N-EO4-OH-terminated surface) suggests that a coupling efficiency of 72% is obtained for this step. The GRGDC-Alexa fluor 647 peptide was conjugated to the modified surface, using DSC/DMAP activation of the 1-amino tetra(ethylene oxide). The XPS analysis is shown in Figure 1C. The similar peaks from the ethylene oxide-terminated surface were observed in the N1s narrow scan; however an increase in the peak at 400 eV, originating from the amide groups within the peptide backbone, provides evidence for the presence of the peptide on the surface. The coupling yield of the GRGDC-Alexa fluor 647 was evaluated by comparing the amide peak within the N1s region with the sulfite peak that originates from the sulfite groups contained in the Alexa fluor 647, within the S2p region. That is, in this case the Alexa fluor 647 dye is used primarily as an XPS label. Unlike the previous step, where there was a linear relationship between the stoichiometric ratio and the coupling efficiency as a result of the absence of any phosphonate groups within the 1-amino tetra(ethylene oxide), the presence of both sulfite groups (4 per GRGDC-Alexa fluor 647 molecule) and amine/amide groups (10 per GRGDC-Alexa fluor 647 molecule) within the GRGDC-Alexa fluor 647 resulted in a logarithmic relationship between


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the S2p (sulfite):N1s (amide) stoichiometric ratio and the coupling efficiency that ranges between zero for a 0% coupling efficiency and 4/11 (the extra amide arises from the link between the PHDA and the H2N-EO4-OH) for a 100% coupling efficiency. The observed ratio of 0.32 suggests that a 47% coupling efficiency is obtained for the conjugation of the peptide-dye conjugate on the DSC/DMAP-activated-1-amino tetra(ethylene oxide) molecule. Combining this efficiency with the 72% efficiency from the modification of acid groups with ethylene oxide molecules, the overall efficiency was calculated to be 34%. That is, 34% of the base PHDA molecules are modified with the GRGDC peptide via the ethylene oxide moiety. The coupling yields of these individual steps on ITO surfaces was consistent with previously reported coupling yields on silicon surfaces.


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Figure 1. C1s, N1s, P2p and S2p XPS analysis of GRGDC attachment on ITO surfaces - (A) carboxyl terminated PHDA SAM on ITO, (B) after 1-amino tetra(ethylene oxide) attachment and (C) after GRGDC-Alexa fluor 647 attachment.

Knowledge of these coupling yields is important as they allow an estimate of the spacing between RGD ligands on a surface. The surface coverage of organophosphonate has been calculated by Pawsey et al.25 to be 4 x 1014 molecules/cm2. Hence with a coupling yield of GRGDC-Alexa fluor 647 per phosphohexadecanoic acid for the 100% EO4-OH surface being 34%, the estimated average spacing between adhesive ligands is 1 nm. Assuming the same coupling yields for surfaces where the H2N-EO4-OH is diluted with the EO4-OCH3 the GRGDCAlexa fluor 647 average spacing is 31 nm for the 1:103 EO4-OH:EO4-OCH3 surface, 968 nm for the 1:106 EO4-OH:EO4-OCH3 surface and 30,597 nm for the 1:109 EO4-OH:EO4-OCH3 surface. Cell-surface interactions. The plating of HeLa cells onto the modified ITO surfaces with different RGD ligand densities is shown in Figure 2. Two important observations can be made from the epi-fluorescent images. Firstly, the cells are most abundant on the 100% EO4-OH, and 1:103 EO4-OH:EO4-OCH3 surfaces and secondly on these same two surfaces the cells are well spread, indicating they are well adhered with prominent focal adhesions. Cells are also abundant and well spread on the 1:106 EO4-OH:EO4-OCH3 surface but with lower RGD ligand density or


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no RGD ligands, the abundance of cells is low and the cells that are visible are rounded. Rounded cells indicate that the cells are poorly adhered, unhealthy or dead.

Figure 2. HeLa cells on ITO surfaces modified with different GRGDC dilutions - (A) 100% EO4-OH, (B) 1:103 EO4-OH:EO4-OCH3 surface, similarly, (C) 1:106, (D) 1:109 (E) 100% EG4OCH3. Quantification of the cell density and cell spreading, as determined from the average area occupied by a cell, are shown in Figure 3. The fact that the cells are rare and rounded on the 100% EG4OCH3 surface shows the tetra(ethylene oxide) layer is effective at being a nonadherent surface for HeLa cells. The low abundance of cells on the 1:109 EO4-OH:EO4-OCH3 surface


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shows that there are insufficient RGD ligands on the surface for cells to form sufficient focal adhesions, primarily because the formation of focal adhesions is dependent on sufficiently closely spaced RGD ligands to allow clustering of integrins to form focal adhesions in the cell membrane. With large spacing there is insufficient RGD ligands density for clusters of integrins to form. Spatz and co-workers26 have suggested the presence of some cells on surfaces such as the 1:109 EO4-OH:EO4-OCH3 surface is indicative of the random distribution of RGD ligands on the surface such that in some regions where there are sufficient RGD ligands for adhesion to occur. Spatz and co-workers26 showed this by comparing surfaces with ordered arrays of ligands versus an average distribution of ligands. Importantly, the density of adherent cells and the area occupied by a given cell is greatest on the 1:103 surfaces which is consistent with previous observations by us5 and others27, 28 which show cells spread well on surfaces with 70 nm spacing where cell spreading and adhesion was highly restricted. The spacing is consistent with the hypothesis that cellular adhesion is optimal when the presentation of cell adhesive ligands is similar to periodicity found in the natural biological system of 67 nm.29 From the perspective of the purpose of the current study, to show that well performing biointerfaces can be prepared on ITO surfaces using organophosphonate self-assembled monolayers, the results in Figure 2 and Figure 3 are very encouraging. This is because the cell adhesion results are consistent with other cell adhesion results on a variety of other surfaces6, 30 which verifies that the strategy we have employed to make biointerfaces on ITO surfaces for the first time gives biointerfaces that perform as well as ‘gold standard’ monolayers on gold31, 32 and silicon4, 5.


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Figure 3. Quantification of the adhesion of HeLa cells on ITO surfaces modified with different GRGDC dilutions (100% EO4-OH, 1:103 EO4-OH:EO4-OCH3, similarly 1:106, 1:109 and 100% EG4OCH3) showing a) the number of adherent cells per area on the different surface and b) the average area each cell occupies on each surface. Conclusions In conclusion, we have shown that biointerfaces can be prepared on indium tin oxide surfaces using organophosphonate self-assembled monolayers. The interfaces are prepared in a stepwise fabrication procedure where a base monolayer is formed followed by the coupling of oligo(ethylene oxide) species and thereafter the interface is given the capability to selectively bind to other proteins and cells in a biological milieu via the attachment of biorecognition species. The density of the biorecognition species can be controlled by varying the ratio of two different oligo(ethylene oxide) species coupled to the base SAM, one of which is amenable to further coupling reactions, the other which is not. The performance of the biointerfaces on ITO was assessed using cell adhesion studies as a model


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application. The cell adhesion studies illustrated that the oligo(ethylene oxide) species were effective at limiting cell adhesion such that the number and spreading of cells is dependent on the density of the cell adhesive GRGDC ligands attached to the surface. Most importantly, what these cell adhesion studies show is that the biointerfaces prepared on ITO perform similar to the equivalent monolayers formed on gold or silicon.

AUTHOR INFORMATION Corresponding Author Justin Gooding: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This research was funded with support from the Australian Research Council. ACKNOWLEDGMENT We thanks the Australian Research Council’s Discovery Projects Funding Scheme (DP1094564), Australian Research Council Centre of Excellence in Coherent Bio-Nano Science and Technology (project number CE140100036) and the University of New South Wales for funding.


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