Patterning of proteins and cells on functionalized surfaces prepared by polyelectrolyte multilayers and micromolding in capillaries

Biosensors and Bioelectronics 22 (2007) 3188–3195

Patterning of proteins and cells on functionalized surfaces prepared by polyelectrolyte multilayers and micromolding in capillaries Hyun-Woo Shim a , Ji-Hye Lee a , Taek-Sung Hwang a , Young Woo Rhee a , Yun Mi Bae b , Joon Sig Choi b , Jongyoon Han c , Chang-Soo Lee a,∗ a

Department of Chemical and Biological Engineering, Chungnam National University, Yuseong-gu, Daejeon 305-764, Republic of Korea b Department of Biochemistry, Chungnam National University, Yuseong-gu, Daejeon 305-764, Republic of Korea c Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Received 20 October 2006; received in revised form 14 February 2007; accepted 21 February 2007 Available online 4 March 2007

Abstract A method for protein and cell patterning on polyelectrolyte-coated surfaces using simple micromolding in capillaries (MIMIC) is described. MIMIC produced two distinctive regions. One contained polyethylene glycol (PEG) microstructures fabricated using photopolymerization that provided physical, chemical, and biological barriers to the nonspecific binding of proteins, bacteria, and fibroblast cells. The second region was the polyelectrolyte (PEL) coated surface that promoted protein and cell immobilization. The difference in surface functionality between the PEL region and background PEG microstructures resulted in simple patterning of biomolecules. Fluorescein isothiocyanate-tagged bovine serum albumin, E. coli expressing green fluorescence protein (GFP), and fibroblast cells were successfully bound to the exposed PEL surfaces at micron scale. Compared with the simple adsorption of protein, fluorescence intensity was dramatically improved (by about six-fold) on the PEL-modified surfaces. Although animal cell patterning is prerequisite for adhesive protein layer to survive on desired area, the PEL surface without adhesive proteins provides affordable microenvironment for cells. The simple preparation of functionalized surface but universal platform can be applied to various biomolecules such as proteins, bacteria, and cells. © 2007 Elsevier B.V. All rights reserved. Keywords: Micromolding in capillaries; Biomolecules patterning; Polyelectrolytes; Poly(ethylene glycol)

1. Introduction The selective patterning of various biomolecules in welldefined microstructures is critical for the development of biosensors and biochips (Mooney et al., 1996; Nicolau et al., 1999; Willner and Katz, 2000; Lee et al., 2003). However, the fabrication of microstructures with well-ordered and spatially discrete forms to provide the patterned surface for the immobilization of biomolecules is difficult because of the lack of distinct physical and chemical barriers separating patterns (Wang et al., 2005). The fabrication of microstructures with physical and chemical barriers that confine various biomolecules, and biological barriers that prevent nonspecific binding of biomolecules, is a prerequisite for constructing highly ordered, reliable biosen-

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Corresponding author. Tel.: +82 42 821 5896; fax: +82 42 822 8995. E-mail address: [email protected] (C.-S. Lee).

0956-5663/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2007.02.016

sors, as well as for densely packed biomolecules (Lee et al., 2003, 2006). Photolithography is the most frequently used technique for fabricating structured physical and chemical barriers at microscopic levels (Wang et al., 2005). This approach can be used to fabricate microwells and increase the density of microstructures for patterns of nanoparticle protein binding. However, the physical adhesion of nanoparticles tends to be unstable and it is generally effective only for hard substrates such as silicon wafers. Another technique is soft lithography with flexible elastomers such as polydimethylsiloxane (PDMS). Microcontact printing (␮CP) with self-assembled monolayers (SAMs), and capillary force lithography for making physical barriers, were used for patterning biomolecules (Cuvelier et al., 2003; Foley et al., 2005; Whitesides et al., 2001). Recently, microreservoirs have been used for protein patterning (Lee et al., 2006). Although well-defined microreservoirs and a surface modification technique was successfully

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demonstrated, this approach required delicate chemical modification steps such as gold sputtering, microcontact printing of polyethylene glycol (PEG), and surface modification of the patterned region. Furthermore, the nonfouling chemicals (PEG) were only applied to the tops of microreservoirs, and they could not prevent nonspecific binding of biomolecules on the sides of microreservoirs. Suh et al. (2004) used capillary force lithography with PEG microstructures for biomolecules patterning. However, when the method is applied to fabricate PEG microstructure at micron level, the retention of PEG may be a problem due to imperfect capillarity in the patterned region. Residual PEG in the patterning region may interfere with the protein and cell patterning because inherent property of PEG prevents biomolecular adhesion. Other potential problems include the instability of the patterned biomolecules owing to simple adsorption without covalent binding between biomolecules and bare surface, washout in the cleaning process, and the low fidelity of the biomolecular patterning because modification of surface could improve bimolecular binding and strengthen the patterning force between biomolecules and surface. In the case of cell patterning, the method also utilized adhesive protein such as fibronectin to increase attachment between cell and surface. However, the adhesive proteins to bind the cells to the surface might be easily degraded. A possible solution to overcome this problem concerns the design of novel interfaces promoting cell adhesion and growth on surface without use of protein coating (Boura et al., 2005; Falconnet et al., 2006). There are thus increasing interests for the fabrication of new functionalized surface aimed to improve cellular adhesion by mimicking extracellular matrix such as collagen or fibronectin (Forry et al., 2006; Reyes et al., 2004). Although micromolding in capillaries (MIMIC) has already been used to obtain micropattern of lipid bilayers (Schuy and Janshoff, 2006) and proteins (Gyoervary et al., 2003) on planar surfaces, MIMIC is not yet intensively used to construct various biomolecule patterns. Thus, we describe a biomoleculepatterning method that is similar to MIMIC and based on the soft-lithographic fabrication of PEG microstructures and the surface modification with polyelectrolyte multilayers. The method creates spatial barriers that prevent nonspecific biomolecule binding. It uses a surface modification technique to selectively bind biomolecules to the patterned region with mainly electrostatic interactions. First, the surface is coated with selfassembled multilayers of polyelectrolyte (PEL), layer-by-layer (LbL), to bind the proteins, bacteria, and cells. Next, the PEG microstructures are fabricated using UV photopolymerization with the MIMIC method. The PEG microstructures are barriers to the adhesion of proteins and cells because the hydrophilic nature of PEG resists biomolecular adsorption. Although ␮CP is relatively a simple and versatile technique for micropatterning, it cannot provide control over surface topology. Therefore, the nonpatterned regions must be modified with biological barriers to prevent protein or cell adhesion. In comparison with ␮CP, PEG microstructures and selectively patterned regions are more easily obtained with our method. Furthermore, MIMIC combined with LbL coating provides a general platform for biomolecular patterning on a broad range of materials because it

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can be easily applied to various substrates such as glass, silicon, silicon dioxide, and polymers. 2. Materials and methods 2.1. Materials Cationic polyallylamine hydrochloride (PAH, Mw 70,000), anionic polystyrene sulfonate ammonium salt (PSS, Mw 200,000), polyethylene glycol-dimethacrylate (PEG-DMA) (Mn = 330) and fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Other chemicals of analytical or research grade were used. 2.2. PEL coatings on glass Glass was cleaned with a piranha solution consisting of a 4:1 mixture of 50% aqueous solution of H2 SO4 and 30% aqueous solution of H2 O2 , rinsed sequentially with deionized water, ethanol, acetone, and finally dried with nitrogen. The cleaned glass was immersed in a solution of PAH (20 mM, pH 9.0) for 20 min, followed by three washes with distilled water. The PAHcoated glass was then immersed in the solution of PSS (60 mM, pH 7.0) for 20 min, followed by three washes. This procedure was repeated until the desired number of PEL layers was assembled on the slide, with a positively charged PAH on the outermost layer. 2.3. The fabrication of PEG microstructures using the MIMIC method PDMS (Sylgard 184, Dow Corning, USA) micromolds and microstamps were fabricated against a complementary relief structure that was prepared by conventional photolithography. The heights of the microstructures in the PDMS blocks were about 20 ␮m. Each PDMS mold was cut such that it formed a network having open ends (Fig. 1(a)). The trimmed PDMS mold was placed on PEL-coated glass to make conformal contact (Fig. 1(b)). When PEG-DMA containing 0.5% (v/v) photoinitiator (DAROCURE D1173, Ciba-Geigy, USA) was placed at the open ends of micromolds, the PEG-DMA spontaneously filled the void spaces by capillary action (Fig. 1(c)). PEG-DMA was cured with an ultraviolet light (250–400 nm, 100 mJ/cm2 ) for 15 min (Fig. 1(d)) and then PDMS micromold was peeled off (Fig. 1(e)). 2.4. Bacteria and cell culture Bacterial cell, E. coli BL21-pET23b-GFP, were grown from a single colony in Luria-Bertani (LB) broth including 100 mg/ml of ampicillin at 37 ◦ C and 200 rpm. When the optical density (OD 600 nm) of the culture reached ca. 1.0, the bacterial cells were centrifuged at 12,000 rpm for 10 min and resuspended in phosphate buffer saline. NIH3T3 fibroblast cells were cultured in Dulbecco’s modified Eagle medium (DMEM), supplemented with 10% fetal

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Fig. 1. A schematic diagram for protein and cell patterning using PEL coating and MIMIC: (a) surface modification with PEL (PAH/PSS), (b) PDMS micromold placement, (c) filling the micromold with a liquid prepolymer by capillary action, (d) the curing of prepolymer by UV radiation, (e) removing the PDMS micromold, and (f) loading of biomolecules onto the fabricated surface.

bovine serum and penicillin–streptomycin. Trypsin-EDTA was used to detach the cells from culture flasks and centrifuged at 800 rpm for 5 min. Fibroblasts was loaded onto the microstructured substrate in a culture chamber.

of the fluorescence images were analyzed by the image software (IPLab, USA).

2.5. Patterning of proteins, bacteria, and cell

The LIVE/DEAD BacLight Bacteria Viability Kits (Molecular Probes, USA) was used to examine the bacteria on the pattered surface. The kit contains a combination of two nucleic acid stains; SYTO9 and propidium iodide. SYTO9 fluorescences green and labels all of the intact bacteria and propidium iodide penetrates only bacteria with damaged membranes. Thus, live bacteria with intact cell membranes stain fluorescent green, whereas dead bacteria with damaged membranes stain fluorescent red. The procedures of determination if the bacteria on the patterned substrates were alive or dead were performed according to the recommended protocols from Molecular Probes.

Finally, a small amount of FITC-BSA (1 ␮g/ml, 10 ␮l), bacteria (1 × 109 cells/ml, 10 ␮l), and fibroblast (2000 cells/mm, 10 ␮l) was dispensed onto the fabricated substrate to cover the entire surface. After binding the proteins and bacteria at room temperature for 10 min, the extent of patterned biomolecules was measured with the fluorescence intensity within patterns and between patterns (batch-to-batch trials) were calculated. In case of NIH3T3, the cells were allowed to grow after being loaded on microstructured substrate at 37 ◦ C in a humidified 5% CO2 incubator. Immediately prior to the experiments, the medium was removed and washed with PBS. 2.6. Contact angle Contact angles of the substrates were determined within 24 h. A Kr¨uss G10 contact angle analyzer (Kr¨uss GmbH, Hamburg, Germany) was used. All the measurements were carried out at room temperature and ambient humidity. Each reported value was the average of five measurements of each contact angle.

2.8. Bacteria viability assay

2.9. Scanning electron microscope (SEM) The topological analysis was performed on a Hitachi S4800 scanning electron microscope with an acceleration voltage of 10 kV. Polymeric microstructures were sputtered with gold (about 100 nm thick) before imaging. 3. Results and discussion 3.1. The fabrication of PEG microstructures

2.7. Fluorescence analysis All fluorescence images were acquired with a fluorescence microscope (NIKON, TE-2000, Japan) equipped with a highresolution CCD camera (Coolsnap, Roper Science, USA). All

Our method used the previously reported MIMIC technique, and the choice of a suitable prepolymer was critical (Kim and Whitesides, 1997). A low molecular weight PEG-DMA (Mn = 330) was used for the fabrication of PEG microstruc-

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tures because PEG-DMA enabled high cross-linking densities, and the resulting microstructures were not washed out with the aqueous buffer. PEG-DMA has several important properties for fabricating microstructures with MIMIC: (1) The prepolymer is liquid and has a low viscosity (6.5 cP at 25 ◦ C). Thus, it fills micromolds quickly, with the trapped air easily escaping by diffusing through the PDMS mold. (2) It is cross-linked by ultraviolet irradiation. This is a simple, fast, and efficient process for fabricating microstructures. (3) It is inert to PDMS molds. It does not react with, swell, or adhere to the PDMS mold. It does not contain organic solvents that cause the PDMS mold to swell. It preferentially adheres to the surface of the substrate. And finally, the elastomeric character of PDMS enables it to make good conformal contact with the substrate because the low interfacial free energy of the surface of PDMS (γ PDMS/air = 21.6 dyne/cm) provides stable adhesion between the mold and the substrate without leaks, but it still allows the PDMS to be easily detached from the fabricated microstructures formed by MIMIC. Capillary force endows a polymer solution with mobility in the void space of the micromold that results in the fabrication of negative replicas of the micromold. In the case of hydrophobic polymers, the polymeric solution is partially wettable for the hydrophobic PDMS micromold or microstamp. Thus, capillary force can be depressed within void space (capillary depression) (Zabow et al., 2002). When the hydrophobic PDMS mold was used in the MIMIC process under our experimental conditions,

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the fabricated microstructures were incomplete and the surfaces of microstructures were very rough because of the nonhomogeneous filling of PEG-DMA (Fig. 2(a) and (b)). Although the contact angle of PEG-DMA (65◦ ) with the bare PDMS indicated PEG-DMA was slightly hydrophilic, we could not obtain perfect capillary filling owing to the lack of hydrophilicity of the PDMS micromold and microstamp. A previous study that improved filling speed by increasing capillarity with a vacuum was unsatisfactory because it required a complex setup and it resulted in a limited resolution due to the collapse of the micromold under the vacuum (Jeon et al., 1999). Thus, the hydrophilic surface treatment of PDMS molds and stamps is essential to facilitate the homogeneous filling of PEG-DMA into spaces in the PDMS. The rapid, simple modification of the surface of the PDMS micromold or microstamp by plasma oxidation to make it hydrophilic was used (Lee and Voros, 2005). Because of the hydrophilic nature of PEG-DMA and hydrophilic modification of the PDMS mold, the mobility of PEG-DMA was sufficient for homogeneous flowing throughout the process. The SEM images of the PDMS microstamp and the fabricated PEG microstructures indicated the resulting microstructures were defect-free replica (Fig. 2(c) and (d)). The PEG microstructure is an accurate negative replica of the relief structure on the surface of the PDMS mold. The patterned microstructures have two distinct regions: the PEG microstructures make a physical, chemical, and biological barrier, and the

Fig. 2. The comparison of capillary filling of PEG-DMA within a bare PDMS micromold and plasma treated PDMS microstamp: (a) the image of PEG-DMA solution in the bare PDMS microstamp with a pattern of 50 ␮m squares, (b) the image of PEG-DMA solution in the bare PDMS micromold with a pattern of 50 ␮m lines, (c) plasma treated PDMS microstamp having 40 ␮m cylinders, and (d) the PEG microstructure that was neatly transferred from the plasma treated PDMS microstamp.

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exposed substrates previously modified with PEL promote protein binding. The hydrophilic surface modification of the PDMS mold resulted in the fabrication and replication of highly stable, precise PEG microstructures with accurately reproduced topological features. The SEM images suggest that our method is suitable not only for fabricating well-defined microstructures with various shapes, including circles, squares, triangles, stars and lines, but also for the easy, quick preparation of surfaces for biomolecular patterning with reliable reproducibility. 3.2. Surface modification with PAH/PSS The sequential adsorption of oppositely charged polyelectrolytes by layer-by-layer deposition technique is well known for an efficient method to easily modify the versatile surfaces. The technique allows controlling structure and film thickness on a molecular level. In this study, we demonstrated a simple procedure to coat glass by self-assembly of the polyelectrolyte multilayers as a platform surface for the fabrication of biomolecules patterning. Prior to biomolecules patterning, the procedure of surface modification with polyelectrolytes should be confirmed. The periodic oscillations in contact angle values were observed for multilayer terminated by positively charged or negatively charged polyelectrolytes (Fig. 3). Odd and even numbers represent the top surface layers of PAH and PSS, respectively. PAH layers (47–51◦ ) are more hydrophobic than PSS layers (28–30◦ ). McCarthy’s group suggested that the distinct oscillation of contact angles was attributed to the periodical change in the surface properties of the top layer and the wettability of multilayers is significantly influenced by the sublayer underneath when PAH/PSS multilayer films were deposited (Wei and Thomas, 1997). As a result, the disappearance of the oscillatory trend in contact angle obtained reflects the multilayers form disordered layers (i.e. mixed layers) composed of interdigitated PAH and PSS segments due to the insufficient surface coverage as well as high surface roughness. Fig. 3 confirmed the stable formation of the top surface layers with sufficient surface coverage as well as with even surface roughness. Thus, the distinct oscillation

Fig. 4. Effect of the thickness of mutilayered polyelectrolyte thin film on binding capacity. A 1 ␮g/ml of FITC-BSA was loaded onto modified surfaces coated with different thickness of polyelectrolyte multilayers.

of contact angles between PAH and PSS could confirm that the surface modification was correctly performed. 3.3. Effect of structural properties of PEL thin film on protein patterning We describe a simple procedure for creating a polymeric thin film with self-assembled, multiple PEL layers as a platform for binding biomolecules. Biomolecules, including DNA, RNA, proteins, bacteria, and cell, are easily immobilized in the PEL thin film by the combination of strong electrostatic interactions, hydrophobic interactions, and entrapment in the porous structure (Haynie et al., 2005). Firstly, we constructed a positively charged surface at the outmost layer for the immobilization of FITC-BSA because the net charge of BSA in phosphate buffer (pH 7.5) is negative considering its isoelectric point (pI = 4.7). To investigate the optimum layer of PEL thin film for successful patterning of biomolecules, FITC-BSA as a model biomolecule was immobilized onto different layers of PEL (PAH/PSS). Fig. 4 showed the typical binding isotherms of FITC-BSA with PEL, the most outer layer of which being constructed by cationic PAH polyelectrolyte. The amount of FITC-BSA that immobilized onto PEL thin film increased with an increase of the number of layers and saturated when the number of layer was around 9. This result correlated with an increase in the number of available binding sites on PEL thin film. Above nine layers, the intensity reached a plateau because the diffusion limitation of protein into PEL network might compensate increase of binding sites under the experimental condition. The trend of thickness effect is in agreement with the previous report on a PEL coating for a protein microarray (Zhou and Zhou, 2006). 3.4. Protein patterning

Fig. 3. Water contact angles measured from PAH/PSS multilayers; odd and even numbers indicated the layers deposited with PAH and PSS, respectively.

PEG microstructures and the PEL coating were examined with pattern (50 ␮m square) and FITC-BSA. The fluorescence intensity is assumed to be proportional to the density of the

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bound proteins. Protein binding is the result of covalent binding, physical adsorption, and specific interactions (Blawas and Reichert, 1998). Although covalent binding is generally the most stable form of bound proteins, it typically involves several stages of surface chemistry to obtain the activated substrates necessary for subsequent protein binding. Some of the detrimental effects of covalent binding are the loss of protein activity due to the randomly orientation of immobilized proteins, direct chemical bond formation with binding site or at an active site on protein, and the loss of flexibility in three-dimensional space. The specific interaction methods include biotin–avidin (Nowall et al., 1998), His tag-nickel (Kato et al., 2005), glutathione-GST (Sehr et al., 2001), and protein A- or G-antibody (Peluso et al., 2003). However, all require multiple treatment steps and all may produce denaturing by the introduction of fusion tag. We describe a simple procedure for creating a polymeric thin film with self-assembled, multiple PEL layers as a platform for binding proteins to a patterned substrate. Biomolecules can be easily immobilized in the PEL thin film by the combination of strong electrostatic interactions, hydrophobic interactions, and entrapment in the porous structure (Haynie et al., 2005). Our preliminary results are consistent with the previous report on a PEL coating for a protein microarray (Zhou and Zhou, 2006). The optimum thickness of the PEL film was about nine lay-

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ers in our experimental condition (Fig. 4). First, FITC-BSA was immobilized onto the 50 ␮m patterned square on a PELcoated surface surrounded by PEG microstructures. Spatially well-ordered, two-dimensional protein patterns were observed with high fluorescence intensity (161.7 ± 4.1), low noise signals (6.7 ± 1.5), and a reliable signal-to-noise ratio (pattern intensity/background intensity = 24.1) (Fig. 5(a)). The variations in the fluorescence intensity between and within patterns were approximately 5%. These results were collected from the analysis of 250 squares per microstamp from three separate batch experiments. Control experiments for the extent of adsorption of protein on nonmodified glass surfaces showed relatively weak fluorescence signals (26.4 ± 11.2) and nonhomogeneous signals having high standard deviations (11.2) for protein patterns and the PEG background (Fig. 5(b)). These measurements suggest that protein adsorption is negligible on fresh glass surfaces. However, nonspecific binding of FITC-BSA was also low on the PEG background in both cases. This result indicates that protein patterning is attributed to the selective binding on the PELcoated surface and the prevention of nonspecific binding on PEG. Compared with the simple adsorption of protein, fluorescence intensity was dramatically improved (by about six-fold) on PEL-modified surfaces. This shows that the immobilization

Fig. 5. Comparison of (a) fluorescence image of PEG patterns on the unmodified glass surface and (b) the PEL-coated surface by electrostatic interaction. (c) Quantitative analysis of images showing signal, noise, and signal-to-noise estimates. All experiments were performed with 1 ␮g/ml of FITC-BSA and a microstamp containing 50 ␮m squares.

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of FITC-BSA was increased as a consequence of the electrostatic interaction between proteins and PEL (Fig. 5(c)). Furthermore, the experiment on protein patterning confirmed that PEL provided high binding capacity and high-adhesion forces between proteins and PEL-coated surfaces because the signal intensity was preserved after several washing steps. It is clear that using the PEG microstructure for the pattern background prevented nonspecific binding of proteins (Elbert and Hubbell, 1998; Suh et al., 2004; Lee et al., 2006), and FITC-BSA could be patterned only on the PEL-exposed regions. As can be seen in the fluorescence image, the intensity of background signals associated with PEG microstructures was very low, and the patterned regions coated with PEL showed strong fluorescence signals. Our experimental results show that the fabricated PEG-DMA microstructures had protein resistance similar to that of PEGterminated SAM molecules because the molecular backbone of PEG was maintained regardless of its density and its cross-links. Our protein-patterning method can be applied to make various patterns such as circles, squares, stars, and lines. Our approach is a simple, reproducible, and stable method for protein patterning without background noise. 3.5. Bacteria and cell patterning Cell patterning on desired areas is an important technique for the development of biosensors, bioelectronic devices, and cell based microsystems. We focused on the effectiveness of the PEG microstructures as a biological barrier to reduce nonspecific binding of cells and PEL coated surface to improve cell immobilization. Bacterial patterning was carried out with GFP expressing E. coli to allow a visualization of their patterns. As expected, bacteria were correctly attached onto only PEL coated surface without nonspecific binding to PEG microstructures because fluorescence signals were observed at only PEL coated region (Fig. 6). In addition, patterned bacteria were treated with a live/dead assay kit; the red fluorescence was not observed from the pattered substrate, which indicated that they were viable on the patterned surface. Although bacterial adhesion to surfaces is not well understood, bacteria irreversibly adhere to surface through the formation of protein–ligand interactions and formation of an extracellular polymer (Vigeant and Ford, 1997; Razatos et al., 1998; Branch et al., 2001). Because most cells including bacteria adhere to surfaces through the formation of protein layer, surfaces that resist the adsorption of proteins are strong candidates as surface that resist the adhesion of bacteria (Razatos et al., 1998; Branch et al., 2001). During the long-term cultivation, the patterned bacteria could be alive and some of growing bacteria could laterally adhere in the PEL region. Thus, the stable preservation of bacterial pattern after 10 days could support our proposed method is relatively reliable in considering of the absence of adhesive proteins in bacterial patterning process. Cells have negatively charged surface because glycolipids and glycoproteins are located on the outer membrane. PAH polymers having amine groups are positively charged at the pH used in this study. Thus, electrostatic interaction between PEL and cell will be happened, which is expected to dramatically

Fig. 6. Bacteria and cell pattern. (a) Optical image of bacterial pattern that is selectively anchored on the 50 ␮m line. (b) Fluorescence image of bacterial pattern. The distinctive difference of fluorescence between PEL region and PEG region with the boundary line was clearly observed. The scale bar indicated 50 ␮m. (c) Optical image of patterned NIH3T3 fibrobrasts culture; after 1 day of culture, cells were spread out. The cell pattern can be stable at least 1 week if suitable culture condition is provided.

improve cell adhesion during the patterning process. As shown in Fig. 6(c), the cells are well attached and stretched out in PEL regions. A morphology of patterned cell indicated healthy cells with good surface attachment. The PEL layer yields a highly charged substrate, as proved by contact angle measurements in Fig. 3. Electrostatic interaction between the polycation and the net negative charge on cell surface probably provides the initial surface interaction, but persistent attachment and growth involves more complicated biochemical interaction. Free protonated amine groups in the final PAH layer may be important, as primary amines have been reported to improve cell adhesion and growth when covalently bound to surface. Although general

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methods employ adhesive proteins such as fibronectin, collagen et al., this method successfully utilized PEL layer as adhesive layer without use of adhesive proteins. This result suggests that the use of PEL could reduce the susceptibility to degradation and provide the greater stability of patterned cells. In addition, the PEG regions did not show attachment of cells although blocking agent did not used to prevent cell adhesion. In conclusion, proposed method proved that fabricated PEG microstructures have potential ability to inhibit bacteria and cell adhesion through the minimization of protein binding and PEL surface efficiently provide adhering layer for bacteria and cells, which result in successful cell patterning at micron scale. In addition, the great simplicity of preparation of functionalized surface offers large possibilities in surface properties controlling such as thickness, roughness, viscoelasticity, and wettability. 4. Conclusions We have demonstrated a method that combines MIMIC for fabricating PEG microstructures with self-assembled PEL multilayers for biomolecular patterning. Highly reliable PEG microstructures with versatile shapes were fabricated using UVpolymerization and this simple MIMIC technique. The PEG microstructures and the exposed PEL-coated surface provided well-ordered physical, chemical, and biological barriers, making this technique suitable for fabricating biosensors and chips with high-density immobilizations of the proteins, bacteria, or cells. Using PEL as mild surface modification increased the efficiency of biomolecules immobilization and resulted in low releases of biomolecules from the coated surface. The successful protein, bacteria, and cell patterning showed the usefulness of PEL coating as universal surface for biomolecule anchoring onto designed area. The preparation of well-ordered two-dimensional microstructures with exposed PEL binding regions may find applications in the development of diversely selective immobilizations for biomolecules such as DNA, RNA, carbohydrates, proteins, cells, and tissues, without the loss of their three-dimensional structures. The demand for strict control over the positioning and the stable immobilization of biomolecules in fabricated structures may result in many applications for the methods we described in this study. Acknowledgment This work was supported by Grants (R01-2005-000-105580) from the Basic Research Program of the Korea Science & Engineering Foundation.

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