Tunable Micropatterned Substrates Based on Poly(dopamine

Mar 7, 2012 - Mireia Guardingo , Elena Bellido , Rosa Miralles-Llumà , Jordi Faraudo , Josep Sedó , Sergio Tatay , Albert Verdaguer , Felix Busqué ...
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Tunable Micropatterned Substrates Based on Poly(dopamine) Deposition via Microcontact Printing Hsiu-Wen Chien,† Wei-Hsuan Kuo,‡ Meng-Jiy Wang,‡ Shiao-Wen Tsai,§ and Wei-Bor Tsai*,† †

Department of Chemical Engineering, National Taiwan University, 1, Roosevelt Rd., Sec. 4, Taipei, 106, Taiwan Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan § Graduate Institute of Biochemical and Biomedical Engineering, Chung-Gang University, Taoyung, Taiwan ‡

ABSTRACT: A simple technique was developed to fabricate tunable micropatterned substrates based on mussel-inspired surface modification. Polydopamine (PDA) was developed on polydimethylsiloxane (PDMS) stamps and was easily imprinted to several substrates such as glass, silicon, gold, polystyrene, and poly(ethylene glycol) via microcontact printing. The imprinted PDA retained its unique reactivity and could modulate the chemical properties of micropatterns via secondary reactions, which was illustrated in this study. PDA patterns imprinted onto a cytophobic and nonfouling substrates were used to form patterns of cells or proteins. PDA imprints reacted with nucleophilic amines or thiols to conjugate molecules such as poly(ethylene glycol) for creating nonfouling area. Gold nanoparticles were immobilized onto PDA-stamped area. The reductive ability of PDA transformed silver ions to elemental metals as an electroless process of metallization. This facile and economic technique provides a powerful tool for development of a functional patterned substrate for various applications. modification of biomaterials.10 Marine mussels bind tightly to various surfaces on which they reside in aqueous environment, which relies on the exhaustively repeated 3,4-dihydroxy-Lphenylalanine−lysine (DOPA-K) motif found in mussel adhesive proteins.11,12 Inspired by the DOPA-K motif, Messersmith’s group recently applied dopamine to create versatile polydopamine (PDA) adlayers on a wide range of organic and inorganic materials.10 Dopamine molecule containing a catechol and an amine simulates the functional moieties of DOPA-K motif. Dipping substrates in an alkaline dopamine solution (e.g., pH 8.5) creates spontaneous coating of a thin adherent film due to oxidative polymerization of dopamine.10 A PDA adlayer is capable of promoting protein adsorption or cell adhesion.13−17 Furthermore, a PDA coating provides surface chemical reactivity for conjugation of a wide variety of molecules. For example, a PDA layer is able to react with thiols and amines via Michael addition or Schiff base reaction,15,18,19 assumedly owing to residual catechols of PDA. In addition, a PDA adlayer is able to reduce metal ions such as sliver and copper ions to metal deposition for electroless metallization.10,20 By reason for PDA’s diverse reactivity, micropatterning of PDA could be a simple and versatile platform to create a wide variety of micropatterned substrates. Several techniques such as photolithography,10 microfluidic technique,21 and microcontact printing (μCP)22,23 have been applied to create PDA

1. INTRODUCTION Microfabrication plays a critical role in microelectronics and optoelectronics and has recently been applied to biomedical applications.1−3 Microfabrication uses a variety of techniques such as photolithography,1,4 soft lithography,2,5,6 and inkjet printing3 to create spatially chemical or topographic micropatterns. Microcontact printing (μCP) is one of the most popular microfabrication techniques for creating chemical micropatterns.2,6−9 This technique is based on contact transfer of the material of interest from a poly(dimethylsiloxane) (PDMS) stamp onto a surface only on the areas contacted by the stamp. A variety of molecules, such as alkanethiols,6 proteins,7 polymers,9 or nanoparticles,8 have been employed as ink to create micropatterned surfaces via μCP. Among the molecules used in μCP, a self-assembled monolayer (SAM) of long-chain alkanethiolates provides a simple route to create a wide variety of chemical patterns depending on the terminal functionality of alkanethiolates. SAMs of alkanethiolates with reactive end-groups such as carboxylic acids or amines could react with other molecules via suitable chemical reactions or ionic interactions to alter the surface properties.5,6 However, a drawback of SAMs is its limitation on substrate types. In general, SAMs form on gold, silver, and metal oxide substrates, but not on polymeric surfaces.2 Besides, alkanethiolates with functional groups are usually costly. Therefore, a more versatile and economic method is needed to fabricate micropatterned substrates with tunable surface properties. Recently, a technique based on marine mussels’ adhesive mechanism provides a facile and versatile tool for surface © 2012 American Chemical Society

Received: January 10, 2012 Revised: February 24, 2012 Published: March 7, 2012 5775

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2.3. Preparation of Polydopamine Patterns by μCP. Polydopamine (PDA) was imprinted onto substrates by using μCP as shown in Figure 1. 3-Hydroxytyramine hydrochloride (dopamine hydrochloride, cat. # H8502, Sigma-Aldrich) was dissolved in 10 mM Tris buffer (pH 8.5) to 2 mg/mL. PDMS stamps were immersed in the dopamine solution for 60 min and then were dried by blowing with a stream of N2. The stamps were placed on substrates and pressed with a 100 g weight for 10 min. After the stamps were lifted, the substrates were rinsed with deionized water. 2.4. Surface Characterization. Flat PDA surfaces fabricated by pressing flat PDMS stamps on substrates were employed for surface analysis. Surface chemical compositions were analyzed by electron spectroscopy for chemical analysis (ESCA) under the VG Microtech MT-500 Spectrometer (UK) with an Mg Kα X-ray source.25 Water contact angle (WAC) measurements were recorded using a goniometer (FTA-125, First Ten Ångstroms) with the sessile drop technique (2 μL drop of deionized water). Surface morphology was scanned by using scanning electron microscope (SEM, JEOL JSM-7600F, Japan) or the atomic force microscopy (NanoScope IIIa, Digital Instruments) in tapping mode using a Si cantilever with a spring constant of 45 N/m at scan speed of 0.8 Hz on the area of 50 × 50 μm.4 2.5. Cell Culture. Prior to cell adhesion experiments, samples were sterilized by soaking in 70% ethanol, followed by rinses with sterilized PBS. L929 cells were suspended in the cell culture medium and then seeded on each sample in 24-well plates at a concentration of 2 × 105 cells/cm2. After 12 h, unattached cells were rinsed away with PBS. Cellular patterns were observed under a phase contrast microscope (Nikon TS100, Japan). Cell adhesion on PDA-patterned area was estimated from the microscopic cell images. The number of cells on each strip was counted (5 strips per image). 2.6. Grafting of Molecules on Imprinted PDA Patterns. Grafting PEG to imprinted PDA was performed on tissue culture polystyrene (TCPS, Corning). O-[2-(3-Mercaptopropionylamino)ethyl]O′-methylpoly(ethylene glycol) (PEG-SH, cat. # 11124, 5 kDa) or methyoxypoly(ethylene glycol)amine (PEG-NH2, cat. # 06679, 5 kDa) was dissolved in 10 mM Tris buffer (pH 8.5) to a final concentration of 5 mg/mL. The imprinted PDA patterns were immersed into either PEG-SH or PEG-NH2 solution of for 2 days, followed by rinses with deionized water. The grafting of PEG to PDA patterns was verified by cell culture. Protein immobilization to imprinted PDA patterns was verified on PEG substrates. The patterned PDA substrates were immersed in FITC-conjugated bovine serum albumin (FITC-BSA, cat. # P8779) solution (5 mg/mL in 10 mM Tris buffer, pH 8.5) for 24 h, followed by rinses with PBS and deionized water. Protein patterning was observed under a confocal microscope (Leica, TCS SP2, Germany). The immobilization of gold nanoparticles (AuNPs) and the reduction of silver ions were performed on PDA-imprinted glass slides. AuNPs were prepared by using a seed-growth method.26 Briefly, 10 μL of 0.5 M HAuCl4 and 3 mL of 1% sodium citrate were diluted in 100 mL of deionized water and then heated to boiling for 30 min. The size of AuNPs was found around 50 nm, determined by transmission electron microscopy analysis and a size analyzer. The PDA-imprinted glass slide was immersed into 50 mM AgNO3 solution or AuNPs solution for 20 h, followed by ultrasonic treatment in deionized water for 5 min. The precipitation of Ag nanoparticles on PDA patterns was observed under the scanning electronic microscopy (SEM, JEOL JSM-7600F, Japan). 2.7. Statistical Analysis. The data were reported as means ± standard deviation (SD) or standard error of the mean (SEM). The statistical analyses between different groups were determined using Student’s t-test. The probabilities of p ≤ 0.05 were considered as significant difference. All statistical analyses were performed using GraphPad Instat 3.0 program (GraphPad Software).

micropatterns. Compared with the former two techniques, μCP is much easier, cheaper, and more convenient to most researchers. Recently, Sun et al. showed that PDA was microcontact printed onto PEG substrates to create micropatterns of cells or bacteria.22,23 In this study, we further demonstrated that PDA patterns could be easily imprinted from PDMS stamps to a wide variety of substrates via μCP. Transfer of PDA from PDMS to substrates was verified by water angle measurement, electron spectroscopy for chemical analysis, atomic force microscopy, scanning electron microscopy, and cellular patterning. Furthermore, the main objective of this study was to investigate whether the imprinted PDA has chemical activities. The reactivity of PDA imprinted toward proteins, thiol- or amine-containing PEG, gold nanoparticles, and reduction of silver ions was evaluated.

2. MATERIALS AND METHODS 2.1. Materials. Reagents were received from Sigma-Aldrich (St. Louis, MO) unless specified otherwise. Ascorbic acid, cetyltrimethylammonium bromide (CTAB), and NaBH4 were purchased from Acros. Polydimethylsiloxane (PDMS; Sylgard 184) was obtained from Dow Corning. Polystyrene (PS) was received from Nihon Shiyaku Industries, Ltd., Japan. L929 mouse fibroblast-like cell line was obtained from Food Industry Research and Development Institute (Hsinchu, Taiwan). Cell culture medium contained alpha minimum essential medium (HyClone) with 10% fetal bovine serum (JRH, Australia) and 2 mg/mL NaHCO3 (Sigma) and supplemented with 0.5% of fungizone (Gibco), 0.25% gentamycin (Gibco), and 0.679% β-mercaptoethanol. Phosphate buffered saline (PBS) contained 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH 7.4. 2.2. Fabrication of PDMS Stamps and Preparation of Substrates. PDMS stamps were fabricated by imprinting technology, according to a previously described procedure.24 Briefly, PDMS stamps were prepared by casting a 10:1 (v/v) mixture of PDMS and curing agent on a micropatterned silicon substrate (ridge/groove/depth: 100 μm/125 μm/5 μm) at 60 °C overnight. Flat PDMS stamps were prepared with the same method by using a flat silicon wafer.

Scheme 1. Schematic Illustration of Preparation of Tunable Micropatterned Substrates Based on Poly(dopamine) (PDA) via Microcontact Printing and Secondary Reactions

Glass slides and silicon wafers were cleaned with piranha solution (7/3 (v/v) of 98% H2SO4/30% H2O2) under ultrasonication. Glass slides sputtered with gold were used as gold substrates. Glass slides coated with PEI-g-PEG/dopamine (1/0.25 mg/mL) solution for 2 h were used as cell-resistant PEG substrates, according to a previous study.18 PS substrates were prepared as previously described.24 Briefly, 5% PS in toluene was dripped on a poly(ethylene terephthalate) (PET) film and pressed by flat PDMS molds. After the solvent was evaporated, the PDMS mold was peeled off from PS substrates.

3. RESULTS AND DISCUSSION 3.1. Characterization of Contract Transfer of PDA from PDMS Stamp to PS Substrate. PDMS stamps were immersed in freshly prepared dopamine solution, and PDA was then developed on the stamps directly. The PDA layer was later 5776

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Figure 1. Schematic illustration of microcontact printing of polydopamine (PDA) patterns on polystyrene.

transferred to another substrate via μCP (Figure 1). The transfer of PDA from PDMS to PS was first examined by WCA measurement and ESCA. PDA was first developed on flat PDMS surfaces for 1−60 min prior to imprinting to PS. The deposition of PDA on PDMS substrates was revealed by decease in surface WCA with time (Figure 2A). The WCA was

Table 1. Surface Elemental Compositions of Polystyrene (PS) Substrates Imprinted with Polydopamine elemental compositions (atomic %) substrate PS 5 min 30 min 60 min

a

C

O

N

100.0 95.9 80.3 80.0

0.0 3.4 14.1 13.9

0.0 0.7 5.6 6.1

a

The time indicates the dopamine incubation of PDMS prior to contact with PS.

the pristine PS to 3.4, 14.1, and 13.9% for the substrates that were in contact with the PDMS treated with dopamine solution for 5, 30, and 60 min, respectively. Similarly, the surface nitrogen contents were increased from 0% for the pristine PS to 0.7, 5.6, and 6.1% for the substrates that were in contact with the PDMS treated with dopamine for 5, 30, and 60 min, respectively. The ESCA results showed that PDA was imprinted onto PS. The above WAC and ESCA data suggest that the transfer of PDA from PDMS is increased with dopamine incubation time and reaches a threshold after 30 min of dopamine incubation. Next, the transfer of PDA micropatterns was identified by several imaging tools. PDMS stamps incubated in dopamine solution for 60 min were in contact with gold-deposited glass slides or silicon wafers. The deposited Au layer was thin enough for visualization of PDA patterns under a light microscope (Figure 3A). The PDA pattern on a silicon wafer was clearly presented on SEM images (Figure 3B). Besides, there are obvious differences in surface topography between the PDA area and the spacing on SEM images and AFM images. The SEM images show that the spacing area was smooth (Figure 3C), compared with the rough PDA area containing many nanoscale aggregates (Figure 3D). Such morphology is similar to the observation in previous studies.17 AFM images also indicated that surface nanomorphology was different between the silicon areas with or without contacting with PDA stamps (Figure 3E). The root-mean-square surface roughness of the noncontacting area (silicon substrate) was 1.40 ± 1.84 nm, while that of the contacting area (PDA deposition) was 6.96 ± 4.26 nm, according to AFM analysis. The key to μCP is that low interfacial free energy and good chemical stability of PDMS leads to that most molecules do not adhere tightly to or react with PDMS.2 Although the mechanism for PDA adherence onto substrates is still not well understood, previous studies proposed several mechanisms on a variety of substrates. Rodriguez et al. suggested that the reaction between catechols and a TiO2 surface results in dehydration and the formation of a charge-transfer complex.27 Anderson et al. suggested that 3,4-dihydroxyphenyl-L-alanine (DOPA) forms hydrogen bonding with mica.28 On the other hand, the interaction between of DOPA and hydrophobic surfaces was supposed via a strong interaction.29 Nevertheless, the stickiness between PDA and PDMS is not sufficient to prevent the transfer

Figure 2. Water contact angles (WCA) on flat (A) PDMS and (B) polystyrene (PS). Dopamine incubation time represents the duration of PDMS or PS in dopamine solution. (A) “Before and after printing” represent the PDMS substrates before and after contact with PS. (B) “Printing” means the PS substrates contacted with polydopamine-coated PDMS, while “coating” means the PS substrates incubated in dopamine solution. Value = mean ± standard deviation, n = 6. *, p < 0.05 vs PDMS; **, p < 0.01, ***, p < 0.001 vs PS.

decreased from 102.34° for the pristine PDMS to 60.29° after 60 min of dopamine incubation. The PDA-coated PDMS surfaces were then in contact with flat PS substrates for 10 min under a 100 g weight. The WAC of PDMS stamps after contacting PS was increased to a level comparable to the pristine PDMS (Figure 2A, no significant difference vs all samples except 60 min). On the other hand, the WAC of the pristine PS was decreased from 90.06° to 80.46° for the PS which made contact with the PDA-deposited PDMS for 60 min of dopamine incubation (Figure 2B), indicating the transfer of PDA from PDMS to PS. Nevertheless, the decrease in hydrophobicity by contact printing was less compared with the PS that was directly incubated in dopamine solution with the same coating time (Figure 2B). The transfer of PDA to PS was further confirmed by ESCA (Table 1). Since dopamine is enriched in oxygen and nitrogen, the appearance of PDA should be revealed by an increase in surface contents of these two elements. The results indicated that the surface oxygen contents were increased from 0% for 5777

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Figure 3. (A) An optical microscopic image of an imprinted polydopamin (PDA) pattern on a gold substrate (magnification 100×). (B−D) SEM images of imprinted PDA patterns on silicon substrates (magnification: B, 170×; C and D, 100000×). (E) An AFM image of an imprinted PDA on a polystyrene substrate (20 μm × 20 μm).

Figure 4. (A) Adhesion of L929 cells on the polystyrene substrate imprinted by PDMS stamps that were incubation in dopamine solution for 1, 5, 10, 30, and 60 min. The phase contrast microscopic images of L929 cellular patterns were taken after 12 h of incubation (magnification 100×). Scale bar = 100 μm. (B) Cell density on the PDA imprinted area. Value = mean ± standard deviation, n = 15.

3.2. Cell Patterning by PDA Imprints. PDA coating has been previously shown to facilitate cell adhesion to substrates.13,14 Recently, Jiang et al. showed that patterned PDA transfer to PEG substrates via μCP created spatially defined anchoring of mammalian cells.22 In this study cell patterning was used to demonstrate the transfer of PDA patterns to PS. PS substrates were imprinted by the PDMS stamps that were coated with PDA for various dopamine-incubation time. Since PS does not support cell adhesion well, L929 cells only appear on the PDA area but not the spacing area. When PDA was transferred from PDMS with dopamine incubation for 1 or 5 min,

of PDA to another substrate. It was reported that hydrophilic surfaces that have high surface free energies are liable to get contamination with less hydrophilic materials to reduce their surface free energies.30 Among the substrates used in this study, the surface free energy of PDMS (γ = 21.6 mN/m)2 was lowest compared with the other substrates: PS (γ = 40.7 mN/m),31 gold (γ = 72.5 mN/m),30 glass (γ = 72.4 mN/m),30 and silicon (γ = 72.5 mN/m).30 The difference in surface free energies may explains that a PDA layer deposited on PDMS is prone to be transferred from PDMS to other substrates such as PS, gold, and silicon via direct contact. 5778

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Figure 5. (A) Schematic illustration of grafting of PEG containing amines and thiols on PDA-imprinted TCPS to create cell patterns. (B) Water contact angle of amine- or thiol-PEG modified substrates. Value = mean ± SD, n = 6. **, p < 0.01, ***, p < 0.001 vs PDA. ☆, p < 0.05. Scale bar = 100 μm. The phase-contrast microscopic images of cells after 12 h of culture on (C) PDA pattern alone, (D) PDA pattern grafted with PEG-NH2, and (E) PDA pattern grafted with PEG-SH (magnification 100×).

PEG (Figure 5A). The conjugation of PEG was first evaluated by WCA measurement. PDA was imprinted to TCPS from flat PDMS with 60 min dopamine incubation. The PDA-stamped TCPS was incubated with PEG-NH2 or PEG-SH for 48 h, the WAC of PDA-coated TCPS was decreased from 77.77° to 61.36° and 56.14°, respectively (p < 0.001, Figure 5B), suggesting that PEG was grafted onto the PDA coated TCPS via interaction with amines or thiols. Cell resistance of PEG-grafted substrates was next evaluated. PDA was transferred from patterned PDMS stamps to TCPS, followed by incubation with PEG-NH2 or PEG-SH. Prior to PEG immobilization, L929 cells covered the whole surface since both TCPS substrate and PDA coating support cell adhesion (Figure 5C). When PEG was grafted using PEG-NH2 or PEG-SH, cell attachment was restrained in the TCPS area but not in the PDA-imprinted area (Figure 5D,E). Our results indicated that PDA retains its reactivity toward amines or thiols after transfer via μCP. 3.4. Protein Immobilization on PDA Imprints. Several previous studies demonstrated that PDA facilitates immobilization of proteins such as trypsin,15 antibody,17 and BSA.16 A protein usually contains amino acid residues with amino or thiol side chains such as lysine or cysteine. Proteins could be conjugated onto PDA via interactions with their exterior amino acid residues containing amino side chains. For example, albumin is enriched in lysine.32 Here, PDA-assisted protein immobilization was tested. PEGylated surfaces were first created by incubation of glass substrates with dopamine/PEI-g-PEG.

few cells adhered on the substrates. Thus, no obvious cell pattern was found on these substrates (Figure 4A). As the dopamineincubation time of PDMS increased to 10 min, cell adhesion was greatly enhanced and cellular patterns could be vaguely identified on the PDA-imprinted PS. After the dopamine-incubation time increased to more than 30 min, cellular patterns were clearly defined. In addition, cell density on patterning area was analyzed. We found that the cell densities were increased from 0.03 × 105% to 3.39 × 105 cells/μm2 for using the PDMS stamps that were treated with dopamine solution for 1 to 60 min, respectively (Figure 4B). The cell adhesion results clearly indicated that PDA patterns were formed on PS via μCP. We found that the PDA pattern created after 30 min dopamine incubation of PDMS is sufficient to mediate cell adhesion. As shown in the WAC measurement and ESCA, the amount of transferred PDA depends on dopamine-incubation time of PDMS. Although overnight incubation of PDMS was used in Jiang’s report for μCP and cell patterning,22 we suggest that 30−60 min of incubation should be sufficient for such a purpose. Therefore, in the rest of this study, 60 min dopamine incubation was applied. 3.3. Conjugation of PEG Containing Amines or Thiols on PDA Imprints. The deposited PDA coating is chemically heterogeneous, and its chemical composition is not precisely known. Nevertheless, catechol/quinone groups are believed present in PDA coating and are capable of covalent coupling to nucleophiles (e.g., amines and thiols).10 We wondered whether the reactivity of PDA remains after transfer to another substrates, which was first tested by thiol- or amine-terminated 5779

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Figure 7A. The appearance of PDA-imprinted glass slides was transparent (Figure 7B). After PDA-imprinted glass slides were incubated with AuNPs solution, purple AuNPs lines were visualized by naked eye (Figure 7C). The microscopic image shows clear margins between the AuNPs area and the spacing area (Figure 7D). 3.6. Reduction of Metallic Ions. One of the notable properties of PDA is its ability to reduce metallic ions to metals such as copper and silver, which is used as an electroless process of metallization for fabrication of electric devices or antibacterial surfaces.10,20,35−37 The possible mechanism for electroless metal deposition on PDA has been suggested that the metallic ions approach the protonated hydroxyl groups in catechol groups of PDA via electrostatic interaction, and then the catechol groups are oxidized to quinone, accompanied by reduction of metallic ions to elemental metal.36,38 It was previously found that the reduced elemental metals were bond to the PDA film as seed precursors for the growth of metal nanopaticles through the atom-by-atom growth with the reduction of metallic ions.36,38 The reductive ability of PDA-imprinted glass slides was evaluated by immersion in AgNO3 solution (Figure 8A). The appearance of glass slides was altered from transparent to yellowish-brown lined structure with metallic luster (Figure 8B). The silver pattern was also clearly demonstrated by SEM imaging (Figure 8C). High magnitude SEM micrographs showed that the PDA-imprinted area was covered with silver nanoparticles (AgNPs, 33.04 ± 17.19 nm in diameter), in contrast to smooth spacing area of glass substrates (Figure 8C). Messersmith’s group demonstrated that PDA deposition directly onto a substrate not only forms a uniform adlayer but also provides a reactive surface for subsequent conjugation.10 PDA is able to react with nucleophiles and reduce metal ions such as silver and copper. PDA can also bind biomolecules such as proteins and cells as well as metal nanoparticles. In this study, we showed that PDA formed on PDMS could be easily transferred to other substrates. The transferred PDA still remains its chemical reactivity. In contrast to long-time incubation of substrates in dopamine solution for 18−24 h prior to formation of PDA patterns by using photolithography,10 microfluidic technique,21 and μCP,22 this study showed that PDA developed for merely 30−60 min was sufficient to transfer to another substrate with the desired properties. Microcontact printing of

We previously showed that codeposition of dopamine/ PEI-g-PEG is a facile method for preparation of low-fouling surfaces on a wide variety of substrates.18 FITC-BSA was incubation with imprinted PDA patterns on PEGylated glass slides (Figure 6A). Prior to incubation with FITC-BSA, no

Figure 6. (A) Schematic illustration of FITC-BSA immobilization on PDA-imprinted PEG substrates to create protein patterns. The fluorescent microscopic images were taken (B) before and (C) after immobilization of FITC-BSA (magnification 200×). Scale bar = 100 μm.

fluorescent image was shown on the imprinted PDA substrate (Figure 6B). After incubation with FITC-BSA, green fluorescence appeared in the PDA regions (Figure 6C). The results indicate that imprinted PDA via μCP can be used to immobilize proteins for creation of protein patterns. 3.5. Immobilization of Metal Nanoparticles. DOPA is previously found to form stable covalent bonds with metals via the two hydroxyl groups of DOPA molecules.33 Recently, Lee et al. reported that catechol-contained polymers could bind gold nanoparticles (AuNPs) to form a nanoparticle assembly with precise control.34 In this study, PDA-assisted immobilization of AuNPs was investigated, schematic illustrated in

Figure 7. (A) Schematic illustration of gold nanoparticles (AuNPs) immobilization on PDA-imprinted glass slides to create AuNPs patterns. The images of (B) a PDA-imprinted glass and (C) a PDA-imprinted glass coated with AuNPs were taken directly from a digital camera. (D) An optical microscopic image of a PDA-imprinted glass coated with AuNPs (magnification 100×). 5780

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Figure 8. (A) Schematic illustration of reduction of silver ions on PDA-imprinted glass slides for electroless metallization. (B) A image of silver patterns was taken from a digital camera. (C) A SEM image of a PDA-imprinted glass coated with AgNPs (magnification 100×). The upper inserted SEM photo displays an enlarged image of the spacing glass area, and the lower inserted SEM photo displays an enlarged image of the AgNPs area (magnification 100000×). (8) Kao, Y. C.; Hong, F. C. Nanotechnology 2011, 22, 185303. (9) Berg, M. C.; Choi, J.; Hammond, P. T.; Rubner, M. F. Langmuir 2003, 19, 2231. (10) Lee, H.; Dellatore, S. M.; Miller, W. M.; Messersmith, P. B. Science 2007, 318, 426. (11) Lane, G. A.; Allinger, N. L. J. Am. Chem. Soc. 1974, 96, 5825. (12) Waite, J. H.; Qin, X. Biochemistry 2001, 40, 2887. (13) Tsai, W. B.; Chen, W. T.; Chien, H. W.; Kuo, W. H.; Wang, M. J. Acta Biomater. 2011, 7, 4187. (14) Ku, S. H.; Ryu, J.; Hong, S. K.; Lee, H.; Park, C. B. Biomaterials 2010, 31, 2535. (15) Lee, H.; Rho, J.; Messersmith, P. B. Adv. Mater. 2009, 21, 431. (16) Wei, Q.; Li, B.; Yi, N.; Su, B.; Yin, Z.; Zhang, F.; Li, J.; Zhao, C. J. Biomed. Mater. Res., Part A 2011, 96, 38. (17) Wan, Y.; Zhang, D.; Wang, Y.; Qi, P.; Hou, B. Biosens. Bioelectron. 2011, 26, 2595. (18) Tsai, W. B.; Chien, C. Y.; Thissen, H.; Lai, J. Y. Acta Biomater. 2011, 7, 2518. (19) Kang, K.; Choi, I. S.; Nam, Y. Biomaterials 2011, 32, 6374. (20) Long, Y.; Wu, J.; Wang, H.; Zhang, X.; Zhao, N.; Xu, J. J. Mater. Chem. 2011, 21, 4875. (21) Ku, S. H.; Lee, J. S.; Park, C. B. Langmuir 2010, 26, 15104. (22) Sun, K.; Xie, Y.; Ye, D.; Zhao, Y.; Cui, Y.; Long, F.; Zhang, W.; Jiang, X. Langmuir 2012, 28, 2131−6. (23) Sun, K.; Song, L.; Xie, Y.; Liu, D.; Wang, D.; Wang, Z.; Ma, W.; Zhu, J.; Jiang, X. Langmuir 2011, 27, 5709. (24) Tsai, W. B.; Lin, J. H. Acta Biomater. 2009, 5, 1442. (25) Kuo, W. H.; Wang, M. J.; Chien, H. W.; Wei, T. C.; Lee, C.; Tsai, W. B. Biomacromolecules 2011, 12, 4348. (26) Jana, N. R.; Gearheart, L.; Murphy, C. J. Chem. Mater. 2001, 13, 2313. (27) Rodriguez, R.; Blesa, M. A.; Regazzoni, A. E. J. Colloid Interface Sci. 1996, 177, 122. (28) Anderson, T. H.; Yu, J.; Estrada, A.; Hammer, M. U.; Waite, J. H.; Israelachvili, J. N. Adv. Funct. Mater. 2010, 20, 4196. (29) Kang, S. M.; You, I.; Cho, W. K.; Shon, H. K.; Lee, T. G.; Choi, I. S.; Karp, J. M.; Lee, H. Angew. Chem. Int. Ed. Engl. 2010, 49, 9401− 9404. (30) Yang, L.; Shirahata, N.; Saini, G.; Zhang, F.; Pei, L.; Asplund, M. C.; Kurth, D. G.; Ariga, K.; Sautter, K.; Nakanishi, T.; Smentkowski, V.; Linford, M. R. Langmuir 2009, 25, 5674. (31) Li, I. T.; Walker, G. C. J. Am. Chem. Soc. 2010, 132, 6530. (32) Chen, S.; Cao, Z.; Jiang, S. Biomaterials 2009, 30, 5892. (33) Weinhold, M.; Soubatch, S.; Temirov, R.; Rohlfing, M.; Jastorff, B.; Tautz, F. S.; Doose, C. J. Phys. Chem. B 2006, 110, 23756.

PDA provides a powerful tool to develop a functional patterned substrate, which could form a wide variety of chemical patterned substrates from organic to inorganic and from biomolecules to cells.

4. CONCLUSIONS This work presented a facile and economic technique to fabricate tunable micropatterned substrates based on PDA chemistry via μCP. PDA imprints were proved to be a powerful platform for further functionalization, such as cell patterning, protein immobilization, conjugation of thiol- or amine-containing molecules, immobilization of gold nanoparticles, and reduction of silver ions into silver nanoparticles. The ordered patterns of proteins, cells, molecules, and metal nanoparticles can be used in biomedical or optical/electric applications.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 886-2-3366-3996; Fax: 886-2-2362-3040; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported from National Taiwan University (Grant 99R70412). The authors acknowledge Dr. Chia-Fu Chou at the Institute of Physics, Academia Sinica, for providing micropatterned silicon wafers.

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REFERENCES

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