Surface patterning with chemisorbed chemical cues for advancing neurochip applications

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Surface patterning with chemisorbed chemical cues for advancing

neurochip applications

Diaz-Quijada, Gerardo A.; Maynard, Christy; Comas, Tanya; Monette,

Robert; Py, Christophe; Krantis, Anthony; Mealing, Geoffrey

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Published: July 28, 2011

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

b

S _{Supporting Information}

ABSTRACT: We are currently developing multisite planar patch-clamp chips capable of recording high resolution electro-physiological 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 mech-anisms 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-Elec-trode Arrays3allow the study of neuronal populations established in cell culture, in brain slices, or in vivo, by measuring intracellular fi_{eld potentials and spiking activity. However, higher resolution} interrogation of electrophysiological function requires the study of underlying ion channel activity. Patch-clamp is widely ac-cepted as the technique of choice to accomplish this task. Patch-clamp is a powerful technique, but requires labor-intensive mani-pulation of a small glass pipet such that an aperture at its apex adheres tightly to a cell’s membrane. In response, planar patch-clamp 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.4,5This approach enables much higher throughput assessment of ion channel activity, which is impor-tant 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 neu-rons (Martina et al. submitted). Applying this technology to

mammalian neurons requires chip surfaces that can attract cells to aperture sites, promote cell membrane/chip substrate adhe-sion, and guide the formation of communicating neuronal net-works. 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

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10030 dx.doi.org/10.1021/ie200358q |Ind. Eng. Chem. Res. 2011, 50, 10029–10035

Industrial & Engineering Chemistry Research ARTICLE

have been corrected for sample charging, with the adventitious C 1s peak (284.6 eV) used as internal reference.

2.2. Deposition of SiO2on Silicon Substrates.SiO2

deposi-tion was accomplished with a Plasmatherm 7000 Plasma En-hanced 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 SiH4and 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. Photolitho-grahic patterning of silicon substrates9was adapted from stan-dard 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 spin-coating 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/cm2UV 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 SiO2surface in

the intended pattern (Figure 1). Alternatively, SiO2coated 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 photolithographi-cally 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 contain-ing two microcentrifuge tubes with 15 μL of 3-aminopropyl-triethoxysilane (APTES) and allowed to react for 4 h under vacuum.10The 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-(3-dimethylaminopropyl)-N0_{-ethylcarbodiimide hydrochloride (EDC,}

Aldrich, Oakville, ON, Canada) and 8.6 mM N-hydroxysuccinimide

(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 sol-ution 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 Interna-tional, Louisville, KY, U.S.A.) and cyclo(RADFK-PEG) (PCI-3954-PI) was carried out on unpattered silicon as follows. Silicon substrates of approximately 1 1 cm having a film of PECVD SiO2film 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.). Amino-modification 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 mML

-gluta-mine (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-D

-lysine 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% CO2humidified incubator (NuAire, Inc., Plymouth, MN).

2.5. Immunofluorescence. Cultured cells plated onto pat-terned samples or coverslips were rinsed with PBS (140 mM 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.

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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 per-meabilized 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 tem-perature 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 sol-ution was prepared in dimethylsulfoxide (DMSO) with vigorous vortexing. The stock solution was then diluted to a 40 μM sub-stock in BM. Cells were subsequently stained using Calcien-AM

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 semi-conductive nature of this material can introduce a shunt in the devices during electrophysiological measurements.7 _To

over-come this issue, the surfaces of our silicon chips7are 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 2. General chemical strategy for the surface modiﬁcation of

immobilization of cell adhesion promoters and chemical patterning.

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the C 1s signals. The spectrum from the pristine silicon surface is consistent with a clean SiO2surface 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 appear-ance 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.12The 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.1315This 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 SiO2interact quite efficiently with polyamines at

pHs lower than the pKaof amines. Consequently, it is

traditio-nal to simply physisorb polylysine on surfaces such as SiO2or

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 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 ﬂ_{uorescence image.}

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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 per-formed by immunostaining with a combination of monoclonal and secondary fluorescently labeled antibodies to identify neu-rons (Map2-red) and astrocytes (GFAP-green) as described in 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).

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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 photolithogra-phy has been demonstrated as a potential technique for chemical patterning of surfaces, our strategy for the chemical immobiliza-tion 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 con-centration 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 chemi-cally modified patterns. Efficient modification of the exposed regions of SiO2require clean surfaces free from residual

photo-resist and/or any other substances that may mask the surface and prevent the reaction with silane reagents. Consequently, all sam-ples were over developed and precleaned with an air plasma generator. It follows that conventional photoresist adhesion promoters (employed in photolithography) such as hexamethyl-disilizane (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 sam-ples in an air plasma prior to the reaction with APTES. However, overexposure resulted in cross-linked photoresist which is diffi-cult to remove at the end of the process; consequently, the opti-mum 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 SiO2surface 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-D

-lysine 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

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 syn-thetic 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 micro-orifices with a network of microfluidic channels that are able to independently address each individual orifice.20

’ ASSOCIATED CONTENT

b

S _{Supporting Information.} Figure SI-1. Chemical

struc-ture of cyclo(RADFK-PEG) which has no cell adhesion proper-ties (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. diaz-quijada@nrc-cnrc.gc.ca.

’ ACKNOWLEDGMENT

The authors would like to thank Mark Malloy for PECVD SiO2depositions and Zhenhua He for the acquisition of the XPS

spectra.

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