Langmuir 2003, 19, 10267-10270
10267
Patterned Generation of Reactive Thiolsulfinates/ Thiolsulfonates on Silicon Oxide by Electrooxidation Using Electromicrocontact Printing Elisabeth Pavlovic,*,†,‡ Arjan P. Quist,†,§ Leif Nyholm,| Angelo Pallin,⊥ Ulrik Gelius,§ and Sven Oscarsson†,‡ Department of Surface Biotechnology, Biomedical Center, Uppsala University, Box 577, S75123, Uppsala, Sweden, Department of Biology and Chemical Engineering, Ma¨ lardalen University, Box 325, S63105, Eskilstuna, Sweden, Department of Physics, Uppsala University, Box 524, S751 20, Uppsala, Sweden, Department of Analytical Chemistry, Uppsala University, Box 531, S75121, Uppsala, Sweden, and Department of Materials Science, Uppsala University, Box 534, S75121, Uppsala, Sweden Received August 6, 2003. In Final Form: September 14, 2003 This work describes a method to produce microstructured PDMS stamps that are metallized in two isolated sections, thus creating the possibility to electroprint a surface using such a stamp as both counter electrode and reference electrode in a surface contact electrochemical cell controlled using a potentiostat. This electromicrocontact printing method was used to perform patterned electroactivation of thiolated surfaces. Patterned electrooxidation of thiols to reactive thiolsulfinates/thiolsulfonates allowed for specific immobilization of thiolated polystyrene particles, showing the potential of this technique to be employed in selective covalent attachment of biomolecules through disulfide bonds.
Introduction 1
Microcontact printing (µCP) is a simple and efficient technique to perform patterned deposition of molecules2,3 or metals,4 by contacting a stamp made of a soft polymer, typically poly(dimethylsiloxane) (PDMS), with a surface. It enables the transfer of patterns onto surfaces in a wide range of sizes, from a few hundreds of microns to a few tens of nanometers,5,6 and the modification of large surface areas. A few variations of this technique are available,4,7 but in many cases, the deposited molecules adsorb to the surfaces through noncovalent interactions.5,6,8 Direct covalent bonding is limited to gold-sulfur interactions.9 In addition, contamination of the patterned surfaces by low molecular weight PDMS from the stamps constitutes a major drawback. * To whom correspondence should be addressed. Phone: +46184713530. Fax: +46184713611. E-mail: elisabeth.pavlovic@ mdh.se. † Department of Surface Biotechnology, Biomedical Center, Uppsala University. ‡ Department of Biology and Chemical Engineering, Ma ¨ lardalen University. § Department of Physics, Uppsala University. | Department of Analytical Chemistry, Uppsala University. ⊥ Department of Materials Science, Uppsala University. (1) Xia, Y.; Whitesides, G. M. Annu. Rev. Mater. Sci. 1998, 28, 153184. (2) Kumar, A.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1994, 10, 1498-1511. (3) Hammond, P. T.; Whitesides, G. M. Macromolecules 1995, 28, 7569-7571. (4) Schmid, H.; Wolf, H.; Allenspach, R.; Riel, H.; Karg, S.; Michel, B.; Delamarche, E. Adv. Funct. Mater. 2003, 13, 145-153. (5) Li, H.-W.; Muir, B. V. O.; Fichet, G.; Huck, W. T. S. Langmuir 2003, 19, 1963-1965. (6) Li, H.-W.; Kang, D.-J.; Blamire, M. G.; Huck, W. T. S. Nano Lett. 2002, 2, 347-349. (7) Gyo¨rvary, E. S.; O’Riordan, A.; Quinn, A. J.; Redmond, G.; Pum, D.; Sleytr, U. B. Nano Lett. 2003, 3, 315-319. (8) Himmelhaus, M.; Takei, H. Phys. Chem. Chem. Phys. 2002, 4, 496-506. (9) Morhard, F.; Pipper, J.; Dahint, R.; Grunze, M. Sens. Actuators, B 2000, 70, 232-242.
Scheme 1. Electrochemical Activation of Thiols to Thiolsulfinates/Thiolsulfonates and Subsequent Binding of Thiolated Particlesa
a
R-SH ) thiolated polystyrene beads.
This work describes a patterning method for covalent immobilization of particles, based on the use of a PDMS stamp coated with a conductive layer of aluminum as a counter electrode and reference electrode to perform an electrochemical activation of a silanized silicon oxide surface. The activation taking place is the electrochemical oxidation of surface thiols to reactive thiolsulfinates/ thiolsulfonates, which can react with thiol groups, resulting in a thiol-disulfide exchange,10 as described in Scheme 1. An essential requirement was proper contacting, from an electrochemical as well as a physical point of view. The fabrication of a conductive PDMS stamp was a central issue to address, since metallized PDMS stamps used so far were designed to achieve deposition of metallic particles on surfaces4 and not to perform as cathodes. It was shown that no PDMS contamination occurs during the process. Thiol-derivatized polystyrene beads were subsequently immobilized onto the electroactivated patterns. The patterned electroactivation and immobilization of polystyrene beads were studied using chemically sensitive lateral force microscopy (LFM) and topographic imaging (10) Pavlovic, E.; Quist, A. P.; Gelius, U.; Nyholm, L.; Oscarsson, S. Langmuir 2003, 19, 4217-4221.
10.1021/la035434x CCC: $25.00 © 2003 American Chemical Society Published on Web 10/28/2003
10268
Langmuir, Vol. 19, No. 24, 2003
Figure 1. Electro-µCP setup. The two separated aluminized regions on the stamp are used as counter electrode and reference electrode, respectively.
in tapping mode. The electrooxidation of surface thiols was analyzed using X-ray photoelectron spectroscopy (XPS). Experimental Section Thiolated silicon oxide surfaces were prepared as described in detail elsewhere.11 P-doped silicon surfaces (Silicon Sense Inc., Nashua, NH) were cleaned using a piranha solution, H2SO4/ H2O2 30% (v/v) 2:1, and rinsed with ultrapure water (18 MΩ, low organic content). The surfaces were dried in an argon flow inside the reactor. Twenty microliters of 3-mercaptopropyltrimethoxysilane (3-MPTMS, ABCR, Karlsruhe, Germany) reagent was introduced in the reactor, which was then closed. The argon flow through the reactor was set to approximately 1 L/min. The silanization was allowed to take place for 1 h. The silanized surfaces were then sonicated, first for 10 min in 99.5% ethanol followed by 10 min in ultrapure water, and dried in an argon flow. The masters for stamp fabrication were prepared using photolithography. The pattern was formed by dry-etching the silicon of the developed areas using an inductively coupled plasma etching system, adjusting the number of etching cycles to obtain a depth-to-width ratio around 1. The masters were subsequently treated with 40% HF to passivate the silicon surface. PDMS stamps were prepared as described elsewhere2 using Sylgard 184 (Dow Corning, Midland, MI). The curing step was carried out at 65 °C for 10 h, followed by 10 h at room temperature. The stamps were peeled from the masters, cut to 1 cm × 1 cm squares, and attached to a 1 cm × 3 cm glass slide to prevent bending of the stamp after metallization. A 1 mm wide strip of aluminum foil was positioned on the stamps before evaporation in order to obtain two isolated Al-coated areas, to be used as counter electrode and reference electrode, respectively. The evaporation was carried out in an Edwards E306 thermal evaporator (BOC Edwards, West Sussex, U.K.). Two 100 nm thick layers of Al were sequentially evaporated on the PDMS stamps (placed 25 cm above the source) at a rate of approximately 5 nm/s. The exposure angle was 60° to the surface normal, with a rotation of the linear pattern of 45° around the surface normal, to obtain metallization only on the outermost surface of the stamp and not on the inside of the patterned structures. The surfaces of Al-coated PDMS stamps were imaged using a Philips XL30 ESEM (FEI, Hillsboro, OR) in the scanning electron microscopy (SEM) mode. Electromicrocontact printing was then performed, see Figure 1. A droplet of 10 mM phosphate buffer, pH 7.0, was placed on the thiol-derivatized surface. The glass-backed stamp was lowered onto the surfaces and pressed down using a 12 g weight. The potential difference was applied for 1 min using a LC-4B (11) Pavlovic, E.; Quist, A. P.; Gelius, U.; Oscarsson, S. J. Colloid Interface Sci. 2002, 254, 200-203.
Pavlovic et al. amperometric detector potentiostat (BAS, USA) between the thiolated surface as the working electrode and the separated aluminized stamp areas as the counter electrode and reference electrode. The surface oxidation was analyzed in a Quantum 2000 Scanning ESCA MicroProbe instrument from Physical Electronics, using monochromized Al KR radiation and a pass energy of 57.8 eV. Spectra were acquired at a 45° takeoff angle. Curve fitting and spectral analysis were performed using Multipak (Physical Electronics). The patterns were imaged using a Nanoscope IIIa MultiMode AFM (Digital Instruments, Santa Barbara, CA), in contact mode for friction imaging and in tapping mode for height imaging. Polystyrene beads, 46 nm in diameter, containing surface amine groups were derivatized with succinimidyl-3-(2-pyridyldithio)propionate (SPDP, Sigma, St. Louis, MO): 0.25 mL of a solution containing 2.5% beads was reacted with approximately 1000 times excess (molecule/bead) of SPDP in 0.1 M phosphate buffer, pH 8.0, for 2 h. The separation of the beads from excess SPDP and cleavage of the pyridyl disulfide to generate a thiol group were carried out as described earlier.12 Immobilization of particles on the patterned surfaces was performed by depositing a 150 µL droplet of 1:500 (v/v) thiolderivatized beads in 10 mM phosphate buffer (pH 7.0) and allowing the beads to react for 90 min. The surfaces were then rinsed under running ultrapure water for 5 min, dried with an argon flow, and imaged using tapping mode atomic force microscopy (AFM). The same procedure and rinsing were used for treatment of the surfaces for 1-3 h with a 50 mM dithiothreitol (DTT) solution in 0.1 M phosphate buffer, pH 8.0.
Results and Discussion In this work, we investigate the possibility of using a microcontact printing setup to perform electroactivation of thiols to thiolsulfinates/thiolsulfonates. It appeared necessary to use a soft polymer stamp, typically made of PDMS, to obtain a conformal contact between the stamp and the surface to be patterned. Since PDMS is a nonconductive polymer, a stamp metallization process had to be optimized. The presence of surface thiols requires the use of an oxide-passivated metal, such as aluminum. The investigation of metallization of siloxane polymer surfaces using Al in the early 1980s13 showed that it is necessary to cool PDMS to the glass transition temperature to obtain conductive metallic films, since PDMS has a low nucleation point for metals. For an uncooled PDMS surface, the first evaporated layer was shown to be nonconductive, probably due to the engulfing of the aluminum by the soft polymer, even at high evaporation rates that speed up nucleation. The Al films obtained after a second evaporation were conductive. Passivation of the silicon masters resulted in an improved quality of the stamps after separation from the master, due to the smaller adhesion of PDMS to such passivated surfaces. The 60° angle to the surface normal together with the 45° rotation to the normal of the linear pattern during evaporation, allowing only for coating of the outermost surfaces of the patterns and not the inner surfaces of the microstructures, is an important parameter since with the use of a buffer solution between the stamp and the silicon surface, the potential is applied over all the areas coated with Al. The high flexibility of the PDMS, allowing bending of the stamp, causes the disruption of the stiff aluminum coating, which means that the stamps must be handled with precaution. Figure 2 shows SEM images of a (12) Batista-Viera, F.; Barbieri, M.; Ovsejevi, K.; Manta, C.; Carlsson, J. Appl. Chem. Biotechnol. 1991, 31, 175-195. (13) Martin, G. C.; Su, T. T.; Loh, I. H.; Balizer, E.; Kowel, S. T.; Kornreich, P. J. Appl. Phys. 1982, 53, 797-799.
Patterned Generation of Reactive Thiol Compounds
Langmuir, Vol. 19, No. 24, 2003 10269
Figure 2. Al-coated nondamaged linear pattern stamp (A) and damaged stamp, with visible cracks (B).
nondamaged Al-coated stamp (A) and a damaged one, with cracks clearly visible in the Al film (B). Hoeppener et al. described the necessity of wetting the silanized surface in a saturated water-vapor atmosphere in order to obtain oxidation when using a transmission electron microscopy (TEM) copper grid as a conductive “hard” stamp.14 In our work, no electrooxidation occurred without introduction of an aqueous medium in the form of a droplet of buffer solution between the stamp and the silicon surface. In our previous study of the electrooxidation of thiols, we determined that thiols start to be oxidized to thiolsulfinates/thiolsulfonates around 0.6 V versus Ag/AgCl.10 The potential of the Al layer was measured to be -1.2 V versus Ag/AgCl. Therefore, a 1.85 V potential difference versus the Al reference electrode was applied to the thiolated silicon surface. Using the setup described in Figure 1, thiolated surfaces were electroactivated using an Al-coated stamp patterned with lines (10 µm wide, spaced 10 µm, Figure 2A). Lateral force microscopy was used to visualize the oxidation patterns after the electroactivation (Figure 3A). Tapping mode imaging of such oxidation patterns showed little to no topography on the surface, indicating that the observed friction images are truly due to chemical differences. The sturdy glass backing of the stamp to prevent bending proved crucial to obtain nondeformed patterns and made the stamps much easier to handle without damaging them. The line width is around 12 µm, possibly resulting from Al evaporated on the sides of the lines which may contact the thiolated surface by compressive deformation or from a water meniscus forming between the Al-coated side of the lines and the surface, thus exposing (14) Hoeppener, S.; Maoz, R.; Sagiv, J. Nano Lett. 2003, 3, 761-767.
Figure 3. Friction image of a thiolated surface electroactivated at a potential difference of 0.65 V versus Ag/AgCl (A); tapping mode image of polystyrene particles immobilized on the linear electroactivated pattern, with a height scale of 40 nm (B).
a wider surface area to the positive potential. Different intensities, corresponding to different degrees of oxidation, are also visible on the electroactivated lines, with darker areas being less oxidized than brighter ones. Possible reasons to explain this observation are a nonhomogeneous Al coating caused by shading of certain areas of the lines by higher ones, for example, line edges in this case, or a compressive deformation, as already mentioned above. XPS spectra of sulfur 2s before (Figure 4A) and after (Figure 4B) the electroactivation indicate that there was no loss of sulfur and consequently no damage to the organic monolayer during the process. Curve fitting of the oxidized region of the S2s spectrum (Figure 4B) revealed two new peaks corresponding to two oxidation states at approximately 231.0 and 233.0 eV. The corresponding S2p binding energies are obtained by subtracting 64.4 eV from the S2s binding energies, with 166.5 and 168.5 eV corresponding to S2p signals from sulfinyl (SO) and sulfonyl (SO2) moieties,10 respectively. Calculations using the integrated areas of the peaks in the electron spectroscopy for chemical analysis (ESCA) spectrum after electroactivation (Figure 4B) show that 18% of the total amount of sulfur is oxidized. Considering the width of the electroactivated lines obtained from the LFM images, a complete activation of the patterns would
10270
Langmuir, Vol. 19, No. 24, 2003
Pavlovic et al.
similar activation level inside the electroactivated lines would result in an oxidation of 24% calculated over the total surface. The lower percentage, 18%, is explained by the nonhomogeneity of the oxidation inside the activated lines. Contamination of the patterned surfaces by low molecular weight PDMS is a major drawback of µCP.15 Low amounts of contamination were detected on the electroactivated surfaces but appear to be hydrocarbon contamination from air, since no signal increase at 102.0 eV, corresponding to polysiloxane (SiO2), was detected. Therefore, it is not necessary to wash the PDMS stamps used in this technique to reduce contamination as is done in µCP.2 No traces of aluminum contamination were detected. Thiol-derivatized polystyrene beads, with a diameter of 46 nm, were immobilized on the patterned surfaces (Figure 3B). Tapping mode images show that the beads are preferentially immobilized on the activated patterns, with a low amount of unspecific adsorption. The beads remained on the surface after treatment with DTT. The inaccessibility of the disulfide bonds linking the particles to the surface has already been reported in our earlier studies10 as the most probable reason to explain this phenomenon. Conclusion
Figure 4. XPS spectra of the sulfur 2s signal before (A) and after electroactivation (B).
result in 30% of oxidized sulfur in the case of homogeneous oxidation. Since the oxidation is nonhomogeneous, the activation level was estimated as approximately 80%, which is comparable to the oxidation level reported previously10 at this potential difference. In this previous report, an activation level of 80% is obtained using a potential difference of 0.65 V versus Ag/AgCl, which corresponds to 40% of the sulfur being oxidized after electroactivation of the total surface area. In the present situation, based on the LFM images, the activated area corresponds roughly to 60% of the total surface area. A
In this work, we have developed aluminum-coated PDMS stamps designed to perform surface electrochemistry using a three-electrode setup. Patterned electroactivation of surface thiols to reactive thiolsulfonates/ thiolsulfinates was shown to result from such an electromicrocontact printing procedure. ESCA and LFM analysis showed the patterned electroactivation was successfully achieved. It was possible to specifically immobilize thiolated polystyrene beads onto the electroactivated patterns. Reversibility of the immobilization using DTT is currently under investigation. Acknowledgment. We thank the Swedish Foundation for Strategic Research (SFF) for financial support. LA035434X (15) Glasma¨star, K.; Gold, J.; Andersson, A.-S.; Sutherland, D. S.; Kasemo, B. Langmuir 2003, 19, 5475-5483.
