Selective Release of DNA from the Surface of Indium−Tin Oxide Thin

Anal. Chem. 2007, 79, 2050-2057

Selective Release of DNA from the Surface of Indium-Tin Oxide Thin Electrode Films Using Thiol-Disulfide Exchange Chemistry Eric J. Moore,*,† Maeve Curtin,† Justin Ionita,‡ Anita R. Maguire,§ Giacomo Ceccone,| and Paul Galvin†

Tyndall National Institute, University College Cork, Cork, Ireland, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, Department of Chemistry, Analytical & Biological Chemistry Research Facility, University College Cork, Cork, Ireland, and Institute for Health and Consumer Protection, Joint Research Centre, I-21020 Ispra (VA), Italy

A new challenge in biointerfacial science is the development of dynamic surfaces with the ability to adjust and tune the chemical functionality at the interface between the biological and nonbiological entities. In this paper we describe fabrication of indium-tin oxide (ITO) electrodes and the design of a ligand that can be switched to enable selectively controlled interactions with DNA. Tailoring the surface composition of the ITO electrode to optimize its optical and electrical properties was also studied. The surface attachment chemistry investigated utilizes thioldisulfide exchange chemistry. This chemistry involved the covalent attachment of a thiol-functionalized silane anchor to a hydroxyl-activated ITO electrode surface. Subsequent reaction with 2-(2-pyridinyldithio)ethanamine hydrochloride formed the disulfide bridge and provided the terminal amine group, which facilitates addition of a cross-linker. DNA was then covalently bound to the cross-linker, and hybridization with the complementary Cy3-labeled target DNA was achieved. Selective release of the attached DNA was demonstrated by both chemical and electrical reduction of the disulfide bond. The surface chemistry was then recycled, and rehybridization of the target DNA was achieved. The ability to control specific release of biomolecules has applications for the development of novel biosensor platforms and a range of medical devices. Biomolecules can adhere and adsorb nonspecifically to most surfaces through a variety of mechanisms (electrostatic interaction, hydrogen bonding, hydrophobic interactions, and/or a combination of these), resulting in biofouling.1 Direct control over immobilization and specific release of biomolecules on electrode surfaces is very important in the development of novel biosensors and biochips. In this paper a dynamic substrate2 which incorporates a switchable ligand that allows for specific release of biomolecules when subjected to either chemical or electrical reduction is described. For many applications it would be beneficial to * To whom correspondence should be addressed. E-mail: [email protected]. Phone: +353 21 490 4451. Fax: +353 21 427 0271. † Tyndall National Institute, University College Cork. ‡ Northwestern University. § Department of Chemistry, University College Cork. | Joint Research Centre. (1) Beech, I. B.; Sunner, J. A.; Hiraoka, K. Int. Microbiol. 2005, 8, 157-168. (2) Yousaf, M. N.; Mrksich, M. J. Am. Chem. Soc. 1999, 121, 4286-4287.

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be able to pattern and immobilize biomolecules at specific locations on an electrode surface, retain their orientation and functionality so that they can further interact with target analytes, and finally remove both the biomolecule and a target from the sensor surface. The development of such tunable surfaces would allow for further control over such types of analysis. Indium-tin oxide (ITO) was used as the thin film conductive material in the biosensor development. Apart from its conductive property3 it has many other advantages such as high optical transmittance,4 a robust nature, the ability for easy patterning, and an excellent adhesion property to substrates. ITO thin-coated films have been used in a wide variety of applications5-9 including sensors, liquid crystal displays, heat-reflective mirrors, solar cells, and optoelectrical devices. The preparation method of the ITO film governs both the optical10 and the electrical properties.11 Formation of stable and highly structured self-assembled monolayers12 (SAMs) on ITO thin films is dependent on both the deposition and pretreatment procedures used in the manufacture.13 SAMs form uniform surfaces and are very useful in materials chemistry14 and in the development of biointerfacial surfaces. The surface chemistry that is described in the development of the dynamic substrate is based on thiol-disulfide exchange chemistry.15 Figure 1 illustrates the reaction profile for the surface chemistry attachment. The attachment involves the formation of a monolayer of (3-mercaptopropyl)trimethoxysilane (3-MPT) on the biosensor surface (ITO). The ITO-coated sample is initially (3) Bhatti, M. T.; Rana, A. M.; Khan, A. F. Mater. Chem. Phys. 2004, 84, 126130. (4) Davenas, J.; Besbes, S.; Ben Quada, H. Synth. Met. 2003, 138, 295-298. (5) Chopra, K. L.; Major, S.; Pandya, D. K. Thin Solid Films 1983, 102, 1-46. (6) Vicente, F.; Garcia-Jareno, J. J.; Benito, D.; Agrisuelas, J. J. New Mater. Electrochem. Syst. 2003, 6, 267-274. (7) Pankove, J. I. Display Devices, Topics in Applied Physics; Springer: Berlin, 1980. (8) Luff, B. J.; Wilkinson, J. S.; Perrone, G. Appl. Opt. 1997, 36, 7066-7072. (9) Kim, J. S.; Granstrom, M.; Friend, R. H.; Johansson, N.; Salaneck, W. R.; Daik, R.; W. J. F. F. C. J. Appl. Phys. 1998, 84, 6859-6870. (10) Kachouane, A.; Addou, M.; Bougrine, A.; El idrissi, B.; Messoussi, R.; Regragui, M.; Be´rnede, J. C. Mater. Chem. Phys. 2001, 70, 285-289. (11) Tsuchiya, T.; Niino, H.; Yabe, A.; Yamaguchi, I.; Manabe, T.; Kumagai, T.; Mizuta, S. Appl. Surf. Sci. 2002, 197-198, 512-515. (12) Finklea, H. O. Self-Assembled Monolayers on Electrodes. In Encyclopedia of Analytical Chemistry; John Wiley & Sons Ltd.: New York, 2000. (13) Asanov, A. N.; Wilson, W. W.; Oldham, P. B. Anal. Chem. 1998, 70, 11561163. (14) Chaki, N. K.; Aslam, M.; Sharma, J.; Vijayamohanan, K. Proc. Indian Acad. Sci. (Chem. Sci.) 2001, 113, 659-670. (15) Carlsson, J. Hind. Antibiot. Bull. 1978, 20, 105-108. 10.1021/ac0618324 CCC: $37.00

© 2007 American Chemical Society Published on Web 01/25/2007

Figure 1. Reaction profile for disulfide attachment chemistry showing (1) attachment of the silane anchor, (2) covalent attachment of PDEA and hence formation of the disulfide bond, (3) covalent attachment of the cross-linker molecule PDITC, (4) immobilization of DNA and hybridization of the complementary DNA strand labeled with the Cy3 fluorophore, and (5) cleavage of the disulfide bond and re-formation of the thiol functionality on the surface.

pretreated by oxygen plasma to functionalize the surface with hydroxyl groups, which facilitates covalent attachment of the silane anchor to the ITO surface.16 Plasma treatment is a nondestructive technique and does not interfere with the physical properties of the thin electrode film. The disulfide bond is formed by reacting the free thiol groups from the 3-MPT with 2-(2-pyridinyldithio)ethanamine hydrochloride (PDEA). The surface is modified further with a cross-linker, 1,4-phenylene diisothiocyanate (PDITC), via covalent attachment to PDEA, by means of a Schiff base reaction. This homobifunctional cross-linker is commonly used in bioconjugate chemistry as a tool for the conversion of supportbound amino groups into reactive isothiocyanates capable of forming a covalent bond with amino groups of free molecules.17 This enables covalent immobilization of amine-functionalized oligonucleotides (DNA). Once immobilization of the DNA is completed, hybridization of a Cy3-labeled complementary DNA was achieved. Application of a reductive potential to the surface effectively cleaves the disulfide bond, leaving the free thiol groups of 3-MPT on the surface, and the biomolecules that were attached can be easily washed off and hence removed from the electrode surface. The attachment chemistry can then be reapplied to the surface by reaction with PDEA, followed by PDITC, etc. The sensor surface can therefore be recycled, and either the same or another oligonucleotide can be immobilized. The reduction of the disulfide ligand can also be achieved with chemical treatment of the surface. Target DNA labeled with paramagnetic beads18 was also hybrid(16) Moore, E.; O’Connell, D.; Galvin, P. Thin Solid Films 2005. (17) Beier, M.; Hoheisel, J. D. Nucleic Acids Res. 1999, 27, 1970-1977. (18) Ko ¨hler, J. M.; Csaki, A.; Reichert, J.; Moller, R. Sens. Actuators, B 2001, 76, 166-172.

ized and selectively released through such chemical reduction using 1,4-dithioerythritol (DTE) as the reducing agent. These paramagnetic beads have been used as labels to detect target DNA in integrated magnetic sensor platforms.19,20 Previous examples of substrates that can release attached biomolecules have relied on thermally responsive poly(N-isopropylacrylamide) films.21,22 It has also been demonstrated that a dynamic SAM could be switched from a state that is initially inert to a state that permits the Diels-Alder-mediated immobilization of ligands.2 Selective and irreversible immobilization of proteins to SAMs of alkanethiolates on gold by using active-site-directed ligands has also been described.23 Current methodology in the development of such tunable surfaces has focused primarily on the following: photolithography,24 solvent-selective switching,25 pH switching control,26 or electrical potential switching.27 Photolithography is perhaps the most commonly used technology for patterning surfaces for both micro- and nanometer scales and involves the process of transferring geometric shapes on a (19) Graham, D. L.; Ferreira, H. A.; Freitas, P. Trends Biotechnol. 2004, 22, 455-462. (20) Baselt, D. R.; Lee, G. U.; Natesan, M.; Metzger, S. W.; Sheehan, P. E.; Colton, R. J. Biosens. Bioelectron. 1998, 13, 731-739. (21) Okano, T.; Yamada, N.; Okuhara, M.; Sakai, H.; Sakurai, Y. Biomaterials 1995, 16, 297-303. (22) Ito, Y.; Chen, G.; Guan, Y.; Imanishi, Y. Langmuir 1997, 13, 2756-2759. (23) Hodneland, C. D.; Lee, Y.; Min, D.; Mrksich, M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5048-5052. (24) Kruger, C.; Jonas, U. J. Colloid Interface Sci. 2002, 252, 331-338. (25) Retsos, H.; Gorodyska, G.; Kiriy, A.; Stamm, M.; Creton, C. Langmuir 2005, 21, 7722-7725. (26) Lupitskyy, R.; Roiter, Y.; Tsitsilianis, C.; Minko, S. Langmuir 2005, 21, 8591-8593. (27) Jiang, X.; Ferrigno, R.; Mrksich, M.; Whitesides, G. M. J. Am. Chem. Soc. 2003, 125, 2366-2367.

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mask to the surface. Soft lithography is another widely used method for pattening surfaces; however, in this case an elastomeric stamp to transfer a pattern to a substrate is utilized. Using this technique and electroactive substrates, it is possible to pattern the attachment of two or more different biomolecules. Solventselective switching has utilized ligands that contain stimuliresponsive amphiphilic Y-shaped structures. These ligands are constructed with dissimilar arms attached to a single grafting point. Switching of surface nanostructures can be achieved upon treatment with selective solvents. pH switching control has also been reported and involves the investigation of a new class of ordered molecular assemblies. These assemblies are stabilized by strong pH-dependent noncovalent interactions. The pH dependency leads to abrupt destabilization of the entire assemblies by acidification, and the ordered layers can thus be repeatedly formed and washed away using one single substrate. The properties of a surface can be controlled by changing the conformation of molecules in a SAM on that particular surface. For example, an electrical potential can be used to reversibly switch an alkanethiolate monolayer between hydrophilic and hydrophobic states. Biomolecular surfaces that release ligands under electrochemical control have also been reported in the literature.28,29 Earlier studies have shown that SAMs of short-chain alkanethiols can be electrochemically desorbed from gold surfaces through the reductive cleavage of the sulfur-gold bond.30,31 It has also been demonstrated that electrochemistry could be used to trigger on demand the release of assembled 350-bp dsDNA and 25-mer ssDNA layers from gold ultramicroelectrodes, with the objective of establishing nonviral controlled delivery of nucleic acids to specific sites.32 This ability to control specific attachment and release of biological components would greatly benefit existing technologies such as biosensors, medical devices, or indeed any substrate exposed to a biological environment. The challenge exists to develop novel dynamic surfaces which can selectively release biomolecules33 in a form of “self-cleaning” that could prevent biofouling. This would be especially effective in automated remote optical sensor devices which operate in aquatic environments where blockage by adhesion of biological materials prevents the sensor from functioning properly and also in the field of biomedical implants (e.g., stents). Biosensors traditionally promise low-cost, rapid, and simple-to-operate analytical tools; however, they are usually only one-shot disposable systems.34 It is envisaged that integration of such dynamic ligands would provide an alternative method of recycling biosensor surfaces for repeated biomolecule adhesion. This would be especially important for on-line sensing applications. There also exists the possibility of linking this technology with other developing concepts such as electrowetting,35,36 where the mobility of biomolecules on the surface can also be controlled. This would (28) Hodneland, C. D.; Mrksich, M. J. Am. Chem. Soc. 2000, 122, 4235-4236. (29) Kim, K.; Yang, H.; Kim, E.; Han, Y. B.; Kim, Y. T.; Kang, S. H.; Kwak, J. Langmuir 2002, 18, 1460-1462. (30) Widrig, C. J. Electroanal. Chem. 1991, 310, 335. (31) Zhong, C. J. Electroanal. Chem. 1997, 425, 147. (32) Wang, J. Langmuir 1999, 15, 6541-6545. (33) Schmid, E. L.; Keller, T. A. Z. D.; Vogel, H. Anal. Chem. 1997, 69, 19791985. (34) Moore, E. J.; Kreuzer, M. P.; Pravda, M.; Guilbault, G. G. Electroanalysis 2004, 16, 1653-1659.

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introduce another level of control to the surface chemistry through manipulation of direction and movement. EXPERIMENTAL SECTION Reagents. Indium-tin oxide powder, (3-mercaptopropyl)trimethoxysilane, 2-(2-pyridinyldithio)ethanamine hydrochloride, 1,4-phenylene diisothiocyanate, 1,4-dithioerythritol were purchased from Sigma. The oligonucleotides used were the 20-mer Tyndall1-NH2 and the complementary Cy3-labeled strand (50-mer Tyndall1-Cy3). Roche streptavidin magnetic particles were obtained from Roche Diagnostics GmbH. The preparation buffers were 1 M Tris/ HCl, pH 7.0, with 1% (v/v) N,N-diisopropylethylamine printing buffer and 0.1% sodium dodecyl sulfate (SDS), respectively. Diethanolamine (DEA) and buffer (pH 9.5), which consisted of 0.1 M DEA, 50 mM KCl (Sigma), and 1 mM MgCl2 (Sigma), were used. Sigma Perfect Hybridization 1× buffer solution was used to store the DNA molecules. Buffers used in the preparation of the blocking solution were Tris-buffered saline (TBS) and phosphate-buffered saline (PBS). All other reagents were of analytical grade or better, and all solutions were prepared daily with nanopure water (grade 18 MΩ) purified using a Maxima Ultra Pure water purification system (di-H2O). Instrumentation. A Hitachi S4000 SEM system, a Topometrix Explorer AFM system, a Dataphysics optical contact angle instrument, a Wentworth PML-8000 probe station for sheet resistance measurements, a Zeiss Axioskop 2 epifluoresence microscope coupled to a CCD camera, a BAS electrochemical workstation (Bioanalytical Systems, BAS), an AXIS ULTRA XPS instrument, and a Leybold LAB600 e-beam evaporator were used. Film Preparation and Surface Chemistry Attachment. The ITO was evaporated in a Leybold LAB600 e-beam evaporator at 230 °C, and the base pressure was kept at 1 × 10-6 mbar. Evaporation was Ar ion-assisted to increase film density and uniformity and improve optical transparency. Ar was introduced to the chamber at 4 sccm and ionized across a W filament (120 V, 16 A). O2 was introduced to the chamber at a flow rate of 7 sccm to improve film stoichiometry. The overall backing pressure was 4.2 × 10-4 mbar. The evaporation rate was 2 Å/s. The ITO film thickness was kept constant at 220 nm. Oxygen plasma treatment was done for 30 min at 50 W to create hydroxyl groups on the surface of the ITO. The ITO-coated electrodes were pretreated by a solvent wash to clean the surface and prepare the samples for silanization. They were first submerged in a beaker of trichloroethylene and sonicated for 10 min. Then they were placed in a separate beaker of acetone at 50 °C for another 10 min, and finally they were sonicated for a further 10 min in a beaker containing isopropyl alcohol. The substrates were then dried with a nitrogen gun prior to silanization. The ITO-coated electrodes were silanized in a toluene solvent containing 1 mM 3-MPT through sonication for 60 min at room temperature (rrt). After this time, the samples were rinsed sequentially with chloroform and di-H2O and then cured at 120 °C for 15 min in a fan-operated oven. The surface was then treated by immersion of the silanized wafers in 1.5 mL of a 20 mM PDEA solution, pH 4, containing 1 M NaCl for approximately 10 min. This reaction forms the disulfide bridge. (35) Walker, S.; Shapiro, B. Lab Chip 2005, 5, 1404-1407. (36) Pfohl, T.; Mugele, F.; Seemann, R.; Herminghaus, S. ChemPhysChem 2003, 4, 1291-1298.

The samples were then washed with di-H2O and dried under a stream of nitrogen. The amino-functionalized surface was then activated for DNA attachment by immersion of the treated samples in 40 mL of a 1 mM PDITC solution containing 10% anhydrous pyridine in dimethylformamide (DMF) for 2 h. The samples were washed sequentially with DMF and 1,2-dichloroethane and dried under a stream of nitrogen. Prior to DNA immobilization a base was added to the probe nucleic acid solution (print buffer) as the cross-linker PDITC has its optimal reactivity in a basic environment.37 The print buffer used was 1 vol % N,N-diisopropylethylamine prepared in Tris/ HCl, pH 7.4, as it is nonnucleophilic and hence does not compete for the reactive sites on the ITO surface. The DNA probe immobilization was done by printing with a micropipet 0.2 µL of the oligonucleotide probe (4 µM Tyndall-1-NH2) onto the surface of the ITO-coated samples and incubation at 37 °C in a humid chamber for overnight. The samples were then washed with diH2O and dried in a stream of nitrogen. The blocking step was then done to prevent nonspecific binding of target DNA. A 1 mL sample of TBS followed by 9 mL of di-H2O were added together in a beaker. A 0.5 g sample of dry skimmed milk (SMA) was then added to this solution and allowed to dissolve. From this solution 1 mL was removed and dissolved in 49 mL of PBS, pH 7.4. The ITO samples were covered and left to block for 2 h at rt. After blocking was completed, they were rinsed in di-H2O and dried with the nitrogen gun. Hybridization of the complementary Cy3labeled target DNA (4 µM Tyndall-1-Cy3) was done at 42 °C for a further 2 h, after which time the ITO electrodes were washed with di-H2O and dried in a stream of nitrogen. The surfaces of the electrodes were then visualized using the epifluorescence microscope to confirm that hybridization occurred. Alternative attachment chemistry38 was used as a control to establish that the selective release of the immobilized DNA was specific to the dynamic ligand. The ITO-coated samples were silanized in a methanol/di-H2O (19:1 ratio) solution containing 3% (3-aminopropyl)trimethoxysilane for 30 min at rt. After this time the samples were rinsed sequentially with methanol and di-H2O and then cured at 120 °C for 15 min in a fan-operated oven. The surface was then activated for DNA attachment by immersion of the silanized substrates in a DMF solution containing 10% pyridine and 1 mM PDITC for 2 h. The samples were washed sequentially with DMF and 1,2-dichloroethane and dried under a stream of nitrogen. The same procedure as described earlier in this section was used to immobilize the probe DNA and hybridize the target DNA. Film Characterization and Analysis of Surface Chemistry. A Wentworth PML-8000 four-point probe station was used to measure the conductivity of the ITO-coated films on Pyrex wafers. Measurements were taken at different positions on the surface and the results averaged (n ) 5 per sample). Optical measurements were also investigated to determine the transmittance (%) and also for verification of the attachment chemistry. Surface topography was also examined using SEM and AFM imaging. Water contact angles were measured with static drops (4 µL) placed onto the surface of the ITO samples. The measurement was done immediately to avoid any loss of water from evaporation (37) Charles, P. T.; Vora, G. J.; Andreadis, J. D.; Fortney, A. J.; Meador, C. E.; Dulcey, C. S.; Stenger, D. A. Langmuir 2003, 19, 1586-1591. (38) Manning, M.; Galvin, P.; Redmond, G. Am. Biotechnol. Lab. 2002, 20, 16.

effects. This was critical to ensure error was to be kept at a minimum. The reported values are from contact angle measurements averaged for n ) 5 repeats and taken at different sites on the ITO-coated surface. This also provided representative results for the uniformity of the film. The standard deviation of the contact angle from spots on the same sample never exceeded 2-4°. XPS was done with an AXIS ULTRA (KRATOS, U.K.). A monochromatic Al KR X-ray source, a 700 × 400 µm2 spot size, and an electron charge neutralizer (V ) -3.9 eV) were used. Analysis was performed with 160 and 20 eV pass energies for survey and high-resolution spectra, respectively. Data analysis was carried out with the CASA XPS program. TOF-SIMS analysis was performed with a TOFIV (ION-TOF GmbH, Germany) equipped with a Ga ion gun, operating at 25 kV. Chemical and Electrical Reduction of the Disulfide Bond. The surface-modified ITO substrates immobilized with the Cy3labeled target DNA were treated with 0.1 M DTE (in di-H2O) for 12 h at rt. This reaction was done in the fume hood. After this time the samples were removed from the reducing agent, washed with di-H2O, dried under a stream of nitrogen, and examined under an epifluorescence microscope, and the image results were recorded. The ITO thin film electrode (working electrode) was connected to the electrochemical workstation via a crocodile clip. The electrode was then held in place in a beaker containing 5 mL of 0.1 M DEA electrolyte solution. A Pt wire was used as the counter electrode and also connected via a crocodile clip to the electrochemical workstation. The distance between the two electrodes was kept constant for all experiments. A potential was applied through the working electrode and maintained for 10 s. After application of the electrical potential the working electrode was washed with di-H2O and then dried under a stream of nitrogen. The surface of the electrode was then visualized using the epifluorescence microscope to confirm the selective release of the Cy3-labeled oligonucleotide. RESULTS AND DISCUSSION Film Preparation. The key factors in the formation of ITOcoated films are the direct control over the film uniformity and optical and conductivity properties. These three areas are essential to manufacture reproducible and reliable films. Conditions were optimized that would ensure a good uniform deposition of ITO, maintain a high optical transmittance of >80%, and also a provide sheet resistance of 80% was achieved with a reproducible sheet resistance of 34 Ω/sq. Figure 2 shows the achieved transmittance results for the ITO thin film electrode coated onto a quartz substrate. SEM and AFM analyses were used to verify the surface topography and to determine ITO film uniformity across the surface, and the results obtained are illustrated in Figure 3. The SEM picture (1) shows that the surface was coarse and compact with grain sizes of approximately 25 nm. Electron-beam deposition and plasma treatment gave a uniform granular surface of ITO, which was reproducible at a thickness of 220 nm. The AFM image (2) clearly illustrates the uniformity of the ITO-coated surface. A value of 0.5455 nm in root-mean-square with a relative standard deviation (RSD) of 6% was obtained for the surface roughness. Analytical Chemistry, Vol. 79, No. 5, March 1, 2007

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Table 1. Contact Angle Measurements Obtained for Surface Attachment Chemistry Modification of the ITO Thin Electrode Films attachment chemistry

contact angle (deg)

RSD (%)

attachment chemistry

contact angle (deg)

RSD (%)

ITO 3-MPTS

18 54

14 7

PDEA PDITC

61 76

6 9

Table 2. Elemental Concentration (atom %) at Two Different Angles Derived from Survey XPS Scans ITO

Figure 2. Transmittance spectra obtained for e-beam-evaporated ITO onto a quartz substrate in the visible region.

Figure 3. (1) SEM picture illustrating the surface topography of the ITO coated thin electrode film. A uniform granular surface was achieved with grain sizes of approximately 25 nm. This image was obtained after O2 plasma treatment and prior to any surface chemistry modification. (2) AFM image of the ITO coated thin electrode film illustrating the uniformity of the ITO coated surface.

Analysis of the surface was also done using contact angle measurements and is discussed further in the next section. Evidence of Surface Modification. Contact Angle Measurements. The attachment chemistry was characterized and verified for each step in the surface modification of the ITO thin film electrodes. The hydroxyl coverage at the surface of the ITO films was increased through pretreatment of the slides with oxygen plasma. This process is nondestructive to the ITO film and is ideal for activating the surface for the silanization step. Treatment of ITO-coated slides by immersion in a strong acid (H2SO4) is not recommended as a means for increasing the amount of active hydroxyl groups at the surface because the ITO is stripped from the surface.39 The achieved results are average values of contact 2054

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C1s O1s In3d Sn3d Si2p S2p N1s

3-MPT

PDEA

PDITC

90°

30°

90°

30°

90°

30°

90°

30°

33.7 46.3 16.4 1.9 1.3

37.0 42.8 16.2 1.8 1.9

e0.5

e0.3

29.4 47.2 14.7 2.3 3.9 2.6