Dual Silane Surface Functionalization for the Selective Attachment

(1-8) The unique physical and chemical properties of pSi including a high surface area, ... dyes or change the media, affording a nonintrusive study o...
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Dual Silane Surface Functionalization for the Selective Attachment of Human Neuronal Cells to Porous Silicon Martin J. Sweetman, Cameron J. Shearer, Joseph G. Shapter, and Nicolas H. Voelcker* School of Chemical and Physical Sciences, Flinders University of South Australia, Adelaide SA 5042, Australia ABSTRACT: Porous silicon (pSi) surfaces were chemically micropatterned through a combination of photolithography and surface silanization reactions. This patterning technique produces discretely defined regions on a pSi surface functionalized with a specific chemical functionality, and the surrounding surface displays a completely different functionality. The generated chemical patterns were characterized by a combination of IR microscopy and the conjugation of two different fluorescent organic dyes. Finally, the chemically patterned pSi surface was used to direct the attachment of neuronal cells to the surface. This patterning strategy will be useful for the development of high-throughput platforms for investigating cell behavior.

’ INTRODUCTION Porous silicon (pSi) is an interesting nanostructured material that has found applications in biosensing, drug delivery, and as a support material for cell cultures.1 8 The unique physical and chemical properties of pSi including a high surface area, tunable pore size, biodegradability, and photonic and photoluminescence effects make this material ideally suited to a diverse range of biological applications.9 13 Porous silicon has also been used for a wide range of nonbiological applications, such as optoelectronic devices, chemical sensors, and molecular separation systems, demonstrating this material’s versatility.14 16 It is now conceivable to develop pSi-based devices for applications where electronic, chemical, and biological systems interface to perform highly specialized tasks. The physical properties that make pSi so well suited to biological applications include the highly tunable pore size, depth, orientation, and morphology as well as a remarkably high internal surface area on the order of 400 800 m2/g.7,17 Control over pore size and depth is of great importance when designing specialized biosensors or drug delivery systems, where each specific application generally requires different pore morphologies. A high internal surface area is a desirable trait in pSi systems, allowing a high internal loading capacity of bioactive or organic materials. For this reason, pSi has been extensively studied as a material for drug delivery because high levels of a therapeutic drug can be incorporated within the porous layer.18,19 There has also been a recent report of stimulus-responsive drug release from pSi films, which allows greater control over drug release and administration.20 Perhaps the most useful property of pSi is the biocompatibility of the material. pSi is completely biodegradable in aqueous media, with the only product of degradation the nontoxic silicic acid.11 The biocompatible and noninflammatory nature of pSi has allowed the material to be used in a range of cell and tissue culture applications, both in vitro and in vivo.6,7,12,21,22 r 2011 American Chemical Society

Porous silicon can also be easily functionalized with a diverse range of chemical species to suit any application. There are two main methods for incorporating functional chemical species into pSi surfaces: hydrosilylation and silanization reactions. Hydrosilylation reactions focus on the attachment of alkene or alkyne species to the Si H-terminated surface of freshly etched pSi.23 These reactions are commonly performed using heat or light to promote the formation of a stable Si C bond on the pSi surface.24,25 Hydrosilylation reactions have been used to incorporate a range of functional groups on pSi surfaces, including carboxylic acids, thiols, and esters.24,26,27 Although this type of functionalization often provides stable pSi surfaces, great care is required to eliminate water and oxygen from the reaction in order to prevent surface oxidation and avoid uneven or low coverage of the hydrosilylated species.28,29 Silanization reactions involve the attachment of alkoxy- or chlorosilane compounds to a hydroxylated (Si OH-terminated) pSi surface.30 A range of functional groups such as amines, isocyanate, and poly(ethylene glycol) (PEG) can be easily displayed on pSi surfaces using this functionalization method.30 33 Silanization reactions are usually performed using milder reaction conditions than hydrosilylation reactions, with the resulting surface functionality providing sufficient stability for applications requiring exposure of pSi to neutral aqueous environments for less than 1 week.34 Selecting the appropriate chemical and physical properties of pSi surfaces is of importance when using this material for cell culture applications. The physical effect of pore size on mammalian cell attachment has been previously demonstrated by Khung et al., where neuronal cells were found to attach preferentially in Received: May 11, 2011 Revised: June 14, 2011 Published: June 16, 2011 9497

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Langmuir pore size regimes of 5 20 nm and 1 3 μm.6 Pore morphology effects have also been investigated by Sun et al., who showed that the pSi morphology has an effect on osteoblast adhesion, metabolic activity, protein synthesis, and mineralization.35 The effect of surface chemistry on mammalian cell attachment to pSi has been investigated by Low et al. Cell attachment to ozone-oxidized, aminosilane-functionalized, and collagen-coated pSi surfaces was compared.7 This investigation revealed that aminosilane and a collagen coating enhanced cell attachment to pSi surfaces. The biocompatibility of pSi surfaces modified by hydrosilylation reactions has also been demonstrated by Alvarez et al., who showed an enhanced cell adhesion of primary rat hepatocytes to undecylenic acid functionalized or collagen-coated, hydrophilic pSi surfaces compared to that of dodecene-functionalized, hydrophobic pSi surfaces.2 Following on from the initial cell surface investigations, pSi has recently been used for the real-time monitoring of different cellular functions such as the excretion of enzymes or cell death in a format termed the “smart Petri dish”.36,37 A considerable advantage of this format is that cell morphological changes can be visualized by conventional light microscopy without the need to add specific dyes or change the media, affording a nonintrusive study of cell behavior. The pSi smart Petri dish studies so far have used homogeneously functionalized pSi surfaces, where cell interaction and function is the same across the entire surface. For the design of higher-throughput variants of the smart Petri dish, it would be highly advantageous to be able to produce patterns of different chemical functionality and hence cell attachment on pSi surfaces. Patterning of chemical species on pSi surfaces has been demonstrated by Stewart et al., where freshly etched pSi was patterned using white-light-initiated hydrosilylation through a photomask.38 However, most recent applications involving patterned pSi focus on generating patterns of etched silicon through various photolithography techniques.39 41 Although these techniques are useful in producing discretely patterned areas of pSi surrounded by flat silicon, there are clear advantages of having patterned chemical functionality on a homogenously etched pSi surface, particularly in the context of smart Petri dish applications. pSi surfaces patterned with bioactive species such as antibodies or DNA could also be used for various microarray applications, where sorting and attachment of specific cells from a mixed cell population are required.42 The ability to pattern the attachment of biological elements on pSi surfaces would be highly advantageous for the further development of pSi-based biosensor devices. In the present work, we demonstrate the ability to produce discretely patterned areas of specific chemical functionality within a pSi surface using a combination of photolithography and silanization. This patterning technique allows two different chemical functionalities, in this case, two different silane compounds, to be conjugated to a single pSi surface. We have demonstrated the functionality of the patterned chemistry by revealing the pattern using fluorescence microscopy after the attachment of two fluorescent dyes. The incorporation of dual chemical functionality also allowed us to generate surfaces that have areas suitable for cell attachment surrounded by a noncell adhesive matrix. Taking the lead from previous investigations into promoting cell adhesion on pSi, patterned areas of aminosilane were generated, with the surrounding pSi surface functionalized with a PEGsilane in order to prevent cell adhesion in these areas.7,33 Indeed, attachment of the human neuronal cell line SK-N-SH on such patterned surfaces was confined to the amine-functionalized patterned areas and was prevented in the PEG-functionalized regions.

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’ EXPERIMENTAL PROCEDURE Fluorescence Microscopy. Fluorescence microscopy was performed on an Eclipse 50i microscope equipped with a D-FL universal epifluorescence attachment and a 100 W mercury lamp (Nikon Instruments, Japan). Fluorescence images were captured with a CCD camera (Nikon Instruments, Japan), and images were analyzed using NISelements v 3.07 software (Nikon Instruments, Japan). IR Spectroscopy. Conventional IR spectroscopy was performed on a Nicolet Avatar 370MCT spectrometer (Thermo Electron Corporation, USA). IR spectra were collected using a transmission accessory, and all spectra were recorded and analyzed using OMNIC version 7 software (Thermo Electron Corporation, USA). Spectra were recorded over a range of 650 4000 cm 1 at a resolution of 2 cm 1 and taken as an average of 64 scans. All samples were corrected to a clean, unetched, nonfunctionalized silicon wafer of the same dopant type and resistivity as the silicon samples used for pSi fabrication. IR microscopy was performed on a Nicolet iN10 infrared microscope (Thermo Electron Corporation, USA). IR microscopy data was collected using transmission mode. IR spectra were captured using an aperture size of 30 μm2 and were recorded over a range of 1200 4000 cm 1 at a resolution of 8 cm 1 and taken as an average of 16 scans. An analysis of the IR microscopy data was performed using OMNIC picta software (Thermo Electron Corporation, USA). Porous Silicon Formation. Low resistivity, boron-doped p-type silicon wafers (0.0008 0.0012 Ω 3 cm, Siltronix, France) were cleaned in methanol (Chem Supply, Australia), acetone (Ajax Finechem, USA), and dichloromethane (Biolab, Australia) before being electrochemically etched using a 2425 source meter (Keithley, USA) as the current source. The following etching parameters were used: etching solution 3:1 HF (48% aq) (Merck, Germany)/EtOH (Merck, Germany), current density 20 mA/cm2, time 300 s. Etched wafers were cleaned in methanol, acetone, and dichloromethane before being dried under a gentle stream of nitrogen gas. Procedure for Silanization and Patterning. Freshly etched pSi surfaces were first oxidized for two hours in a flow of ozone (3.25 g/h). Following oxidation, the first silanization reaction was performed using the silane with functionality appropriate for the areas surrounding the patterned regions (silane 1). Oxidized pSi surfaces were reacted in a solution of 50 mM silane 1 in 5 mL of anhydrous toluene. The reaction was allowed to proceed for 10 min at room temperature with occasional agitation, after which time the pSi surface was removed, washed with chloroform, acetone, and water, and dried under nitrogen. Following the first silanization, the patterning procedure schematically depicted in Figure 1 was performed as follows. Positive tone photoresist AZ1518 (Microchemicals, Germany) was spin coated at 3000 rpm for 30 s onto the pSi surface at a thickness of ∼2 μm and soft baked at 100 °C for 1 min (Figure 1a). The photoresist was patterned by exposure to an Omnicure S1000 100 W ultraviolet lamp (EXFO Life Sciences and Industrial Division, Canada) for 10 s through a chrome-on-glass photomask (Figure 1b). Following UV exposure, the photoresist was immediately developed in AZMIF326 developer (Microchemicals, Germany) for approximately 10 s and then washed with Milli-Q water and dried under a stream of nitrogen gas. The patterned photoresist was then hard baked at 115 °C for 2 min (Figure 1c). Following patterning and hard baking of the photoresist, the silane in the exposed regions was removed by rinsing the pSi surface in 1:1 HF(48% aq)/EtOH. HF-treated surfaces were washed with water to remove any residual HF, and the resulting Si H surface of the patterned regions was reoxidized by exposure to a flow of ozone (3.25 g/h) for 2 h (Figure 1d). Reoxidized pSi surfaces were then subjected to the second silanization reaction, with silane 2, following the same general procedure outlined previously, except this time using the silane with the desired chemical 9498

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Figure 1. Surface-patterning procedure illustrating the patterned conjugation of dual silane species. Silanes 1 and 2 correspond to the silanes used for the first and second silanization reactions, respectively. For illustration purposes, silane 1 has been shown as N-(triethoxysilylpropyl)-poly(ethylene oxide) urethane (PEG-silane), and silane 2 has been shown as 3-aminopropyltriethoxysilane (APTES) (The schematic is for illustration purposes only and is not to scale.) functionality for the patterned regions (Figure 1e). Following this silanization, the photoresist was removed by washing the surface with copious amounts of acetone and drying under a stream of nitrogen gas (Figure 1f). Functionalization of Patterned Surfaces. Single Fluorescent Dye Labeling (Pattern A). The silanes used for patterning were silane 1 (i.e., 3-aminopropyltriethoxysilane, Sigma-Aldrich, USA) and silane 2 (i.e., 3-mercaptopropyltrimethoxysilane, Sigma-Aldrich, USA). Fluorescein-5-maleimide (Invitrogen, USA) dye (100 μg/mL) in phosphatebuffered saline (PBS) at pH 7.4 was reacted with the patterned surface for 1 h at room temperature with agitation. Following the reaction, the surface was washed with PBS and Milli-Q water and dried under a stream of nitrogen gas. Dual Fluorescent Dye Labeling (Pattern B). The silanes used for patterning were silane 1 (i.e., 3-aminopropyltriethoxysilane) and silane 2 (i.e., 3-isocyanatopropyltriethoxysilane; Fluka, USA). Carboxy-5-fluorescein (Sigma-Aldrich, USA) dye (100 μg/mL) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 20 mM, Sigma-Aldrich, USA) in PBS (pH 7.4) were reacted with an APTES-functionalized pSi surface for 1 h. The surface was washed with PBS and Milli-Q water before being subjected to photolithographic patterning as described previously. The patterned regions of the surfaces were functionalized with an isocyanatosilane. Dye lissamine (rhodamine B ethylenediamine, 100 μg/mL, Invitrogen, USA) in PBS (pH 7.4) was reacted with the patterned surface for 1 h at room temperature with agitation. Following the reaction, the surface was washed with dimethylformamide (EMD Chemicals, Belgium), PBS, and Milli-Q water and dried under a stream of nitrogen gas. Patterned Surfaces for Cell Culture (Pattern C). The silanes used for patterning were silane 1 (N-(triethoxysilylpropyl)-o-polyethylene oxide urethane (PEG-silane, Fluorochem, U.K.) and silane 2 (i.e., 3-aminopropyltriethoxysilane). SK-N-SH cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, JRH Bioscience, USA) containing 5 mM L-glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin sulfate (all from Invitrogen, USA), and 10% v/v fetal bovine serum

(FBS) (Bovogen Biologicals, Australia) and maintained at 37 °C in 5% CO2. To investigate cell attachment to the biologically functionalized surfaces, cells were incubated in contact with the surfaces at a density of 6.58  105 cells/cm2 in DMEM containing 5 mM L-glutamine, 100 IU/mL penicillin, 100 μg/mL streptomycin sulfate, and 10% v/v FBS for a period of 5 h. During the final 30 min of the incubation period, Hoechst 33342 (Molecular Probes, USA) was added to the culture solution for each surface to a final concentration of 2 μg/mL. Following Hoechst staining, each surface was rinsed with Dulbecco’s phosphate-buffered saline (dPBS, pH 7.4, 100 mg/L CaCl2, 100 mg/L MgCl2 3 6H2O) to remove any nonspecifically or weakly attached cells. Cells were then fixed with 4% (w/v) paraformaldehyde (Electron Microscopy Sciences, Australia) in PBS for 10 min, washed with PBS, and permeablized with 0.1% Triton-X100 (Sigma-Aldrich, USA) in PBS for 5 min. Finally, cells were stained with 0.38 μM Phalloidin (tetramethylrhodamine B isothiocyanate conjugated, Sigma-Aldrich, USA) solution in PBS for 30 min in the dark and then washed with PBS. Each surface was mounted on a glass slide, covered with gel mount media, and protected with a coverslip.

’ RESULTS Surface Characterization. Infrared spectroscopy and microscopy were used to characterize the chemically patterned pSi surfaces. Figure 2 displays a series of conventional IR spectra and two IR microscopy graphs for a patterned surface (pattern C), along with a schematic displaying the corresponding surface chemistry. In the case of Figure 2a, the ozone-oxidized pSi surface displays characteristic silicon oxide vibrational modes at 1100 and 3700 cm 1 corresponding to stretching vibrations of the silicon oxide (Si O) and silanol (SiO H) functional groups, respectively.43 47 Once the oxidized pSi surface has been reacted with the PEG-silane, CH2 methylene vibrational peaks appear at 9499

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Figure 3. Fluorescent images of fluorescein-5-maleimide conjugated to 3-mercaptopropyltrimethoxysilane (mercaptosilane) patterns on a pSi surface, with APTES silane functionality in the surrounding areas (a and b). A schematic of the corresponding surface chemistry, with the dye attached, is also displayed in part c.

Figure 2. IR characterization of patterned pSi surfaces (pattern C). (a) IR spectra displaying ozone-oxidized, PEG-silane-functionalized, APTES-functionalized, and APTES/PEG silane-patterned pSi surfaces. (b) IR correlation intensity map of IR spectra matching that of APTES for a patterned area (c) displaying a similar correlation map for IR spectra matching that of the PEG-silane, over the same area. (d) Schematic of the corresponding surface chemistry for the patterned pSi surface area from which the IR maps were obtained.

approximately 2900 cm 1, as well as a carbonyl stretching vibrational peak at 1700 cm 1 and amide II vibrational peak at 1550 cm 1 both associated with the urethane bond within the silane.25,33,48 There is also a broad vibrational peak at around 3300 cm 1 associated with the terminal OH group of the PEG silane. The IR spectrum in Figure 2a corresponding to an APTES-functionalized pSi surface again displays the methylene vibrational peaks at 2900 cm 1. There are also amine vibrational peaks at 3330, 3280, and 1590 cm 1.49 The final IR spectrum displayed in Figure 2a corresponds to a pSi surface that has been patterned with PEG-silane and APTES (pattern C) to generate the dual silane functionalization. Vibrational peaks for the PEGsilane functional groups are all present in this spectrum, along with an amine vibrational peak at 1590 cm 1 corresponding to the terminal amine functional group of the APTES.50 The detection of peaks corresponding to both silanes confirms the successful incorporation of each silane into the pSi surface. This spectrum also indicates that the chemical functional groups associated with each silane are preserved during the patterning procedure.

Three-dimensional IR intensity maps of a 400  400 μm2 area of pattern C encompassing four squares are displayed in Figure 2b,c. (See Figure 2d for a schematic of the pattern.) The map in Figure 2b shows the correlation intensity of all IR spectra collected within the patterned area with respect to that of a typical IR spectrum for APTES. It can be seen that the highest correlation intensity to the APTES spectrum occurs within the four squares, where APTES is expected to be located (silane 2, as indicated by the schematic in Figure 2d). IR spectra from the areas surrounding the squares show virtually no correlation to APTES, indicating confinement of the APTES functionality within the patterned areas. It should also be noted that the APTES functionality within the patterned areas appears to be homogeneous, with a similar correlation intensity across each of the squares. Figure 2c displays an IR map of the same patterned area. However, in this case the correlation intensity with respect to that of an IR spectrum for the PEG silane has been graphed. As expected, the maximum correlation intensity corresponds to the areas outside of the square patterns, where the PEG-silane is expected to be located. Again, the coverage of this silane is homogeneous and is very well contained to the areas outside of the pattern. By comparing Figure 2b and Figure 2c, it can be seen that there is virtually no crossover of either silane. In other words, there is no PEG silane in the APTES region, and there is no APTES in the PEG region. This means that well-defined, discretely patterned regions of dual chemical functionality have indeed been generated on the pSi surface using this patterning approach. Figure 2 shows the characterization of four square patterned features of 100  100 μm2 size. Feature sizes with these dimensions were selected for characterization by IR microscopy because of the resolution limit of the instrument of approximately 10 μm. By mapping larger features and using an aperture of 30  30 μm2, high-quality, well-resolved patterned features could be mapped. This surface-patterning technique, however, is capable of producing much smaller features, with the fluorescent images in Figure 3 displaying patterned features of approximately 25 μm2. 9500

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Figure 5. Patterned attachment of SK-N-SH cells on APTES/PEGsilane-patterned pSi surfaces. The patterned areas/shapes are highlighted to aid the visualization of the patterned cell attachment. Cells are stained with Hoechst 33342 and tetramethylrhodamine B-labeled phalloidin.

Figure 4. Dual silane-functionalized pSi surfaces, with different fluorescent dyes conjugated to each silane compound. (a) Patterned conjugation of lissamine to an isocyanate silane. (b) Carboxy-fluorescein conjugated to APTES. (c) Schematic of the corresponding surface chemistry of the two dyes conjugated to isocyanatosilane and APTES inside and outside of the patterned regions.

Surface Functionalization. To visualize the patterned areas, fluorescent dyes were covalently attached to a chemically patterned pSi surface (pattern A). Figure 3 shows the obtained fluorescence microscopy images for the conjugation of the fluorescein-5-maleimide dye to the patterned pSi surfaces. In this case, the maleimide-functionalized dye was attached via conjugation to the mercaptosilane that was present in the patterned regions. The fluorescence microscopy images show excellent confinement of the dye to the patterned areas, with no observable fluorescence in the regions surrounding the patterns. Consistent with the IR maps, the surface coverage of the dye appears to be homogeneous throughout the patterned areas, with each feature showing a sharp, well-defined boundary with respect to the surrounding pSi surface. Because our patterning technique allows two chemical functionalities to be present on a single surface, the conjugation of two different fluorescent species with different excitation and emission wavelengths was also investigated. The reactive chemical functionality of each dye was also carefully selected to minimize the cross-reaction of each dye within the patterned and surrounding areas. On the basis of these two criteria, the following organic dyes were chosen: carboxy-5-fluorescein and lissamine (rhodamine B ethylenediamine). These dyes fluoresce in the green (521 nm) for carboxy fluorescein and in the red (610 nm) for lissamine, with a carboxylic acid functionality present on the carboxy-5fluorescein and an amine functionality present on the lissamine dye. This allows the dyes to be selectively conjugated to a pSi surface patterned with APTES and an isocyanatosilane (pattern B). Conjugation of these two dyes to a chemically patterned pSi surface (pattern B) was performed. After the conjugation reactions of both dyes, it was observed that only the rhodamine dye was immobilized on the pSi surface. The reactivity of APTES toward the carbodiimide coupling of carboxy fluorescein may have been lost after it had come into contact with the photoresist. This is not the case for the isocyanate reactivity because this

functional group never comes into contact with the photoresist during the surface-patterning procedure. The carboxy fluorescein was therefore first coupled to an APTES-functionalized pSi surface, and the patterning procedure was then performed. Following patterning with the isocyanatosilane, the lissamine dye was conjugated to the surface and viewed by fluorescence microscopy. Figure 4 displays the images obtained for the dual dye-functionalized surfaces, where Figure 4a corresponds to the fluorescence of the rhodamine dye in the patterned isocyanate regions and Figure 4b corresponds to the carboxy-5-fluorescein in the APTES region. From the fluorescence images in Figure 4, it is obvious that there is again excellent confinement of the dyes to the respective regions. It can also be seen that there is complete removal of the carboxy-5-fluorescein functionality in the patterned areas, suggesting that the HF treatment of the exposed patterned regions during the modification process is sufficient to remove all silane groups. This leads to very well defined chemical boundaries between the patterned regions and the surrounding areas. The demonstration of conjugation of two different organic dyes to the patterns shows that it is possible to pattern the pSi surfaces discretely with the desired contrast in chemical functionality. Patterned Attachment of Mammalian Cells. Following the patterned attachment of different fluorescent dyes, mammalian neuronal cell line SK-N-SH was cultured on dual silane-patterned pSi surfaces (pattern C, Figure 1d). This pattern was chosen because of the ability of APTES-functionalized pSi to promote the attachment of the neuronal cells and PEG silane to prevent the attachment of mammalian cells.7,33 An aminosilane coating of surfaces has been previously found to promote the attachment of different mammalian cell lines,51,52 and PEG surface coatings have been shown to prevent the nonspecific attachment of a range of biomolecules and mammalian cells.53,54 The pore size and topographical features of the pSi surfaces used here were fabricated in order to promote cell attachment, with work by Khung et al. demonstrating enhanced cell attachment to pSi with pore radii of 5 20 nm.6 This means that the prevention of cell attachment to the nanostructured, chemically functionalized surface should be based on surface chemistry cues and not on topographical effects. Cells were cultured in contact with the patterned pSi surfaces. After the incubation period, each surface was gently washed with buffer to remove weakly bound cells. Cells were fixed and stained with the nucleus stain, Hoechst 33342, and the tetramethylrhodamine B-labeled cytoskeleton-binding phalloidin stain. Figure 5 displays fluorescence microscopy images of neuronal cells (SK-N-SH) attached to the dual functionalized pSi surfaces. The dashed lines in the images in Figure 5 are a guide to the eye and outline the patterned areas on the surfaces. It is apparent that 9501

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Langmuir the attached cells are almost exclusively located within the APTES patterned regions on the pSi surfaces (only 2% of cells are located outside of the APTES patterns). The PEG-silane-functionalized areas outside of the APTES patterns are able to prevent the adsorption of proteins and cell attachment, consistent with previous findings on nonpatterned surfaces.55,56 As can be seen in Figure 5a, cell patterns down to approximately 100 μm in diameter have been successfully produced. The feature size and shape of surface patterns are important factors when investigating cell attachment, interaction, and proliferation on chemically modified surfaces.54,57 59 By using the surfacepatterning technique presented here, feature sizes smaller than those of microprinted microarrays have been produced, with a range of feature shapes also being possible.42,60 The patterning of mammalian cells on pSi surfaces, along with the ability to incorporate multiple chemical species, allows the possibility of the development of highly specialized biosensors and cell culture platforms including smart Petri dishes.

’ CONCLUSIONS A method for producing discretely patterned areas of chemical functionality on pSi surfaces has been demonstrated. A combination of silanization and photolithography procedures afforded pSi surfaces chemically patterned with two different silane compounds. The overall patterning procedure is versatile because the photolithographic and silanization techniques are commonly employed in laboratories around the world. As shown, this technique is also suitable for the incorporation of a range of silane compounds, allowing very selective surface chemistry to be patterned on pSi surfaces. Chemically patterned surfaces have been characterized by conventional IR spectroscopy and IR microscopy, which revealed the chemical patterns to be well-defined on the pSi surface, with virtually no crossover between chemically functionalized regions. The incorporation of multiple silane compounds with different chemical reactivity has also allowed the patterned incorporation of two different fluorescent dyes. The patterned attachment of mammalian cell line SK-N-SH on an APTES/PEG-silane-patterned pSi surface was also demonstrated. We obtained greater than 98% total cell attachment within the APTES-patterned regions. ’ AUTHOR INFORMATION Corresponding Author

*E-mail: nico.voelcker@flinders.edu.au.

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