Poly(dimethylsiloxane) Substrate Fabricated by

May 5, 2009 - In this paper, we present a novel approach for preparing patterned Au/poly(dimethylsiloxane) (PDMS) substrate. Chemical gold plating ins...
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Patterned Au/Poly(dimethylsiloxane) Substrate Fabricated by Chemical Plating Coupled with Electrochemical Etching for Cell Patterning Hai-Jing Bai, Min-Ling Shao, Hong-Lei Gou, Jing-Juan Xu, and Hong-Yuan Chen* Key Laboratory of Analytical Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China Received March 18, 2009. Revised Manuscript Received April 8, 2009 In this paper, we present a novel approach for preparing patterned Au/poly(dimethylsiloxane) (PDMS) substrate. Chemical gold plating instead of conventional metal evaporation or sputtering was introduced to achieve a homogeneous gold layer on native PDMS for the first time, which possesses low-cost and simple operation. An electrochemical oxidation reaction accompanied by the coordination of gold and chloride anion was then exploited to etch gold across the region covered by electrolyte. On the basis of such an electrochemical etching, heterogeneous Au/PDMS substrate which has a gold “island” pattern or PDMS dots pattern was fabricated. Hydrogen bubbles which were generated in the etching process due to water electrolysis were used to produce a safe region under the Pt auxiliary electrode. The safe region would protect gold film from etching and lead to the formation of the gold “island” pattern. In virtue of a PDMS stencil with holes array, gold could be etched from the exposed region and take on the PDMS dots pattern which was selected to for protein and cell patterning. This patterned Au/PDMS substrate is very convenient to construct cytophobic and cytophilic regions. Self-assembled surface modification of (1-mercaptoundec-11-yl)hexa(ethylene glycol) on gold and adsorption of fibronectin on PDMS are suitable for effective protein and cell patterning. This patterned Au/PDMS substrate would be a potentially versatile platform for fabricating biosensing arrays.

Introduction In vivo, cells are cuddled by a complex microenvironment. Cell patterning could be used as an efficient tool to simulate the microenvironment of cells and to probe cell-cell and cellmicroenvironment interactions. It has potential applications in fundamental research of cell biology, biosensing, medical diagnostics, and tissue engineering.1-10 A series of methods have been founded to achieve cell patterning in the past decades, such as photolithography,11-13 soft photolithography,14-16 *Corresponding author. E-mail: [email protected]; Tel/Fax: +86 25-835 94862. (1) Pancrazio, J. J.; Whelan, J. P.; Borknolder, D. A.; Ma, W.; Stenger, A. Ann. Biomed. Eng. 1999, 27, 697–711. (2) Bhadriraju, K.; Chen, C. S. Drug Discovery Today 2002, 7, 612–620. (3) John, P. M. S.; Davis, R.; Cady, N.; Czajka, T.; Batt, C. A.; Craighead, H. G. Anal. Chem. 1998, 70, 1108–1111. (4) Yap, F. L.; Zhang, Y. Biosens. Bioelectron. 2007, 22, 775–788. (5) Nahmias, Y.; Schwartz, R. E.; Verfaillie, C. M.; Odde, D. J. Biotechnol. Bioeng. 2005, 92, 129–136. (6) Gadegaard, N.; Martines, E.; Riehle, M. Q.; Seunarine, K.; Wilkinson, C. D. W. Microelectron. Eng. 2006, 83, 1577–1581. (7) Zhu, A. P.; Chen, R. Q.; Chan-Park, M. B. Macromol. Biosci. 2006, 6, 51–57. (8) Ito, Y. Biomaterials 1999, 20, 2333–2342. (9) Veiseh, M.; Veiseh, O.; Martin, M. C.; Asphahani, F.; Zhang, M. Q. Langmuir 2007, 23, 4472–4479. (10) Ziauddin, J.; Sabatini, D. M. Nature 2001, 411, 107–110. (11) Bhatia, S. N.; Yarmush, M. L.; Toner, M. J. Biomed. Mater. Res., Part A 1997, 34, 189–199. (12) Revzin, A.; Tompkins, R. G.; Toner, M. Langmuir 2003, 19, 9855–9862. (13) Hui, E. E.; Bhatia, S. N. Langmuir 2007, 23, 4103–4107. (14) Xia, Y. N.; Whitesides, G. M. Angew. Chem., Int. Ed. 1998, 37, 550–575. (15) Whitesides, G. M.; Ostuni, E.; Takayama, S.; Jiang, X. Y.; Ingber, D. E. Annu. Rev. Biomed. Eng. 2001, 3, 335–373. (16) Suh, K. Y.; Seong, J.; Khademhosseini, A.; Laibinis, P. E.; Langer, R. Biomaterials 2004, 25, 557–563. (17) Miller, J. S.; Bethencourt, M. I.; Hahn, M.; Lee, T. R.; West, J. L. Biotechnol. Bioeng. 2006, 93, 1060–1068. (18) Lee, S.-H.; Moon, J. J.; West, J. L. Biomaterials 2008, 29, 2962–2968. (19) Liu, Y.; Sun, S.; Singha, S.; Cho, M. R.; Gordon, R. J. Biomaterials 2005, 26, 4597–4605. (20) Iwanaga, S.; Akiyama, Y.; Kikuchi, A.; Yamato, M.; Sakai, K.; Okano, T. Biomaterials 2005, 26, 5395–5404.

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laser-scanning lithography,17,18 laser ablation, 19,20 hot-embossing imprint lithography,21 and so on. For cell patterning, preparation of a patterned substrate containing addressable factors for cells is the key. On gold substrate, the self-assembled monolayers (SAMs) of alkanethiolates, as an ordinary strategy of surface chemistry, are employed extensively. Combining SAMs with microcontact printing (μCP), chemical etching, or electrochemical manipulation, patterned gold substrates have been well fabricated. Whitesides et al. used a pen to write patterns of hexadecanethiolate as a monolayer on gold substrate and etched underivatized Au as well as underlying Ti with etchant which consisted of 1 M KOH and 0.1 M KCN.22 Crooks and co-workers used μCP to print hexadecanethiol locally on gold substrate and filled 11-sulfanylundecanoic acid in the space, resulting in a patterned surface exhibiting excellent cell adhesion and spatial definition.23 Jiang et al. reported a method for patterning multiple types of adherent cells on gold substrate by electrochemical desorption of SAMs in local areas defined by a microfluidic system.24 Compared with chemical etching and μCP, this patterning method based on electrochemistry has some remarkable advantages. First, a dynamic substrate has been introduced into cell patterning. Transformation of surface properties could be triggered during cell culture within a quite short process time. Only several minutes and even a few seconds are required for electrochemical reaction. Converting hydroquinone groups to quinine groups is very easy to be realized by electrochemical treatment. Mrksich et al. have used this electrochemical (21) Charest, J. L.; Eliason, M. T.; Carcı´ a, A. J.; King, W. P. Biomaterials 2006, 27, 2487–2494. (22) Kumar, A.; Biebuyck, H. A.; Abbott, N. L.; Whitesides, G. M. J. Am. Chem. Soc. 1992, 114, 9189–9191. (23) Amirpour, M. L.; Ghosh, P.; Lackowski, W. M.; Crooks, R. M.; Pishko, M. V. Anal. Chem. 2001, 73, 1560–1566. (24) Li, Y.; Yuan, B.; Ji, H.; Han, D.; Chen, S. Q.; Tian, F.; Jiang, X. Y. Angew. Chem., Int. Ed. 2007, 46, 1094–1096.

Published on Web 05/05/2009

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reaction to induce Deils-Alder-mediated immobilization of RGD-Cp peptide for mediating a second type of cells to attach.25 Second, the pattern could be formed easily without physical confinement. This property has been exploited perfectly in the typical work of Nishizawa and his co-workers. They introduced microelectrochemical lithography to produce cell patterning and guide cell migration in situ.26,27 HBrO which was produced by electro-oxidation of Br- in electrolyte was employed to destroy BSA adsorbed on substrate previously. The broken region was just the diffusion region of HBrO. Wittstock et al. adopted a similar means to remove OEG modified on gold substrate.28 Furthermore, the cost of electrochemical patterning including requisite devices is lower and it is convenient to be developed in a general chemical laboratory. However, up to now, only few electrochemical reactions have been exploited for cell patterning on gold substrate, and the patterned gold substrate was commonly constructed on glass or silica which was not a better choice for biochips due to its absent biocompatibility or optical transparency. At present, poly (dimethylsiloxane) (PDMS) is one of the most popular polymers for bio-MEMs and microfluidic devices due to its well-known advantages such as chemical inertness, flexibility, optical transparency, permeability, and simple machining.29,30 Therefore, constructing conductive gold film on biocompatible PDMS would be ideal. Metal evaporation and sputtering have been the routine approaches to achieve gold film on many kinds of materials including PDMS.31 In view of their high cost and low feasibility for ordinary chemical laboratories, chemical plating would be a good substitute. Chemical gold plating which took place on polymers such as polycarbonate32 and poly(ethylene terephthalate)33 have been reported. However, there still is not any report of chemical gold plating on PDMS to date. Here, we describe a novel method for fabricating a patterned Au/PDMS substrate by chemical plating coupled with electrochemical etching strategy. A firm and conductive gold film on PDMS via chemical plating is for the first time realized, and a twostep reduction mechanism is proposed which is obviously different from chemical gold plating on other polymers.32,33 And the electrochemical reaction accompanied by the coordination of gold and chloride anion was exploited in a cell patterning application. On the basis of such an electrochemical etching, a heterogeneous Au/PDMS substrate which has a gold “island” pattern or PDMS dots pattern was fabricated. The fabricated heterogeneous substrate with pattern possesses great convenience on surface modification to induce cell patterning.

Experimental Section Materials and Reagents. Sylgard 184 (including PDMS monomer and curing agent) was purchased from Dow Corning (Midland, MI). (1-Mercaptoundec-11-yl)hexa(ethylene glycol) (EG6) and Cy3-anti-IgG were purchased from Sigma (St. Louis, MO). Fibronectin (FN) was from Biological Industries (25) Yousaf, M. N.; Houseman, B. T.; Mrksich, M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5992–5996. (26) Kaji, H.; Kanada, M.; Oyamatsu, D.; Matsue, T.; Nishizawa, M. Langmuir 2004, 20, 16–19. (27) Kaji, H.; Tsukidate, K.; Matsue, T.; Nishizawa, M. J. Am. Chem. Soc. 2004, 126, 15026–15027. (28) Zhao, C.; Witte, I.; Wittstock, G. Angew. Chem. 2006, 118, 5595–5597. (29) McDonald, J. C.; Duffy, D. C.; Anderson, J. R.; Chiu, D. T.; Wu, H. K.; Schueller, O. J. A.; Whitesides, G. M. Electrophoresis 2000, 21, 27–40. (30) Sia, S. K.; Whitesides, G. M. Electrophoresis 2003, 24, 3563–3576. (31) Feng, J. T.; Zhao, Y. P. Biomed. Microdevices 2008, 10, 65–72. (32) Kong, Y.; Chen, H. W.; Wang, Y. R.; Soper, S. A. Electrophoresis 2006, 27, 2940–2950. (33) Hao, Z. X.; Chen, H. W.; Zhu, X. Y.; Li, J. M.; Liu, C. J. Chromatogr., A 2008, 1209, 246–252.

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(Beit-Haemek, Israel). Acridine Orange (AO) was from Amresco (Solon, OH). HAuCl4 3 4H2O was from Shanghai Chemical Reagent (Shanghai, China). Glucose was from Shanghai Bio Life Science & Technology (Shanghai, China). All other chemicals were of analytical grade and used without further purification. Milli-Q grade water (Millipore Inc., Bedford, MA) was used for all solutions and cleaning steps. Ten millimolar phosphate-buffered saline (PBS, pH 7.4) was prepared for protein dissolution and rinsing. Fabrication of Gold Film on PDMS Chip. Native PDMS was prepared as before34 (mass ratio of PDMS monomer to curing agent was 10:1) and then cut into 3  3 cm2 chips. The solution for chemical gold plating containing 1% (w/v) HAuCl4, 200 g L-1 KHCO3, and 2% (w/v) glucose (v/v, 2:1:1) was prepared just before use. A poly(methyl methacrylate) (PMMA) frame with inner area 2  2 cm2 was sandwiched between two native PDMS chips named “cover chip” and “base chip”, and then the solution for chemical gold plating was injected into this sandwich architecture. It was then held at room temperature for about 3 h to ensure elemental gold deposit completely.

Electrochemical Etching for Patterned Au/PDMS Substrate. Pattern of Gold “Island”. In virtue of aluminum conductive adhesive tape, the gold/PDMS cover chip could be prepared as the working electrode. Drops of electrolyte, 100 mM NaCl solved in 10 mM PBS solution (pH 7.4), were added onto the surface of the working electrode. Pt auxiliary electrode and Ag/AgCl reference electrode were inserted into the electrolyte droplet vertically. A positive potential of E = 0.9 V (vs Ag/AgCl) was applied for electrochemical etching. Pattern of PDMS Dots. A PDMS stencil with a thickness of 1.5 mm was self-made. Holes with a diameter of ca. 800 μm were punched through by using a sharpened needle tip (∼1.2 mm in diameter). And the interspaces of holes were controlled within 1 mm. The gold/PDMS cover chip was sealed with PDMS stencil and then cleaned by air plasma (PDC-32G, Harrick) for 60 s to drive out air captured by the holes. Drops of electrolyte were added to fill the holes. The three-electrode system and etching conditions mentioned above were then used. Pt auxiliary electrode and Ag/AgCl reference electrode were placed on the surface of the PDMS stencil covered with electrolyte, keeping away from the region of holes array. A positive potential of E = 0.9 V (vs Ag/AgCl) was applied. In this case, a Pt wire auxiliary electrode with a diameter of 250 μm was adopted. Cell Culture. Human gastric carcinoma BGC-823 cells (a gift from Gulou Hospital, Nanjing, China) were maintained in RPMI medium 1640 (Gibco Invitrogen Corp.) supplemented with 10% fetal bovine serum (Gibco Invitrogen Corp.) at 37 °C and 5% CO2 environment. Protein/Cell Patterning. SAMs of EG6 on heterogeneous Au/PDMS substrate with PDMS dots pattern were prepared by immersing chips in 2 mM solution of EG6 dissolved in deoxygenated, absolute ethanol for 4 h at room temperature. After rinsing with absolute ethanol and Milli-Q water and then drying with high purity nitrogen, fluorescence protein Cy3-anti-IgG (40 μg mL-1 in PBS) was used as a probe to show the cytophilic region defined by EG6. The adsorption time of Cy3-anti-IgG on PDMS was 1 h, followed by rinsing with PBS thoroughly. For cell patterning, we incubated patterned substrate with FN solution (20 μg mL-1 in PBS) after EG6 passivation. Drops of the protein solution were evenly distributed onto the surface of the patterned Au/PDMS. After storing at room temperature for 30 min, the substrate was rinsed with PBS for several times before cell seeding. Confluent BGC-823 cells were trypsinized and washed. They were then resuspended in RPMI medium 1640 supplemented with 10% fetal bovine serum, and directly seeded on the patterned substrate at a density of ca. 5  105 cells mL-1. (34) Dou, Y. H.; Bao, N.; Xu, J. J.; Chen, H. Y. Electrophoresis 2002, 23, 3558– 3566.

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Figure 1. Sandwich-type framework for chemical gold plating on PDMS. (a) Cover chip, PMMA frame, and base chip with similar size were placed layer by layer. Solution for chemical gold plating was injected into the sandwich architecture. (b) Photos of cover chip (left) and base chip (right) coated with gold film. (c) Microscope images of gold film on cover chip (left) and base chip (right). Scale bar = 50 μm. The substrate was incubated in a CO2 incubator for 1 h, followed by washing with PBS moderately before observation. AO (100 μg mL-1 in PBS) was diluted with cell medium as 1:50 (v/v) for staining live cells. Microscopy and Imaging. A DMIRE2 Inverted fluorescence microscope (Leica, Germany) equipped with a DP71 CCD (Olympus, Japan) was used for microimaging (bright-field and fluorescent images). Photos were acquired by using a Nikon camera 4300. Instruments. Scanning electron microscopy (SEM) was performed on a FEI Sirion 200 field emission scanning electron microscope (FEI, USA) at an accelerating voltage of 5 kV. X-ray powder diffraction (XRD) patterns were taken with a XRD-6000 instrument (Shimadzu, Kyoto, Japan) using a monochromatized X-ray beam with nickel-filtered Cu KR radiation. A CAM 200 contact angle goniometer (KSV Instruments Ltd., Helsinki, Finland) was used to measure water contact angles. Electrochemical measurements were performed on a CHI 750C electrochemical workstation (Shanghai Chenhua Apparatus Corp., China).

Results and Discussion Fabrication of Gold Film on PDMS by Chemical Plating. We used chemical gold plating to fabricate a continuous gold layer on PDMS, which is low-cost and convenient compared to routine methods such as metal evaporation and sputtering. In an attempt to obtain a desirable gold layer on PDMS which is smooth, shining, and conductive, a sandwich-type framework was adopted. The process is illustrated in Figure 1a. After holding at room temperature for about 3 h, gold grew on both chips, but color and luster were notably different (Figure 1b). The PDMS cover chip coated with gold film looked more like a shining mirror. The experimental observation under microscope showed that the gold film on the cover chip was flat, while on the base chip it was obviously made of two portions: a loose layer composed of floccule deposited randomly on a flat layer (Figure 1c). As a result, the thickness of the gold layer on the base chip drastically increased to micrometers. The gold film on both chips was firm enough for washing with water and ethanol, but scraping should be avoided as much as possible. The growth of gold on PDMS was considered to follow a twostep reduction mechanism (Figure 2). It was reported in our previous work35 that the residual Si-H in PDMS can reduce HAuCl4 directly. When PDMS film is dipped in the HAuCl4 solution, gold nanoparticles can be produced in the PDMS (35) Zhang, Q.; Xu, J. J.; Liu, Y.; Chen, H. Y. Lab Chip 2008, 8, 352–357.

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Figure 2. Schematic diagram for a two-step reduction mechanism in the growth of gold on a PDMS chip.

matrix. Thus, in the first step, gold nanoparticles would be formed on the surface and even embeded into the PDMS surface layer adjacent to the solution which contains HAuCl4. In the second step, the resulting gold nanoparticles on the surface would act as seeds to induce deposition of gold produced by the reduction of HAuCl4 with glucose on PDMS and form a continuous gold layer. This two-step reduction mechanism has been proven by two simple experiments. In experiment 1, the PDMS chip which was half protected by a mask was pretreated by air plasma to oxidate residual Si-H groups on the exposed surface. In the result, gold film appeared only on the protected region. In experiment 2, after removing the gold layer thoroughly, the PDMS chip was used for a second time. Whereas gold film could not be achieved anymore, it may be because the gold nanoparticles have been taken off in the pretreating process and cannot be formed again (Si-H groups on the surface had been almost consumed in the first time). Obviously, this two-step reduction mechanism depended strongly on the existence of residual Si-H groups on the PDMS surface, which would avoid the cumbersome activation process required to produce functional groups for succeeding catalyst formation.32,33 It could be looked as an orderly deposition which led to the formation of a compact gold layer on both PDMS chips. An additional deposition of gold conglomeration driven by gravity would take place on base chip, which led to the appearance of a loose layer sitting on the compact layer and would be responsible to the difference between the gold film on the cover chip and base chip (Figure 1c). Characterization of Gold Film on PDMS Cover Chip. Due to the uniformity, a gold/PDMS cover chip was selected as substrate for patterning in the latter step. Its surface properties were investigated by SEM, water contact angle measurements, XRD, and cyclic voltammetry (CV) (Figure 3). SEM images showed that the gold layer was composed of coherent nanoparticles and its thickness was about 100 nm. Figure 3c shows the XRD pattern of the gold layer on the cover chip. The diffraction peaks demonstrated that the gold layer was composed of pure crystalline gold with a face-centered cubic structure (JCPDS card No. 04-0784). The higher intensity of the (111) diffraction peak indicated that the deposited gold structure has a tendency to grow with the surfaces dominated by the lowest energy (111) facet. In addition, this gold substrate presented approximate hydrophobicity (water contact angle was 110.6°) to native PDMS. The conductivity of the gold/PDMS cover chip can be demonstrated by CV in 0.5 M H2SO4. Typical oxidative and reductive peaks corresponding to the electrochemical behavior of Au could be observed clearly in cyclic voltammograms. Langmuir 2009, 25(17), 10402–10407

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Figure 3. Characterization of gold on a cover chip. SEM photographs of the surface morphology (a) and cross section (b) of the gold layer. The inset in panel (a) is the shape of the water droplet on the surface of the gold/PDMS cover chip. Scale bar in (a) is 1 μm. Scale bar in (b) is 500 nm. (c) XRD pattern of a gold layer on the cover chip. (d) Cyclic voltammogram of a gold electrode made of a gold/PDMS cover chip in 0.5 M H2SO4. Scan rate is 100 mV s-1.

Electrochemical Etching. For electrochemical etching, every gold/PDMS cover chip used as working electrode was cleaned thoroughly in 0.5 M H2SO4 previously. At neutral potential, the aqueous electrolyte showed itself hemispherical drop on the hydrophobic surface of working electrode. Pt wire auxiliary electrode and Ag/AgCl reference electrode were inserted in the drop vertically. A potential of E = 0.9 V (vs Ag/AgCl) was applied to etch gold. During the treatment process, the gold film under the coverage of electrolyte gradually changed to transparent in 30 s. Simultaneously, the colorless electrolyte got glaucous. In the end, the oxidation current became zero and the circuit was disconnected. A clear borderline appeared at the edge of the electrolyte and delimited the electrochemically treated region and the untreated region which correspond to the dim half and the bright half, respectively, in Figure 4. It was evident that most gold nanoparticles have been peeled off. When the gold electrode was exposed to Cl- free solution (100 mM KNO3 solution), there were not any changes under the same experiment conditions. So, chlorine anion in the electrolyte was critical to this event. Gingery et al. have employed Cl- to realize Au dissolution for making sharp gold scanning tunnelling microscopy tips.36 The electrochemical reaction was as shown below: AuCl4 - þ 3e - fAu þ 4Cl - ,

EΘ ¼ 1:002 V ðvs RHEÞ ð1Þ

A working potential range of 0.9-1.1 V was suitable for etching. At higher potential (1.23 V, vs RHE), oxygen evolved in water electrolysis would overflow drastically from the gold surface and consequently tear the gold film. Preparation of Patterned Au/PDMS Substrate. Two approaches were introduced for preparing Au/PDMS substrate with different patterns. Both of them were founded on the phenomenon relevant to reaction 1. One made use of hydrogen bubbles generated on the auxiliary electrode, which went along :: (36) Gingery, D.; Buhlmann, P. Rev. Sci. Instrum. 2007, 78, 113703-1–113703-4.

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Figure 4. SEM image of the boundary between the electrochemically treated region (dim) and untreated region (bright). Scale bar is 10 μm.

with reaction 1, to form a gold “island” pattern. The other utilized the stencil as physical confinement to define the region which would make contact with electrolyte and realized local electrochemical etching. The details are elucidated in Figure 5 and following paragraphs. Pattern of Gold “Island”. Since the potential required by reaction 1 was a little higher than the potential of hydrogen generation (-0.83 V, vs RHE) in water electrolysis, the electrochemical etching at the working electrode was accompanied by the hydrogen overflow at Pt auxiliary electrode for certain. This implied that a safe bubbles region would be constructed under the Pt auxiliary electrode when the auxiliary electrode faced the gold substrate vertically. Pt wire electrodes with different diameters were employed as auxiliary electrodes successively, and the vertical distance between the Pt wire electrode and gold working electrode was controlled around to 1.2 mm. As we anticipated, DOI: 10.1021/la900944c

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Figure 5. Schematic diagram of two approaches for constructing Au/PDMS substrate with different patterns. (a) Gold/PDMS cover chip was used to make patterned Au/PDMS substrate. It was fitted with aluminum conductive adhesive tape as lead wire previously. Pattern of gold “island” was formed as follows: (b1) Drops of electrochemical etchant were added onto the cover chip. The magnified schematic graph indicates the key part for electrochemical etching. (c1) Gold under the coverage of electrolyte was etched with an “island” reserved. The upper photos correspond to gold “islands” saved under Pt auxiliary electrode with 500 μm diameter (left) and 250 μm diameter (right). The actual diameter of gold “island” is shown below its photo. The lower microscope image points to a local gold “island”. Scale bar is 200 μm. The pattern of PDMS dots was formed as follows: (b2) A PDMS stencil was placed on the cover chip and they were cleaned by air plasma together. (c2) Electrochemical etchant was added onto the structure and filled the holes in the stencil. A three-electrode system was prepared. (d2) Application of appropriate potential helped gold removal by electrochemical etching. PDMS dots appeared on the cover chip at the positions corresponding to the holes of the stencil. A gray level image of dots is presented. Scale bar is 1 mm.

during electrochemical etching, hydrogen bubbles overflowed and the gold film covered by the bubbles region was protected from damage. As a result, an “island” of gold was reserved (Figure 5). And from the image of the gold “island”, we could observe concentric rings with different colors obviously. They were corresponding to the increase of transparency from the center to the rim and the gas diffusion gradients from the brim of anxiliary electrode. In addition, the size of gold “islands” was different as the diameter of the auxiliary eletrode changed, which resulted from the variation of generated hydrogen volume caused by different electrode areas. The results suggested that gold “islands” with different size could be achieved by modulating some factors such as the size of the auxiliary electrode, the applied potential, the vertical distance between the auxiliary electrode and substrate, as well as the coverage area of the electrolyte. Pattern of PDMS Dots. For patterning, utilizing stencils with holes of specified shape and size is long-standing. As early as 1967, Carter had used a nickel stencil to make cellular micropatterns.37 Afterward, metallic stencils were replaced gradually with stencils made of elastomeric material such as PDMS, due to the compact touch between elastomeric stencils and substrates.38,39 In this work, a PDMS stencil with holes array was used as a physical confinement for selective gold etching, which could be well bound with the Au/PDMS cover chip reversibly (Figure 5). Since the PDMS stencil and Au/PDMS cover chip are similar in hydrophobicity, bubbles trapped in the holes are difficult to remove with increased depth and decreased diameter of holes. Air plasma treatment was employed to change (37) Carter, S. B. Exp. Cell Res. 1967, 48, 189–193. (38) Folch, A.; Jo, B.-H.; Hurtado, O.; Beebe, D. J.; Toner, M. J. Biomed. Mater. Res., Part A 2000, 52, 346–353. (39) Ostuni, E.; Kane, R.; Chen, C. S.; Ingber, D. E.; Whitesides, G. M. Langmuir 2000, 16, 7811–7819.

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its hydrophobicity to hydrophilicity. The time of electrochemical treatment was prolonged to 5 min to guarantee gold removal from all holes to the best. Thus, dots with similar size to stencil holes were produced. The formation of the above-mentioned “islands” did not emerge in this case because the auxiliary electrode was placed on the blank region of the PDMS stencil covered with electrolyte but not the region of the holes array. Protein/Cell Patterning on Patterned Au/PDMS Substrate. Patterned Au/PDMS substrate fabricated with the help of the stencil was prepared for protein and cell patterning. Since the thickness of the gold film is about 100 nm, contrasting with the size of BGC-823 cells which are ca. 15 μm in suspension, a dot circled with gold could not be considered as a well. This meant that there is no physical barrier and cells could ignore the “ridge” entirely. Here, the special properties of gold and PDMS were used to build contrast regions which could be distinguished by cells (Figure 6a). EG6 was reported by many research groups as an inert material to resist adsorption of proteins and adhesion of cells efficiently.40,41 Its assembly on gold could conduce to cytophobic regions. It is well-known that PDMS is strong to adsorb the hydrophobic biomolecules.42,43 To demonstrate the applicability of this strategy, we employed fluorescence protein Cy3-anti-IgG as a probe to show cytophilic region defined by EG6. Experimental results (Figure 6b) showed that red fluorescence appeared as a dot pattern. Thus, exposed PDMS due to electrochemical etching still plays the role of proteins adsorption. (40) Mrksich, M.; Chen, C. S.; Xia, Y. N.; Dike, L. E.; Ingber, D. E.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10775–10778. (41) Nelson, C. M.; Raghavan, S.; Tan, J. L.; Chen, C. S. Langmuir 2003, 19, 1493–1499. (42) Makamba, H.; Kim, J. H.; Lim, K.; Park, N.; Hahn, J. H. Electrophoresis 2003, 24, 3607–3619. (43) Liu, J. K.; Lee, M. L. Electrophoresis 2006, 27, 3533–3546.

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Figure 6c, cells (stained with AO) have adhered on electrochemically treated regions selectively.

Conclusions

Figure 6. Strategy for cell patterning. (a) Assembly of EG6 on gold and adsorption of FN on PDMS were adopted to build a contrast region which could be distinguished by cells. (b) Fluorescence image of Cy3-anti-IgG pattern. (c) Fluorescence image of AOlabeled BGC-823 pattern. The scale bar in each case is 200 μm.

Cy3-anti-IgG was replaced by extracellular matrix (ECM) protein fibronectin to construct a cytophilic region for cell patterning after EG6 passivation. The patterned substrate was immersed into a BGC-823 cell suspension. After 1 h incubation in media, a cell pattern was achieved as anticipation. As shown in

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A novel approach which used chemical plating coupled with electrochemical etching strategy has been successfully used to fabricate patterned Au/PDMS substrate. This heterogeneous substrate is beneficial to build contrasting cytophilic/cytophobic regions for cell patterning applications because self-assembly based on gold and adsorption based on PDMS are both typical in surface modification. Compared to gold foil in common use which is based on silica or glass substrate, introduction of PDMS which is the most general material in biochips is significant to contact cell patterning based on gold with biochips indeed. The research of renewable electrodes and complex integrated biochips with more elaborate division of the conductive gold layer by electrochemical etching presented here is being carried out in our laboratory. Acknowledgment. This work was supported by the National Natural Science Foundation of China (No. 20890021, 20775033), the National Natural Science Funds for Creative Research Groups (20821063), and the 973 Program (2007CB936404, 2006CB933201).

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