pubs.acs.org/Langmuir © 2010 American Chemical Society
Electrodes Combined with an Agarose Stamp for Addressable Micropatterning Soichiro Sekine,† Shinya Nakanishi,† Takeo Miyake,†,‡ Kuniaki Nagamine,†,‡ Hirokazu Kaji,†,‡ and Matsuhiko Nishizawa*,†,‡ †
Department of Bioengineering and Robotics, Graduate School of Engineering, Tohoku University, 6-6-01 Aramaki, Aoba-Ku, Sendai 980-8579, Japan, and ‡JST, CREST, Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan Received February 18, 2010. Revised Manuscript Received April 19, 2010
We have combined a topographically patterned agarose microstamp with an electrode substrate to develop a novel printing device that internally contains an electrochemical system for a controlled supply of reactive ink to the stamp surface. The 10 wt % agarose gel containing 0.1 M PBS þ 25 mM KBr showed suitable elasticity for forming stamps and served as the electrolytic medium for the electrochemical oxidation of Br- to generate HBrO. The electrode substrate patched with an agarose stamp having 50-μm-high bumps was used for the spatially confined detachment of heparin/ polyethyleneimine precoated on glass substrates, followed by micropatterned adsorption of fibronectin. Using the microelectrode array, the addressable micropatterning of protein by the controlled delivery of HBrO to each bump was demonstrated.
Introduction Considerable effort has been and continues to be devoted to the advancement of a new method of surface micropatterning. Soft lithography with microstamps has been widely used as one of the most convenient patterning methodologies in various research fields from electronics to biology.1-5 Polydimethylsiloxane (PDMS) elastomer is the typical stamp material owing to its moderate elasticity for accurate stamping, optical transparency, and chemical inertness. In addition to PDMS stamps, hydrogelbased stamps have recently attracted attention, especially for biochip preparations because hydrogels provide a biocompatible surface that makes it possible to print proteins, bacteria, and even mammalian cells directly.6-8 As another advantage of hydrogels, reactive chemicals can be reserved within the gel and gradually delivered to the stamp surface. Grzybowski et al. reported the etching of metals, glass, silicon, and semiconductors using hydrogel stamps impregnated by an appropriate etchant.9-13 For example, the HF-impregnated agarose stamp was used for the surface micropatterning of glass and silicon. Hansen et al. used an *Corresponding author. E-mail:
[email protected]. (1) Xia, Y.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 550–575. (2) Parashkov, R.; Becker, E.; Riedl, T.; Johannes, H.-H.; Kowalsky, W. Adv. Mater. 2005, 17, 1523–1527. (3) Benor, A.; Hoppe, A.; Wagner, V.; Knipp, D. Thin Solid Films 2007, 515, 7679–7682. (4) Werner, O.; Persson, L.; Nolte, M.; Fery, A.; Wagberg, L. Soft Matter 2008, 4, 1158–1160. (5) Nishizawa, M.; Takoh, K.; Matsue, T. Langmuir 2002, 18, 3645–3649. (6) Mayer, M.; Yang, J.; Gitlin, I.; Gracias, D. H.; Whitesides, G. M. Proteomics 2004, 4, 2366–2376. (7) Weibel, D. B.; Lee, A.; Mayer, M.; Brady, S. F.; Bruzewicz, D; Yang, J.; DiLuzio, W. R.; Clardy, J.; Whitesides, G. M. Langmuir 2005, 21, 6436–6442. (8) Stevens, M. M.; Mayer, M.; Anderson, D. G.; Weibel, D. B.; Whitesides, G. M.; Langer, R. Biomaterials 2005, 26, 7636–7641. (9) Grzybowski, B. A.; Bishop, K. J. M. Small 2009, 5, 22–27. (10) Campbell, C. J.; Smoukov, S. K.; Bishop, K. J. M.; Baker, E.; Grzybowski, B. A. Adv. Mater. 2006, 18, 2004–2008. (11) Campbell, C. J.; Smoukov, S. K.; Bishop, K. J. M.; Grzybowski, B. A. Langmuir 2005, 21, 2637–2640. (12) Smoukov, S. K.; Bishop, K. J. M.; Klajn, R.; Campbell, C. J.; Grzybowski, B. A. Adv. Mater. 2005, 17, 1361–1365. (13) Smoukov, S. K.; Grzybowski, B. A. Chem. Mater. 2006, 18, 4722–4723.
11526 DOI: 10.1021/la100735e
oxidant (HClO) for the direct patterning of conducting polymer films.14 Such reactive inks (etchant or oxidant) are continuously supplied to the stamp/substrate interface from the bulk, and thus the reaction proceeds continuously for longer periods than in the case of the PDMS stamp, where the surface is simply inked. In the present work, we attempted to develop a hydrogel-based microstamp device that internally contains an electrochemical system for a controlled supply of reactive ink to the stamp surface. A hydrogel such as agarose is known to serve as an inert medium for electrochemical etching and plating.15,16 We fabricate an agarose stamp combined with a microelectrode system for the controlled supply of HBrO by an electrochemical reaction, as illustrated in Figure 1. The supplied HBrO will detach the heparin layer precoated on substrates and allow the selective adsorption of proteins to the exposed substrate surfaces.17-19 In particular, by combining the stamp with a microelectrode array, we demonstrate spatially addressable micropatterning.
Experiments Agarose (Dojindo, Kumamoto, Japan), polyethyleneimine (PEI, average Mw 50 000-100 000, MP Biomedicals, Solon, OH), heparin (sodium salt, Wako Pure Chemical Industries, Osaka, Japan), fibronectin (FN, from human plasma, CHEMICON International, Temecula, CA), fluorescein sodium (Tokyo Chemical Industry, Tokyo, Japan), and all other chemicals were used without further purification. FN was conjugated to Cy2 dye (FN-Cy2) in accordance with instructions from GE Healthcare. The agarose stamps were prepared by pouring the melted 10 wt % agarose solution (containing 0.1 M phosphate-buffered (14) Hansen, T. S.; West, K.; Hassager, O.; Larsen, N. B. Adv. Mater. 2007, 19, 3261–3265. (15) Zhang, L.; Zhuang, J. L.; Ma, X. Z.; Tang, J.; Tian, Z. W. Electrochem. Commun. 2007, 9, 2529–2533. (16) Tang, J.; Zhuang, J. L.; Zhang, L.; Wang, W. H.; Tian, Z. W. Electrochim. Acta 2008, 53, 5628–5631. (17) Kaji, H.; Hashimoto, M.; Nishizawa, M. Anal. Chem. 2006, 78, 5469–5473. (18) Sekine, S.; Kaji, H.; Nishizawa, M. Anal. Bioanal. Chem. 2008, 391, 2711–2716. (19) Sekine, S.; Kaji, H.; Nishizawa, M. Electrochem. Commun. 2009, 11, 1781–1784.
Published on Web 05/06/2010
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Figure 1. Concept of the electrode-combined hydrogel stamp. A reactive oxidant, HBrO, is generated at the working electrodes (WE) by applying a voltage pulse of 1.7 V between the counter electrode (CE), and it diffuses to the stamp surface. The electrochemical reaction is the oxidation of Br- to Br2, followed by reaction with H2O to generate HBrO. saline (PBS) and 25 mM KBr) over the SU-8 master that had 50-μmhigh relief microstructures. The thickness of the base (150 μm) was regulated by a silicone rubber spacer. The gel stamp, with a total thickness of 200 μm, was peeled off of the master and then stored in PBS containing 25 mM KBr. Prior to use, the gel stamps were dried in a stream of N2 for 15 s and attached to the electrode substrate. A glass substrate was washed with acetone, ethanol, and distilled water in an ultrasonic bath. The cleaned substrate was treated with an oxygen plasma asher, LTA-101 (1 Torr, 100 W, 1 min; Yanaco, Tokyo, Japan) to add hydroxyl groups to its surface. An electrostatic bilayer of heparin and PEI (Hep/PEI) was layered onto the substrate by dipping it sequentially into solutions of PEI (5 mg mL-1) and heparin (2 mg mL-1) for 30 min each. This procedure was repeated three times to make the blocking layer more robust.20 The Hep/PEI treatment acts to block protein adsorption. Pt microelectrodes were fabricated on a glass slide by a series of photolithography-based microfabrication technologies. The wiring parts of the electrodes were insulated with a photoresist (3 μm height). A voltage pulse of 1.7 V, supplied by a potentiostat (HA1010 mM2S, Hokuto Denko, Tokyo, Japan), was applied between the arrayed anodes (Pt) and a common larger cathode (Pt). This procedure resulted in the electrochemical oxidation of Br- to Br2 (E0 = 1.07 V vs NHE), followed by reaction with H2O to generate HBrO. We have previously shown that this twoelectrode system with a large counter electrode showed enough reproducibility for biopatterning over an area of a few micrometers.18,19 The electrochemically treated substrate was immersed in a solution of FN-Cy2 for 15 min and was then washed with PBS. The substrate was subsequently imaged using an inverted fluorescence microscope (IX71, Olympus, Tokyo, Japan). We examined the detachment of the Hep/PEI layer after treatment with HBrO by XPS17 and AFM,19 but the detailed mechanism of the detachment is still unclear.
Figure 2. Cyclic voltammograms of a Pt electrode in PBS containing 25 mM KBr with various concentrations of agarose. The measurements were conducted with a Pt counter electrode and an Ag/AgCl reference electrode at a scan rate of 100 mV/s.
Results and Discussion Figure 2 shows voltammograms obtained in agarose gels containing 0.1 M PBS and 25 mM KBr. Gel concentrated to greater than 10 wt % was too viscous for making stamps. Although a slight decrease was observed in the current amplitude at 1.7 V versus Ag/AgCl at higher agarose concentration, all gels showed almost the same voltammetric shape as that in the PBS solution. This suggests that an agarose gel containing 0.1 M PBS can serve as an inert electrolytic medium for Br- oxidation. We also studied other biocompatible hydrogels such as collagen, alginate acid, and fibrin. However, an additional unknown redox peak appeared at around 1 V in these gels probably because of the electrochemical degradation of the gels themselves. In addition, (20) Ji, J.; Tan, Q.; Fan, D.-Z.; Sun, F.-Y.; Barbosa, M. A.; Shen, J. Colloids Surf., B 2004, 34, 185–190.
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Figure 3. (a) Schematic illustration of the experimental setup used to observe the electrogeneration of HBrO. The agarose gel additionally contains fluorescein sodium. (b) Fluorescent images of tetrabromofluorescein generated by the reaction with HBrO. (c) Fluorescence intensity vs the distance along the broken line in b.
all of them were found to be too soft for forming stamps. Therefore, we have decided to use 10 wt % agarose gel containing 0.1 M PBS þ 25 mM KBr as an electrolytic medium for the present printing device. DOI: 10.1021/la100735e
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Figure 5. (a) Micrograph of 3 3 array of working electrodes fabricated on the base plate of the stamp. (b) Micrograph of the gel stamp. (c) Fluorescence micrograph of FN-Cy2 selectively patterned on the Hep/PEI substrate after 50 s of electrolysis at electrodes 1-3 and 5. Figure 4. (a) Schematic illustration of the experiments using an agarose stamp combined with a single large electrode. (b) Micrograph of the gel stamp and the fluorescence micrograph of FN-Cy2 locally adsorbed on the electrochemically treated region of the Hep/PEI substrate after 120 s of electrolysis.
To visualize the electrogeneration and diffusion of HBrO, a 500-μm-thick electrolyte agarose gel film that additionally contained 1 mM fluorescein sodium (mixed before gelation) was placed on a pair of Pt electrodes as shown in Figure 3a and a voltage of 1.7 V was applied between the electrodes. The electrogenerated HBrO is known to react with fluorescein to form red fluorescent eosin (tetrabromofluorescein) by the electrophilic substitution of Br.21,22 Therefore, the appearance of a red fluorescent region would indicate the electrochemical generation of HBrO and its diffusion into the gel. Figure 3b is a fluorescent micrograph obtained before and after electrolysis, and Figure 3c shows fluorescence intensity profiles. Even before electrolysis, the fluorescence on the electrode is higher than that in the surroundings because of the optical reflection of the Pt electrodes. After electrolysis, red fluorescence was intensified on the anode (left electrode) but no change was observed on the cathode (right electrode). The results suggested that the generated HBrO diffused ca. 500 μm into the gel phase during 300 s of electrolysis, indicating that the diffusion coefficient of HBrO (or eosin) is in the range of 10-10 m2/s, which is consistent for small molecules. We have designed the gel stamp to have 50-μm-high bumps on a 150-μm-thick base that is necessary to handle with tweezers. The total diffusion length from the electrode to the substrate is thus roughly 200 μm, that is, within the diffusion layer formed after a few minutes electrolysis, as judged from Figure 3. Figure 4 shows experiments using the KBr-containing agarose stamp combined with a single large electrode (7 mm 7 mm). The agarose stamp actually has 25 bumps 25 bumps in a 5 mm 5 mm area. (21) Fishman, M. J., Friedman, L. C., Eds. Methods for Determination of Inorganic Substances in Water and Fluvial Sediments; Techniques of Water-Resources Investigations of the United States Geological Survey, 3rd ed.; U.S. Geological Survey: Lakewood, CO, 1989; Book 5, Chapter A1. (22) Grinbaum, B.; Freiberg, M. Bromine. In Kirk-Othmer Encyclopedia of Chemical Technology, Wiley: New York, 2001.
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The stamp/electrode assembly was put on a glass substrate coated with Hep/PEI, followed by the application of 1.7 V for 120 s to generate HBrO electrochemically. Then the substrate was immersed in a solution of FN-Cy2 for 15 min. Figure 4b shows the micropatterns of FN-Cy2 adsorbed on the glass surface exposed by the detachment of Hep/PEI. As evidenced by the micrograph, the patterns of FN-Cy2 adsorption directly reflect that of the gel stamp. However, there is a tendency for the FN-Cy2 patterns to show brighter edges. This is probably because the oxidant produced at the gap between bumps can diffuse along the side wall of the bumps and concentrate at the edges. The HBrO remaining within the stamp is gradually decomposed back to Br- by a light-catalyzed reaction22 within 10 min under laboratory conditions, which is the important advantage enabling the repeated use of a stamp. It is worth noting that just stamping without voltage application did not cause FN-Cy2 adsorption at all, indicating that the contact of the agarose stamp itself does not damage the Hep/PEI blocking layer. These results demonstrate that the present gel/electrode assembly can serve as a novel stamp system with a controllable ink supply. We attempted the further improvement of the stamp system by employing an electrode array for the selective delivery of HBrO to each bump, as proposed in Figure 1. Figure 5a shows (a) 3 electrodes 3 electrodes (50 μm 50 μm each) separated by 200 μm intervals to be combined with (b) the stamp having 75 μm 75 μm square bumps. The gel/electrode assembly was put onto a Hep/PEI-coated substrate, and electrodes 1-3 and 5 were turned on for 50 s of electrolysis and then quickly removed from the substrate. From a preliminary experiment using a 200-μm-thick flat gel sheet without bumps (Supporting Information), we knew that the extension of the modified area on the substrate for 50 s would be around 100 μm, corresponding to the size of a bump. Figure 5c shows the micropatterns of adsorbed FN-Cy2, demonstrating that selective delivery to each bump was possible. It is of practical importance that in this case the patterned FN-Cy2 adsorption was uniform, in contrast to the slightly edged patterns seen in Figure 4, because of the absence of oxidant generation in the gaps between bumps. The stamp that we used here is 200 μm thick, which was required for handling with tweezers. Therefore, electrogenerated HBrO should diffuse 200 μm through the gel to Langmuir 2010, 26(13), 11526–11529
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reach the substrate surface. This chemical diffusion occurs isotropically and possibly affects the neighboring bumps. In fact, there is slight unwanted adsorption on the opposite corners of neighboring bumps (4 and 6) that is probably due to the overdiffusion of oxidant through the gel base between bumps. It will be an attractive trial to use a thinner stamp that shortens the diffusion path and will allow finer patterning. As we have already noted, because HBrO is unstable and decomposes back to Br-, the conditions will be initialized for repeated stamping.
for the selective delivery of ink to each bump. We demonstrated that the supplied HBrO locally detached Hep/PEI precoated on a glass substrate, which is an important step in protein and cell micropatterning. Other applications include the inactivation and etching of a conducting polymer for organic flexible electronics.14 We are preparing a flexible PET film-based electrode substrate to make the present system applicable to a curved surface.
Conclusions
Acknowledgment. This study was partially supported by Grants-in-Aid for Scientific Research B (no. 20310070) from the Ministry of Education, Science, and Culture, Japan.
We have developed an agarose-based microstamping device that internally contains an electrochemical system for the controlled supply of reactive HBrO ink to the stamp surface. The internal inkgeneration system made repeat patterning without inking possible. The system was further improved by employing an electrode array
Supporting Information Available: Patterning protein using a 200-μm-thick flat gel sheet without bumps. This material is available free of charge via the Internet at http://pubs. acs.org.
Langmuir 2010, 26(13), 11526–11529
DOI: 10.1021/la100735e
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