ARTICLE pubs.acs.org/IECR
Surface Patterning with Chemisorbed Chemical Cues for Advancing Neurochip Applications Gerardo A. Diaz-Quijada,*,† Christy Maynard,† Tanya Comas,‡ Robert Monette,‡ Christophe Py,§ Anthony Krantis,|| and Geoffrey Mealing‡ Steacie Institute for Molecular Sciences, ‡Institute of Biological Sciences, and §Institute for Microstructural Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Ontario K1A 0R6, Canada Centre for Research in Biopharmaceuticals and Biotechnology, University of Ottawa, Ottawa, Ontario, Canada
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bS Supporting Information ABSTRACT: We are currently developing multisite planar patch-clamp chips capable of recording high resolution electrophysiological function from individual neurons established in culture. This capability provides a unique opportunity to establish and study communication between synaptically connected neurons on-chip at the level of ion channel function. Critical to realizing this goal for mammalian cell applications are chip surface functionalization strategies that will promote attraction and adhesion of cells to on-chip interrogation features as well as facilitate in the guidance of connectivity between neurons. Here, a chemical strategy is presented that has been adapted to be compatible with standard photolithography techniques for chemical patterning of amine rich cell adhesion promoters on silicon-based surfaces. This chemisorption approach will not only enable electrophysiological studies of neuronal networks but also allow patterning of small peptides such the ones containing the RGD motif, known to induce cell adhesion via molecular recognition of integrin receptors on cell membranes and also stimulate other important biological processes.
1. INTRODUCTION Advances in our understanding brain function and the mechanisms underlying communication between neurons have been largely dependent upon technical advances in interrogating neuronal activity from the network level down to the high resolution study of individual cells and synapses.1,2 Multi-Electrode Arrays3 allow the study of neuronal populations established in cell culture, in brain slices, or in vivo, by measuring intracellular field potentials and spiking activity. However, higher resolution interrogation of electrophysiological function requires the study of underlying ion channel activity. Patch-clamp is widely accepted as the technique of choice to accomplish this task. Patchclamp is a powerful technique, but requires labor-intensive manipulation of a small glass pipet such that an aperture at its apex adheres tightly to a cell’s membrane. In response, planar patchclamp devices have been developed to simplify and expedite the recording process. They are composed of a flat substrate with a micrometer-sized aperture separating two fluidic chambers, such that the chip aperture replaces the function of the apex of the glass pipet, and isolated cells in suspension are delivered to the aperture by aspiration.4,5 This approach enables much higher throughput assessment of ion channel activity, which is important for studying ion channel pharmacology and for drug development. However, unlike conventional patch-clamp, the technique is limited to use with isolated cells, which lack communication with neighboring cells. We have recently developed planar patch-clamp chips capable of recording from molluscan neurons established in cultured directly over on-chip apertures,6,7 and have demonstrated that these neurons form functional synapses with neighboring neurons (Martina et al. submitted). Applying this technology to r 2011 American Chemical Society
mammalian neurons requires chip surfaces that can attract cells to aperture sites, promote cell membrane/chip substrate adhesion, and guide the formation of communicating neuronal networks. We have previously patterned chemical modification of SiN surfaces with poly-D-lysine transferred from PDMS stamps to promote adhesion and guidance of cryo-preserved primary rat cortical neurons.8 While this approach was effective it is not suitable for multiple stamping required for large scale chip fabrication. The present study describes a generalized approach for the chemical modification and patterning of cell adhesion promoters on silicon substrates using standard photolithographic techniques which is suitable for large scale production. We not only demonstrate that chemisorption is required when dealing with small peptides but also the chemical strategy has been adapted to be compatible with standard photoresist technologies.
2. EXPERIMENTAL SECTION 2.1. X-ray Photoelectron Spectroscopy (XPS). Spectra were acquired on a PHI-5500 spectrometer (Physical Electronics Inc., Chanhassen, MN) using monochromatized Al KR radiation (hν = 1486.6 eV) from an anode operating at 13.6 kV and 350 W. The survey spectra (01400 eV) were recorded at a pass energy of 187.85 eV and for the high-resolution scans for the elements of interest at 11.75 eV. The eV/step of the spectrometer was 0.8 eV for survey scans and 0.05 eV for high-resolution scans. All spectra Received: February 22, 2011 Accepted: July 28, 2011 Revised: July 11, 2011 Published: July 28, 2011 10029
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Figure 1. Schematic representation of the steps involved in chemically patterning surfaces using conventional photolithography. Exposed areas after development are chemically modified sequentially with amino groups, carboxylic acid groups, and finally with the desired cell adhesion promoter.
have been corrected for sample charging, with the adventitious C 1s peak (284.6 eV) used as internal reference. 2.2. Deposition of SiO2 on Silicon Substrates. SiO2 deposition was accomplished with a Plasmatherm 7000 Plasma Enhanced Chemical Vapor Deposition instrument (Plasmatherm, Petersburg, Florida) operating at 350 °C and 40W power with gas flow rates set at 500 sccm N2O, 54 sccm SiH4 and 1100 sccm He, and a pressure of 1500 mTorr. The deposition rate was approximately 350 Å/min to give 200 nm films. 2.3. Chemical Patterning of Silicon Substrates. Photolithograhic patterning of silicon substrates9 was adapted from standard procedures employed in microelectronics. Since surface modifications are extremely sensitive to surface contaminants, well-known photoresist adhesion promoters such as HMDS were avoided. To this end, PEVCD SiO2 coated wafers were precleaned with a 50 W oxygen plasma generated by a Jupiter III etcher (Norson-March, Concord, CA, U.S.A.) and dehydrated by sintering over a hot plate at105 °C for 10 min prior to spincoating Microposit S1813 (Shipley Co., Marlborough, MA, U.S. A.) at 5000 rpms followed by heating at 110 °C for 5 min to give a 1.3 μm photoresist film. These substrates were exposed through a photomask to 100 mJ/cm2 UV radiation using a contact aligner (Karl Suss America, Waterbury, VT, U.S.A.). The pattern was overdeveloped without loss of fidelity with a 10 min immersion in Microposit MF321 developer (Shipley, Marlborough, MA, U.S.A.) followed by a rinse in deionized water. Overdevelopment is necessary to ensure the UV exposed (positive) resist is completely removed thereby ensuring complete exposure of the SiO2 surface in the intended pattern (Figure 1). Alternatively, SiO2 coated silicon substrates were prefunctionalized with 3-aminotriethoxysilane as described below prior to patterning with photoresist using the same procedure with the exception of omitting the plasma precleaning step prior spin-coating the resist. Prior to chemical functionalization of the photolithographically patterned silicon wafers, the substrates were cleaned in a Plasma Prep II (Structure Probe Inc., West Chester, PA, U.S.A.) set at 50 W for 30 s at an air base pressure of 7 102 mTorr. Freshly cleaned substrates were placed inside a chamber containing two microcentrifuge tubes with 15 μL of 3-aminopropyltriethoxysilane (APTES) and allowed to react for 4 h under vacuum.10 The resultant amino modified substrates were reacted with a freshly prepared solution of 10 mg/mL poly(methacrylic acid) (MW = ∼100,000, Cat. # 00578, Polysciences Inc., U.S.A.) in 0.1 M borate buffer pH = 8 in the presence of 9.6 mM N-(3dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDC, Aldrich, Oakville, ON, Canada) and 8.6 mM N-hydroxysuccinimide
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(NHS, Aldrich, Oakville, ON, Canada). The substrates were washed with a solution of 0.1% sodium dodecylsulfate (SDS) in PBS pH = 7.4 (Aldrich, Oakville, ON, Canada), rinsed in Milli-Q water, and dried under a stream of nitrogen after the 2 h reaction. Immobilization of poly-D-lysine was carried out by reacting the surface with a 20 mg/mL solution of poly-D-lysine hydrobromide (Aldrich, Oakville, ON, Canada) in borate buffer pH = 8 in the presence of 9.6 mM EDC and 8.6 mM NHS for 2 h. The reaction was quenched by immersing the substrate in a 1 M aqueous solution of ethanolamine (Aldrich, Oakville, ON, Canada) pH = 8.5 for 4 min followed by washing in Milli-Q water and drying with a stream of nitrogen. Removal of the photoresist was accomplished with an acetone (Aldrich, Oakville, ON, Canada) wash followed by immersion for 30 min and sonication at 40 °C for 15 min in Nano Remover PG (Microchem, Newton, MA, U.S.A.). To facilitate the visualization of patterned polylysine, test samples were selected prior to removal of the photoresist, and they were treated with a 5 μM solution of Alexa Fluor555 reactive dye (Invitrogen Canada Inc., Burlington, ON, Canada) in the presence of 13.9 mM NHS and 15.4 EDC in 0.16 M borate buffer pH = 8. 2.4. Chemical Immobilization of Peptides That Contain the RGD and RAD Sequence. The covalent immobilization of cyclo(RGDFK-PEG) (Cat. # PCI-3696-PI, Peptides International, Louisville, KY, U.S.A.) and cyclo(RADFK-PEG) (PCI3954-PI) was carried out on unpattered silicon as follows. Silicon substrates of approximately 1 1 cm having a film of PECVD SiO2 film were precleaned at 120 °C for 4 h in a piranha solution made from 4 parts conc. sulfuric acid (Mallinckrodt Baker, Inc., Phillipsburg, NJ, U.S.A.) and 1 part 30% hydrogen peroxide (Mallinckrodt Baker, Inc., Phillipsburg, NJ, U.S.A.). Aminomodification of these substrates was carried out with APTES in the same manner as described as before. Subsequently, the modified surfaces were reacted for 1 h with a 0.12 M glutaric anhydride solution in a 9:1 mixture of N-methylpyrrolidone (Aldrich, Oakville, ON, Canada)/borate buffer pH = 8. Finally, a 1 μL droplet of a solution containing the desired cyclopeptide at the concentration of 1.7 mM in 0.1 M borate buffer pH = 8 in the presence of 9.6 mM EDC and 8.6 mM NHS was delivered to the center of the wafer. The reaction is carried out for 2 h in a humid chamber followed by washing with a 0.1% SDS in PBS solution. 2.4. Neural Cell Culture. Cryopreserved rat cortical neurons (QBM Cell Sciences, Ottawa, ON, Canada) were thawed in a 37 °C water bath for 2.5 min. Cells (4 106) contained in 1 mL of the thawed solution were resuspended prior to transferring to a 15 mL sterile tube. To avoid osmotic shock, 9 mL of Neurobasal Medium (Invitrogen, Carlsbad, CA) containing 2 mM L-glutamine (Sigma, St. Louis, MO), B-27 (Invitrogen), and 5% fetal bovine serum (Gemini, West Sacremento, CA) was added over a period of 2.5 min. Cells were then resuspended with a 10 mL pipet and inverted twice to ensure complete mixing of cells and media prior to plating. The resultant cell suspension (1 mL) was added to each well of a 24 well plate (VWR Canlab, Mississauga, ON, Canada) containing either a patterned sample or a poly-Dlysine coated glass coverslip (Bellco Glass, Vineland, NJ). The media was exchanged with 1 mL of serum-free Neurobasal Medium after 4 h. Subsequent media exchanges were performed biweekly by replacing half of the media with fresh Neurobasal Medium. No antimitotic drugs were added to reduce glial proliferation. Cell cultures were maintained at 37.5 °C in a 5% CO2 humidified incubator (NuAire, Inc., Plymouth, MN). 2.5. Immunofluorescence. Cultured cells plated onto patterned samples or coverslips were rinsed with PBS (140 mM 10030
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Industrial & Engineering Chemistry Research NaCl, 4 mM KCl, 0.5 mM Na2HPO4, 0.15 mM KH2PO4) then fixed for 30 min at room temperature in freshly prepared 4% paraformaldehyde. Cells were then washed twice in PBS and permeabilized by incubation in 0.25% Triton-X for 10 min followed by two more washes in PBS. To reduce nonspecific binding of antibodies, blocking agent (Dako, Mississauga, ON, Canada) was added to cells for 1 h at room temperature. Mouse monoclonal Map2 antibody (1:200 dilution) and goat antirabbit GFAP antibody (1:400 dilution) (both antibodies from Sigma) were incubated with cells overnight at 4.0 °C. The next day cells were washed twice with PBS and incubated in the following secondary antibodies: Alexa Fluor 568 goat antimouse (1:400) and Alexa Fluor 488 goat antirabbit (1:800) (both secondary antibodies from Invitrogen, Burlington, ON, Canada) for 1 h at room temperature followed by rinsing twice with PBS. Patterned samples were inverted onto coverslips containing a drop of fluorescent mounting medium. Mounted samples were stored at 4.0 °C until imaging. 2.6. Live Cell Staining. Bath Medium (BM) was prepared as follows: 140 mM NaCl, 3.5 mM KCl, 0.4 mM KH2PO4, 20 mM HEPES, 5 mM NaHCO3, 1.2 mM MgSO4, 1.3 mM CaCl2, and 15 mM glucose. Calcein-AM (MolecularQ2 Probes, C-3100) was used as an indicator to access cell viability. A 5 mM stock solution was prepared in dimethylsulfoxide (DMSO) with vigorous vortexing. The stock solution was then diluted to a 40 μM substock in BM. Cells were subsequently stained using Calcien-AM
Figure 2. General chemical strategy for the surface modification of immobilization of cell adhesion promoters and chemical patterning.
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at 5 μM. A 10 mM stock solution of RH-237 was prepared and subsequently diluted to 50 μM in BM for cells staining. Each sample was placed in an individual well of a 24-well plate. A 350 mL portion of 5 mM calcein was placed in each well before incubation for 30 min at 37 °C in the dark. Calcein was then replaced by the same volume of RH-237, followed by incubation for 10 min before rinsing to minimize nonspecific background fluorescence. Finally, 450 mL of NBM was added to each well. 2.7. Cell Imaging. Fluorescence and reflection images of samples were obtained using an LSM-410 Zeiss (Thornwood, NY) confocal microscope equipped with a Krypton/Argon laser (Melles Griot, Carlsbad, CA) and an LSM Tech, Inc. (Etters, PA) objective inverter. For each dye, excitation wavelength and emission filter were appropriately selected, and images of both dyes were collected sequentially. Calcein was excited with the 488 nm wavelength of the laser, and an emission filter with a bandwidth of 515540 nm was used. For the RH-237 conjugate, the excitation wavelength of 568 nm and a long-pass emission filter with a cutoff at 610 nm were used. Reflection images were collected using the 568 nm line of the laser with no filter in front of the photomultiplier tube (PMT). Reflection and fluorescence images of the same field were merged using Northern Eclipse software (Empix, Mississauga, ON, Canada)
3. RESULTS AND DISCUSSION 3.1. Chemical Modification of Silicon Wafers. Silicon is the preferred material because of its well established micromachining techniques in the microelectronic industry. However, the semiconductive nature of this material can introduce a shunt in the devices during electrophysiological measurements.7 To overcome this issue, the surfaces of our silicon chips7 are passivated with thin films of PEVCD-deposited insulating layers of SiO2. Since these surfaces can be readily modified covalently with silane reagents, we reacted the silicon chips with APTES to give amino-modified surfaces which are subsequently converted to carboxylic acid-modified surfaces with glutaric anhydride, as depicted in Figure 2. Such surfaces are highly versatile since they can be activated and made reactive toward a variety of amine rich biomolecules (proteins) or cell adhesion promoters (Figure 3). For instance, the terminal carboxylic acid groups can be activated in the presence of EDC and NHS as succinimidyl esters (Figure 2) which are known to react efficiently with amines in an aqueous environment to form a stable amide linkage.11 The stepwise chemical modification was characterized by X-ray Photoelectron Spectroscopy (XPS) as illustrated in Figure 4 with a set of surveyed spectra and an inset with line deconvolution of
Figure 3. Chemical structures for cell adhesion promoters, poly-D-lysine and cyclo(RGDFK-PEG). 10031
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Figure 4. XPS spectra of pristine, amino-, and COOH-modified silicon surfaces illustrating the presence of nitrogen after the modification with APTES, as expected. Inset set of spectra depicts line deconvolutions for C 1s, and they corroborate the presence of carboxylic acid groups on the COOH-modified surface with the appearance of the new peak (D) at 288.2 eV.
Figure 5. Direct comparison of the effects of physisorption and chemisorptions of large molecules such as poly-D-lysine. In this example, physisorption was carried out by delivering a droplet of fluorescently labeled (AlexaFluor 555) poly-D-lysine on plasma cleaned silicon wafer. Chemisorption was performed in the same manner with the exception that the reaction of polylysine was carried out in the presence of NHS and EDC. Subsequent washings indicate that physisorption leads to not only smearing of the polylysine but also a low concentration of physisorbed polylysine as visualized from its fluorescence image.
the C 1s signals. The spectrum from the pristine silicon surface is consistent with a clean SiO2 surface exhibiting peaks due to O 1s and Si 2s and Si 2p and a trace C 1s signal that corresponds to adventitious carbon. Reaction with APTES is corroborated by the appearance of the N 1s signal at 400.9 eV with a concomitant increase of the C 1s peak. Line deconvolution indicates the appearance of a new peak at 285.9 eV which is due to *CH2NH2. The subsequent reaction of the amino-modified surface with glutaric anhydride yielded substantial changes in the C 1s signal. In this case, two new signals at 288.2 eV (D) and 287.6 eV (C) appeared, and they are due to the carbonyl carbon in carboxylic acids and amides, respectively.12 The peak at 285.4 eV is likely due to overlapping signals from *CH2C(dO)OH and *CH2C(dO)-NH-. 3.2. Physisorption versus Chemisorptions. It is well-known that polymers and large biomolecules such as proteins can physisorb on surfaces, as it is the case for Bovine Serum Albumin.1315 This is due to ionic, dipole, and van der Waals interactions between molecules and surfaces. Such interactions
may be weak (van der Waals), but their additive effects in large molecules can lead to substantial effects. Negatively charged surfaces such as SiO2 interact quite efficiently with polyamines at pHs lower than the pKa of amines. Consequently, it is traditional to simply physisorb polylysine on surfaces such as SiO2 or glass.16,17 Such modifications are not entirely stable over time in environments of high ionic strength. A comparative study of physisorbed and chemisorbed poly-D-lysine indicates that in the physisorbed case poly-D-lysine not only smears out during washing with PBS buffer but also yields a rather low surface density of poly-D-lysine. Whereas in the chemically immobilized case (Figure 5), the poly-D-lysine remains bound to the surface with a much higher surface density. It is also very important to establish that physisorption is not a viable method for the modification of surfaces with small molecules. This is illustrated with short peptide sequences such as the cyclo(RGDFK-PEG) peptide (PCI-3696-PI) which consists of 4 amino acids in a cyclic arrangement linked to a PEG chain that has a terminal primary amino group. This peptide contains the RGD sequence which is 10032
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Figure 6. (A) Importance of chemisorptions when using small molecules is illustrated by culturing cryopreserved rat cortical neurons on surfaces that have circular regions of physisorbed and chemisorbed cyclo(RGDFK-PEG). Cells were visualized with a cell membrane dye (RH-237). The negative control experiment with cyclo(RGDFK-PEG) was carried out to corroborate molecular recognition of the RGD motif. (B) Immunofluorescence staining of cells for GFAP and Map2 to corroborate the presence of neurons on the cyclo(RGDFK-PEG) modified surface.
Figure 7. Proof-of-concept demonstration of chemical patterning of fluorescently labeled (Alexafluor 555) poly-D-lysine using conventional photolithography, followed by culture of cryopreserved rat cortical neurons (QBM). In this case, cells were visualized by staining with calcein (viability, green) and RH-237 (membrane dye, red).
known to be the minimum sequence that will be recognized by the integrin receptors on cell membranes, thereby inducing cell adhesion via a molecular recognition event.18 In contrast, the negative control peptide, cyclo(RADFK-PEG) (PCI-3954-PI), which differs by only one methyl group (structure in the Supporting Information, Figure SI-1) does not induce cell adhesion. The results from culturing QBM cells on physisorbed
and chemisorbed cyclo(RGDFK-PEG) and chemisorbed cyclo(RADFK-PEG) are illustrated in Figure 6a using RH-237 (red) as an indicator for the presence of cell membranes. Further characterization of the cells on cyclo(RGDFK-PEG) was performed by immunostaining with a combination of monoclonal and secondary fluorescently labeled antibodies to identify neurons (Map2-red) and astrocytes (GFAP-green) as described in 10033
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Industrial & Engineering Chemistry Research the Experimental Section (Figure 6b). It is corroborated in this experiment that physisorption is not feasible with such small peptides and that cell adhesion with this cyclic RGD peptide involves molecular recognition. 3.3. Chemical and Cell Patterning. Although photolithography has been demonstrated as a potential technique for chemical patterning of surfaces, our strategy for the chemical immobilization of cell adhesion promoters was modified and optimized to be compatible with common photoresists such as Shipley S1813 which swell or dissolve in polar organic solvents. Consequently, the preparation of carboxylic acid surfaces was performed with poly(acrylic acid) instead of glutaric anhydride. This polymeric reagent has the advantage of not only producing a high concentration of carboxylic acid groups but also allowing the reaction with the amino terminated surfaces to be carried out in aqueous media in the presence of EDC and NHS. The following key points were found to be important to achieve successful chemically modified patterns. Efficient modification of the exposed regions of SiO2 require clean surfaces free from residual photoresist and/or any other substances that may mask the surface and prevent the reaction with silane reagents. Consequently, all samples were over developed and precleaned with an air plasma generator. It follows that conventional photoresist adhesion promoters (employed in photolithography) such as hexamethyldisilizane (HMDS) need to be avoided. In this case, wafers are dehydrated at 105 °C prior to spin-coating the photoresist to dehydrate the SiO2 surface. An alternative approach which proved to have a higher success rate is based on spin-coating the photoresist on amino-modified wafers. As mentioned above, it is necessary to preclean photolithographically patterned samples in an air plasma prior to the reaction with APTES. However, overexposure resulted in cross-linked photoresist which is difficult to remove at the end of the process; consequently, the optimum conditions were exposure for 30 s at 50 W and an air pressure of 7 102 mTorr. The undesirable cross-linking effect is very likely due to the generation of hydroxyl and carboxylic acid groups in the photoresist, and these can react with each other to form ester groups in the presence of EDC and NHS which are necessarily employed in subsequent steps. In addition to the above key points, we observed that the reaction of APTES with the photoresist patterned SiO2 surface needs to be limited to 4 h in the gas phase to prevent swelling and detachment of the photoresist. To illustrate the concept with our chemical strategy, test silicon wafers were photolithographically patterned to have squared regions of 100 μm with a pitch of 200 μm or 400 μm. Poly-D-lysine was chemically immobilized and subsequently labeled with Alexafluor 555 in situ, and its fluorescent image is presented in Figure 7 after the removal of the photoresist. Cryopreserved rat cortical neurons were cultured for 1419 days on a wafer that had been patterned with unlabeled poly-Dlysine in the same manner as before. Staining of the cells with calcein for cell viability and RH-237 for cell membranes allow the visualization of the patterned cells along with their processes to enable connectivity (Figure 7). These proof-of-concepts cell patterns set the stage for not only cell placement over the micro-orifice of a patch-clamp but also the fabrication of predefined synthetic networks where cells may be placed over an array of microholes and/or a predefined pattern of cells are placed in close proximity to a cell over a micro-orifice. One can also envision many other opportunities that can be explored which may include taking advantage of the
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presence of microfluidic channels that are connected to each individual micro-orifice.
4. CONCLUSIONS A chemical approach for the immobilization of cell adhesion promoters on silicon substrates is presented for guiding neuronal adhesion and growth. In contrast to polymeric cell adhesion promoters, it is demonstrated that chemisorptions is necessary when immobilizing small molecules such as short peptides. The concept is illustrated with a cyclopeptide that has the RGD motif which is known to interact specifically with the integrin receptors in cell membranes and therefore promote cell adhesion. To prove that molecular recognition is at play, the negative control is carried out where cultured cryopreserved rat cortical neurons do not adhere to surfaces that had been modified with a cyclopeptide that possesses the RAD motif which only differs by one amino acid. RGD induced cell adhesion is a rather important outcome since it is known that integrin receptors are in close proximity to ion channels, and this could be used to position ion channels at or near the orifice in our planar patch-clamp.19 It is also important to note that the presented chemical strategy has been adapted to be compatible with a modified version of standard lithographic techniques which are suitable for industrial scale processes. Proof-of-concept is illustrated by patterning silicon substrates with 100 μm squares of polylysine with a pitch of 200 μm and subsequently culturing cryopreserved rat cortical neurons. The resultant pattern illustrates neurons on the squares with processes extending to neighboring neurons in an organized manner. The presented techniques will enable the study of synthetic networks defined by patterning of chemical cues. Moreover, the level of complexity of such synthetic networks will be increased with planar patch-clamp chips that possess an array of microorifices with a network of microfluidic channels that are able to independently address each individual orifice.20 ’ ASSOCIATED CONTENT
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Supporting Information. Figure SI-1. Chemical structure of cyclo(RADFK-PEG) which has no cell adhesion properties (negative control). This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*Phone: 613-949-4409. Fax: 613-991-2648. E-mail: gerardo.
[email protected].
’ ACKNOWLEDGMENT The authors would like to thank Mark Malloy for PECVD SiO2 depositions and Zhenhua He for the acquisition of the XPS spectra. ’ REFERENCES (1) Greene, A. J. Making connections. Scientific American Mind 2010, 21 (3), 22–29. (2) Kotaleski, J. H.; Blackwell, K. T. Modelling the molecular mechanisms of synaptic plasticity using systems biology approaches. Nat. Rev. Neurosci. 2010, 11 (4), 239–251. (3) Taketani, M., Baudry, M., Eds.; Advances in Network Electrophysiology: Using Multi-Electrode Arrays; Springer: New York, 2006. 10034
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