Specific Neuron Placement on Gold and Silicon Nitride-Patterned

Banker , G. ; Goslin , K. Culturing Nerve Cells; MIT Press, 1998. There is no corresponding record for this reference. 25. Vogt , A. K.; Wrobel , G.; ...
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Specific neuron placement on gold and silicon nitride patterned substrates through a two-step functionalization Andrea Mescola, Claudio Canale, Mirko Prato, Alberto Diaspro, Luca Berdondini, Alessandro Maccione, and Silvia Dante Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b01352 • Publication Date (Web): 07 Jun 2016 Downloaded from http://pubs.acs.org on June 11, 2016

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Specific neuron placement on gold and silicon nitride patterned substrates through a two-step functionalization. Andrea Mescola,a*† Claudio Canale,a Mirko Prato,b Alberto Diaspro,a Luca Berdondini,c Alessandro Maccione,c£ and Silvia Dantea£ a

Department of Nanophysics, Istituto Italiano di Tecnologia (IIT), Via Morego 30, 16163, Genova, Italy.

b

Department of Nanochemistry, Istituto Italiano di Tecnologia (IIT), Via Morego 30, 16163, Genova, Italy.

c

Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia (IIT), Via Morego 30, 16163, Genova, Italy.

£

Co-last authorship

ABSTRACT: The control of the neuron-substrate adhesion has been always a challenge for neuron-based cell chip and in particular for Multi Electrode Arrays (MEAs) devices which allow investigating the electrophysiological activity of neuronal networks. The recent introduction of high density chips based on CMOS technology integrating thousands of electrodes improved the possibility to sense large networks and raised the challenge to develop newly adapted functionalization techniques to further increase neuron electrode localization to avoid the positioning of cells out of the recording area. Here, we present a simple and straightforward chemical functionalization method that leads to the precise and exclusive positioning of the neural cell bodies onto modified electrodes and inhibits, at the same time, cellular adhesion in the surrounding insulators areas. Differently from other approaches this technique does not require any adhesion molecules as well as complex patterning techniques such as µ-contact printing. The functionalization was first optimized on gold (Au) and silicon nitride (Si3N4) patterned surfaces. The procedure consisted of the introduction of a passivating layer of hydrophobic silane molecules (PTES) followed by a treatment of the gold surface by 11-amino-1-undecanethiol hydrochloride (AT). On model substrates, well-ordered neural networks and an optimal coupling between single neuron and single micrometric functionalized gold surface were achieved. In addition we presented preliminary results of this functionalization method directly applied on a CMOS-MEA: the electrical spontaneous spiking and bursting activities of the network recorded for up to four weeks demonstrate an excellent and stable neural adhesion and a functional behavior comparable with what expected by using standard adhesion factor as polylysine or laminin, thus demonstrating that this procedure can be properly considered a good starting point to develop alternatives to the traditional chip coatings in order to provide selective and specific neuron-substrate adhesion.

INTRODUCTION

Surface modification represents a widespread tool to investigate cell-biomaterial interaction; over the years, a number of innovative approaches have been used to control the cell adhesion properties of biomaterials including physical adsorption and self-assembly, covalent coupling, cross-linking as well as the synthesis of novel graftcopolymers with targeted functional groups.1–7 Generally, cellular responses to functionalized biomaterials are strictly dependent on the cell type8 and consequently on the application field. In particular, chemical surface modification of neuron-substrate interfaces assumes crucial importance in neuron-based cell chip design9–11 that includes all those applications, such as bioelectronics and biochip technologies, in which a synthetic substrate represents an interfacial layer between a live neural cell and the electrical compounds. In this context, numerous bioactive molecules, such as for example cell adhesion pep-

tides12 and extracellular matrix (ECM) proteins,13 have been used to functionalize substrates with the aim to tune neuronal adhesive properties. Usually in neural-chip design, substrates are composed of metal electrodes separated by inorganic or organic insulators;14 metal electrodes are mostly of platinum, titanium or gold while silicon dioxide and silicon nitride are generally used as inorganic insulators. In order to promote neural adhesion exclusively on metal electrodes several surface modification have been developed depending on the materials involved: for example, insulators were mostly functionalized by organosilane molecules with different functional groups15 while metal electrodes were modified with various bioactive molecules, such as adhesive peptides, polyaminoacids or polycations16–19 to promote the selective adhesion of neurons. Despite the impact of this recent progress, the interdisciplinary scientific community is constantly looking for alternative ways that provide a se-

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lective, specific and stable neural adhesion. For example, in the field of Multi Electrode Arrays (MEAs), the recent introduction of CMOS-technology allowed the integration of thousands of metal electrodes in few hundreds of µm,20 dramatically changing geometrical and physical constraints of the substrates interfacing neuronal cultures, thus making difficult the use of standard pattering techniques as µ-contact printing (due to spatial resolution limitation)21 or microdropping technique (long preparation time for thousands of electrodes)22 to promote selective adhesion of neurons on the sensing elements. In this paper, we present a two-step chemical functionalization method that allows for a selective neuron positioning on gold electrodes without the need of using any further adhesion molecules or patterning technique. In particular, the optimization of the surface chemistry was carried out onto non-conductive model substrates made up of the same materials involved in the largest part of neuron-based devices. To explore neuronal adhesion a first one-step functionalization has been performed onto gold substrates treated with amino thiol (AT) molecules by dip coating and by patterning through a benchtop microdropper. After validation of a stable adhesion over time and the precise positioning of cells onto treated gold substrates, we applied the procedure on ad hoc fabricated silicon nitride substrates that faithfully reproduce the active areas of CMOS Multi Electrode Arrays (MEA) device, both in geometry and chemical composition (see Materials and Methods). On these substrates, we optimized the functionalization by introducing a preliminary step to passivate the silicon nitride layer from undesired cell adhesion (two-step functionalization) by exploiting the hydrophobic properties of propyltriethoxysilane (PTES) molecules able to inhibit the neuronal attachment. Surface chemico-physical analysis was performed to monitor the change of the surface properties occurred after the functionalization steps: water contact angle (WCA) measurements were executed to provide a rapid feedback on wettability change, while X-ray photoelectron spectroscopy (XPS) analyses were carried out to characterize the chemical composition of the substrates before and after modification and to ensure the compatibility of the selected modifications. As a result of this functionalization, engineered neural networks only on the gold squares were obtained and their development was observed up to four weeks. Neuronal selectivity and adhesion were monitored by using optical and fluorescence imaging as well as electron microscope techniques to get a deeper insight on the structure of neural network. Finally, a conductive CMOS-MEA device with electroless-deposited gold electrodes was functionalized with this procedure and cultured with hippocampal neurons from rat embryos in order to characterize the functional electrical behaviour of the developed network. The electrophysiological activity recorded at different Days In Vitro (DIVs) reveals a development analogue of what can be obtained by using standard adhesion molecules as polylysine or laminin, and suggests therefore, that the proposed method represent a good starting point in the advancement of alternatives to

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traditional adhesion factors to provide selective and specific neural-substrate adhesion. MATERIALS AND METHODS

Substrates preparation. All solvents and ordinary chemical reagents were purchased as high purity reagent grade materials from Sigma-Aldrich and were used as received. Gold substrates were prepared by evaporation onto silicon wafer at a rate of 0.5 Å ∙ s-1 up to reach a final thickness of 150 nm; silicon wafers were previously covered by a thin chrome layer (5 nm), i.e., a primer, that ensures an excellent gold adhesion23. Silicon nitride-gold (Si3N4–Au) patterned substrates were prepared by ordinary photolithography technique: first, a silicon nitride wafer was coated with a photoresist layer (S1813, at 4000 rpm, baked at 90°C for 4 min) and then exposed to UV light, placing above it a predesigned mask; the developed resist was thus covered by 50 nm of gold layer by using ebeam evaporator. As a result of the lift off process, a series of gold flat squares were obtained onto silicon nitride substrates. The designed mask consists of a matrix of 64 x 64 micrometric gold squares each of them measuring 20 x 20 µm2 covering a total area slightly larger than 7 mm2 in order to resemble the CMOS-MEA active area. Immediately before the use, all the substrates were cleaned with a piranha solution (3:1 (v/v) of 98% H2SO4 and 30% H2O2) for 15 min, Milli-Q water, anhydrous ethanol and finally dried under clean nitrogen flow at room temperature. (Caution! Piranha is a very corrosive solution and a vigorous oxidant therefore it was handled with extreme caution). AT functionalization of Au substrates (One-step functionalization). A set of chemically functionalized uniform gold surfaces was prepared by incubating gold substrates in a 1 mM aqueous solution of 11-amino-1undecanethiol hydrochloride (Sigma-Aldrich) and stored for approximately 24 h at room temperature to form a self-assembled monolayer (SAM). At the end of the incubation the samples were rinsed in Milli Q water and dried under nitrogen flow. A second set of samples was prepared by depositing on the gold surface microsized drops of AT arranged in a patterned structure resembling the electrode displacement of the CMOS-MEA; the patterning was performed by using a benchtop microprinter system (NanoEnabler)equipped with SPT-C60 R cantilevers (Bioforce, USA). Briefly, using the conditions detailed in Supporting Information (SI),multiple square spot arrays with constant pitch were deposited (spot dimension 25 ± 5µm, pitch 60 µm, as shown in the inset of Figure 1B). After the molecule deposition all the substrates were sterilized under UV light for 30 min and stored at room temperature in sterile environment before the neuronal adhesion test. PTES-AT functionalization of patterned Si3N4–Au substrates (Two-step functionalization). Si3N4–Au patterned substrates, prepared by photolithography as described, were soaked for 2 h at room temperature in ethanol containing propyltriethoxysilane (PTES - Sigma Aldrich) at a final concentration of 10 mM. The silanized

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substrates were thoroughly rinsed with ethanol, dried with a nitrogen flow and afterwards they were held at 70°C for 30 min in order to promote the formation of siloxane bond by eliminating water molecules. Then the functionalization of the gold with the AT was performed. Water Contact Angle measurements (WCA). To obtain a quick feedback about both functionalization steps, WCA measurements were carried out using a KSV CAM 200 instrument. The measurements were performed on a silicon nitride substrate half-covered with a gold layer; the samples were tested immediately after surface modifications. Five random and different spots on each slide side were analyzed and two slides for each sample were tested. The sessile drop method was employed using Milli-Q with a typical drop volume of 1 μL. All the WCA measurements were performed at equilibrium, 30 s after the droplet deposition. X-ray photoelectron spectroscopy (XPS). In order to verify the effectiveness of the functionalization process and to get more insights on the chemical nature of the neuron-substrate interface, X-ray photoelectron spectra were recorded on a Kratos Axis Ultra DLD spectrometer, using a mono-chromatic Al Kα source (15 kV, 20 mA). All the XPS measurements were carried out on a silicon nitride substrate half-covered with a gold layer; the measurements were performed after both functionalization with both steps (PTES and AT) molecules. Highresolution narrow scans were performed at constant pass energy of 10 eV and steps of 0.1 eV. The photoelectrons were detected at a take-off angle of Φ = 0° with respect to the surface normal. The pressure in the analysis chamber was maintained below 7∙10−9 Torr for data acquisition. The data were converted to VAMAS format and processed using CasaXPS software, version 2.3.15. The binding energy (BE) scale was internally referenced to the C 1s peak (BE for C-C = 284.8 eV). The spectra in N 1s and Si 2p regions were fitted with pseudo-Voigt profiles. Atomic Force Microscopy (AFM). The surface roughness of gold and silicon nitride areas was investigated by atomic force microscopy (AFM) before and after the functionalization steps. AFM images were performed using a Nanowizard II (JPK Instrument, Germany) in tapping mode, in air. Uncoated silicon cantilevers (PPP-NCH-50, NanoWorld, Switzerland) with typical resonance frequency of 300 KHz and with nominal tip radius of curvature value around 10 nm were used. Neuronal cultures. Culturing media and additional compounds were acquired from Gibco-Invitrogen. Primary hippocampal neurons were dissociated from E18 rat embryos following the protocol described in previous works.24,25 Successively, a 100 µL drop containing dissociated neurons at a nominal concentration of 500 cells/µL was plated on functionalized substrates as well as the APS-MEA chips. After 3 h, Neurobasal supplemented with 2% of B27, 1% of Glutamax was added. Samples were maintained in an incubator at 37°C in 5% of CO2. Considering the relatively low cell concentration and the consequently sparseness of the forming network, feeder tech-

niques were applied to sustain culture growth. For the functionalized substrates, four paraffin drops were deposited, in sterile conditions, at the bottom of multi-well plates (Corning, Lowell, MA, USA); the plates were then coated with 0.1 mg/mL Poly-D-Lysine (PDL) and seeded with hippocampal cultures at 1000 cells/µL concentration. Three hours after seeding, when cells adhered to the substrates, gold and silicon nitride-gold (Si3N4–Au) patterned substrates were transferred into the wells and placed on the paraffin drops with the cell culture side facing the feeder culture. In the case of APS-MEAs instead of feeder cultures, media was mixed 50% with conditioned one. For all the samples, 50% of the medium was changed every week. Immunofluorescence protocol and optical images. Cells were washed once in 1X Phosphate Buffered Saline (PBS) solution, fixed with 4% (w/v) paraformaldehyde at room temperature for 30 min and rinsed three times in 1X PBS. Fixed samples were permeabilized with 0.1% (v/v) Triton X-100 in 1X PBS for 15 min. Blocking solution (PBS 1X, 1% BSA, 5% FBS) was added for 45 min at room temperature to block nonspecific reactions. Chicken antiβTubulin III able to detect endogenous levels of neuron specific β-Tubulin III without cross-reaction with other beta tubulin isotypes and mouse anti-NeuN (Millipore, Billerica, MA, USA), were used as primary antibodies for staining, following the appropriate dilutions in blocking solution (respectively 1:500, 1:100). After incubation (3 h at room temperature), samples were washed and incubated with the secondary antibodies, (respectively, Alexa488 labeled goat anti-chicken antibody and Alexa647 labeled goat anti-mouse antibody) for 45 min at room temperature. Samples were mounted on glass microscope slides using Prolong anti-fade reagent containing DAPI (Invitrogen), and then stored at 4°C. Fluorescence images were acquired with a confocal inverted microscope (Nikon A1) equipped with a 60X oil immersion objective and with multiple laser lines. Three-channel images were acquired by exciting at 405, 561 and 633 nm and by collecting the emitted fluorescence using the suitable band pass filter. Scanning electron microscopy (SEM) imaging. Primary hippocampal neurons were fixed for 1 h in a solution of 1.2% glutaraldehyde (Sigma) in 0.1 M cacodylate buffer (pH = 7.4) at 4°C. After fixation, neurons were extensively washed in the same buffer, and postfixed for 1 h on ice in a solution of 1% osmium tetroxide (Sigma) in cacodylate buffer 0.1 M. After several washes in ice-cold Milli-Q water, fixed samples were rinsed for 5 min in increasing concentrations of filtered ice-cold ethanol (30%, 50%, 70%, 90% and 96%), followed by 2 × 15 min rinses with ice-cold 100% ethanol. The dehydration with ethanol was followed by gradual replacement with ice-cold hexamethyldisilazane (Sigma) that was allowed to evaporate in a fume hood overnight. Samples were finally coated with 10 nm Au/palladium and observed with a JEOL JSM-6490LA variable pressure scanning electron microscope, working in high vacuum mode at 30 kV. Electrophysiological validation of the PTES-AT functionalization onto gold-modified CMOS-MEA.

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The two-steps functionalization procedure (PTES-AT) was then tested on commercially available CMOS-MEA (3Brain.com). This monolithic chip, working in principles as a complementary metal oxide semiconductor (CMOS) camera but sensing cellular voltage potentials, provides an active area of 64 x 64 (4096) squared microelectrodes, with a recording size of 21 µm and a pitch of 42 µm. Each single electrode integrates an amplifier directly beneath the sensing area and can be sampled simultaneously with all the other electrodes at 7.022 kHz.26 Since this device is produced by standard CMOS technology the bare electrode is composed of aluminum alloys. To cover the electrode with a gold layer necessary for the functionalization steps, we utilized an electroless deposition technique by using a commercially available gold cyanide plating solution, as described by Berdondini et al.27 Primary dissociated hippocampal neurons were grown onto the CMOSMEAs for about 4 weeks as described in the previous section (cf. Neuronal cultures). Spontaneous electrical activity was recorded for 10 minutes at 18 and 25 DIVs to monitor the functional development of the network. Spike events (i.e. fast signal oscillation of about 1 msec duration) were detected to generate the raster plots of Figure 4 by using the Precise Timing Spike Detection (PTSD) algorithm described in Maccione et al.28 RESULTS AND DISCUSSION

Neural adhesion on AT functionalized Au substrates. The effect of specific chemical functionalization on adhesion and outgrowth of primary hippocampal neurons was investigated using chemically functionalized surfaces prepared by SAMs of amino terminated alkanethiolate on gold substrates by exploiting a wellestablished thiol-based chemistry;29–33 thiol-gold SAMs with various functional groups and different lengths have been well documented34 and by choosing the proper functional group it has been possible to tune surface adhesion properties. The first goal was to demonstrate the not trivial possibility of growing a neural network onto a thiolated gold surface, and, in particular, onto a patterned gold surface resembling the electrode surface of a CMOS-MEA, both for geometry and material. Gold samples were thus chemically functionalized using 11-amino-1-undecanethiol hydrochloride (referred to as AT-Au) either uniformly on the whole surface by dip coating technique or in a patterned geometry. Primary rat hippocampal neurons cultured on both functionalized gold substrates show an excellent adhesion property after 7 DIV giving rise also to neurite outgrowth. In particular, on the AT-Au substrates homogeneously functionalized by dip coating technique, neural networks develop randomly onto the whole surface uniformly coated with the positively charged amino groups (Figure 1A,), sign that the molecule of choice was satisfactory. This result is important in order to show adhesion and growth of delicate primary neurons on gold without the use of a common adhesion factor, such as poly-D-lysine and/or laminin, commonly used to coat MEA devices. The further test carried out on micropatterned gold was necessary to demonstrate that AT func-

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tionalization is effective in geometrically confined micrometric areas: neurons seeded onto the patterned substrates found favourable conditions for adhesion and growth; in particular, cell nuclei appeared to be placed exactly onto the AT spots, being arranged in a regular geometrical order. Moreover, the distance between adjacent spots is well below the threshold that hinders neuron connect-ability:35 neurons were able to connect to each other, with their dendrites, therefore, bypassing the not functionalized gold areas between the AT micrometric spots, giving rise to a rather ordered neural network, as clearly shown in confocal images in Figure 1B. We stress out however that this test was carried out as a proof of principle to evaluate neural survival in such an unusual configuration (somata confinement and no common adhesion factor). Given these encouraging results, and having the future perspective to functionalize the active surface of a conductive CMOS-MEA, we tested the AT functionalization onto a surface faithfully replicating the CMOS-MEA active area: a 64 x 64 matrix of micrometric gold squares reproduced on silicon nitride substrates (Si3N4–Au). Trying to avoid time-consuming microdropping technique on such a large number of electrodes, the patterned Si3N4–Au substrates were functionalized by dip coating in AT, supposing that only functionalized gold areas could offer a positive adhesion cue to neural cells. We noticed instead that the silicon nitride insulator surrounding the gold areas is moderately neuron-adhesive: in fact, at 7 DIV neural cells tend to adhere randomly on the AT/Si3N4–Au substrate leading to a indiscriminate adhesion both on gold squares and on the surrounding silicon nitride areas (see Figure S1 and S2 in Supporting Information). Therefore, a further chemical treatment able to prevent neural adhesion on silicon nitride insulators was required. Neural adhesion on PTES-AT functionalized Si3N4Au substrates. One of the most used approaches to prevent cells-substrate interaction is to expose “repulsive molecules” having specific cues able to avoid the interaction with membrane proteins. We therefore added a preliminary functionalization step through the introduction of a hydrophobic silane as “repulsive molecule” which passivates the silicon nitride insulators regions. In particular, Si3N4–Au substrates were first functionalized with propyltriethoxysilane (PTES) that was immobilized on silicon nitride regions via silanization; only after this step, the PTES functionalized substrates were dipped in a solution of AT molecules, to obtain PTES-AT functionalized Si3N4–Au substrates (PTES-AT/Si3N4–Au). Rat embryo hippocampal neurons were then plated on PTES-AT/ Si3N4–Au and their growth were investigated through optical microscopy techniques. At 7 DIV samples were fixed and then examined by both confocal and SEM for higher resolution inspection of neurite outgrowth on the functionalized areas. Immunofluorescence images at 7 DIV (Figure 1 C,D) show a well ordered neuron positioning; in particular, the cell soma are mainly placed on the gold squares, and neurons are well connected to neighbouring cells; this demonstrate the capability of axons to explore

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the surrounding environment feeling the adhesion cues provided by molecules placed on the next gold areas, separated by 20 µm, despite the presence of a molecule inhibiting the adhesion. SEM images (Figure 1 E,F) indicates the optimal coupling between single neuron and gold square; the zoomed area shows the whole connection between different squares on which are placed different neurons. Surface characterization of functionalized substrates. To have a rapid feedback about the resulting functionalized substrates, WCA measurements were realized. The measurements were performed on silicon nitride substrate half-covered with a gold layer on which both functionalization steps (PTES and AT) were consecutively performed. The reactions were indirectly verified by measuring the change of hydrophilicity before and after each step of the functionalization, i.e., after PTES and after PTES-AT functionalization. In particular, Figure 2 shows WCA measurements performed on gold and silicon nitride areas through sessile drop method; on gold the contact angle values were not significantly changed after the PTES treatment (Fig.2A,B), meaning that the silanization process does not affect the wettability of the metal region; instead, WCA on gold increases from 17° to 34° after the PTES-AT functionalization steps: the exposure to PTES does not hinder the AT reaction at the gold surface, and, eventually, the WCA values are kept in a well suitable range for cell adhesion purposes. Similarly, wettability measurements on the silicon nitride area were performed to prove the PTES–Si3N4 interaction. The change of the drop shape after the reaction with silane molecules is shown in the bottom of Figure 2: the panels clearly show the increase of the WCA from 14° up to 68° after the reaction with PTES molecules. This increase is due to the inert propyl groups exposed over the silicon nitride after the silanization that enhance the hydrophobic properties of the surface. Moreover, the WCA values detected after the PTES-AT steps (Figure 2F) confirm that the silicon nitride area is not affected by the subsequent treatment with AT. To investigate the chemical structure of the surfaces in contact with neurons, XPS characterization was carried out with the aim to demonstrate that both AT-Au and PTES -Si3N4 reactions took place and, at the same time, to ensure that the insulator passivation performed by the PTES functionalization did not affect the gold substrate. The data acquired on a PTES-AT functionalized substrate (Si3N4 wafer half covered with an Au layer) are compared in Figure 3 with those obtained on a bare one. On the Au side, the two-step functionalization had several effects. First of all, analyses on S 2p and N 1s regions (data reported in Figure 3B and 3C, respectively) show the appearance of new peaks that could be related to the presence of AT molecules. Indeed, the doublet of peaks centred at 162.1 and 163.3 eV36(Figure 3B) indicates the formation of thiolate (Au–S) bonds, while the broad peak centred between 398 and 403 eV (Figure 3C) can be reasonably attributed to the presence of amine groups; the line shape asymmetry suggests the occurrence of two sub-components centered at 400 ± 0.2 eV (green line) and 401.7 ± 0.2 eV

(red line) that could be respectively assigned to amine nitrogen (NH2)37–39 and protonated amine nitrogen (NH3+)40–43. The presence of both peaks suggests a double nature of the immobilized groups: a first neutral, related to the amine nitrogen groups at the end of alkyl chains stabilized after the thiol-gold reaction and a second one acidic, to correlate to the acidic nature of AT molecule, due to residual HCl. This behaviour represents a sign that the positive charge on amine groups was partially kept despite the several cleaning treatments. Also, the intensity of the Au 4f doublet, characteristic of Au0 (Au 4f7/2 component centered at 84 eV, see Figure 3A) appeared reduced after the functionalization process; following a standard approach,44,45 from the attenuation of the Au 4f spectra we obtained a layer thickness around 1.4 nm (see SI for details). This value is in good agreement with the current literature,46 and confirms the uniformity of the coverage. Concerning the Si3N4 side, the XPS analysis suggests that the AT-functionalization was not effective there, since there is no trace of thiol S and of amine N peaks on the data acquired over the S 2p and N 1s energy regions (see Figure 3E and 3F). The N 1s peak shown in Figure 3F is centred at 397.4± 0.2 eV, and it is typical of the nitride moiety of the substrate.47 The formation of a PTES layer on Si3N4 insulator region was confirmed by XPS analysis in Si 2p region (Figure 3D). The decrease of total integrated spectral intensity of the Si 2p peak, after the treatment with PTES, is an expected result due to the presence of an organic layer, which attenuates the photoelectrons emitted from the substrate. The intensity of Si 2p peak after the functionalization with silane molecules decreased at ≈ 90% of its initial value that corresponds to a layer thickness of ≈ 0.5 nm, as detailed in the SI. In addition to the silicon nitride peak at 101.6 eV, other two sub-components were detected at 99.4 eV and 103.2 eV (green and orange lines of Figure S4, see SI); whereas the peak at lower binding energy can be associated with Si0 of the substrate, the higher binding energy can be correlated with native silicon dioxide layer. After the PTES treatment a slight decrease of the signal associated with Si0 was noted, reasonably related to the presence of organic layer which attenuates the photoelectrons emitted from the substrate. At the same time, the component relative to the silicon dioxide was weakly increased: this behavior could be related to the fact that silicon contributions from silane molecules and from native silicon dioxide show very similar binding energy values.48 Finally, the roughness at the nanoscale of the surfaces (RMS) was checked by AFM before and after functionalization. The measured RMS values were 0.6 nm on Au, before and after PTES-AT treatment, while on Si3N4 a value of 0.5 nm was measured on the bare substrate and of 0.6 nm on the PTES-AT coated surfaces. From this further analysis we can claim that in our experiments the nanoscale roughness does not influence the neural adhesion since no significant change before and after the functionalization was detected (see Figure S5 in Supporting Information).

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Electrophysiological validation of the two-steps functionalization onto gold-modified CMOS-MEA. Spontaneous electrical activity of a hippocampal neuronal culture grown on the gold coated and functionalized CMOS-MEA was recorded up to four weeks. As shown in Figure 4, the dissociated culture formed a functionally active network expressing both spiking activity as well as synchronous network burst events. In particular, raster plots in Figure 4A show how the temporal dynamic of the burst events changes by comparing 10 minutes recording at 18 DIVs (left) with one at 25 DIVs (right). At 18 DIVs the network is rapidly growing increasing the number of connections and expressing frequent and short in time bursts. On the opposite at 25 DIVs, connectivity reached a stable and mature stage, providing a more complex dynamic resulting in less frequent bursts but more sustained for number of spikes and duration. This physiological behaviour is comparable with what can be recorded from networks that have been grown on CMOS-MEA coated with standard adhesion molecules, as shown for example in Figure S6. The increasing of burst duration and number of events can also be observed in Figure 4B by looking at the raw data of three illustrative channels. While at 18 DIVs signal amplitude is low and not sustained, at 25 DIVs spikes are higher (200-500 µVolt) and show a tonic and synchronous behaviour as for hippocampal cultures coated with polylysine (e.g. red inset of Figure 4B). No evident S/N ratio improvement has been noticed: the thickness of the two methods (two-step functionalization vs. polylysine) is rather comparable (about 1nm), so we cannot expect a tighter cell/electrode coupling to justify an increasing of signal amplitude. Finally in Figure 4C, it is possible to observe the spatial propagation of a burst event recorded from all the 4096 electrodes of the CMOSMEAs. The time lapse shows how the event arises from the bottom right side and then propagate through the entire array in about 300 msec.

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gold-modified active areas of commercial and custom post-processed CMOS-MEA leading to promising results: in particular, we were able to let a culture grow on the CMOS-MEA without the use of any adhesion molecules. Furthermore, by recording the electrical spontaneous activity of the neural network we demonstrated that the adhesion was excellent and stable up to four weeks and the culture was fully functional providing both spiking as well as bursting activity comparable with cultures coated by using standard molecules as polylysine. The results provided here clearly show the potential application of the proposed two-steps functionalization methodology: even if the entire procedure requires further investigations, it might be applied for the further development of these emerging devices, and represents therefore, a valid alternative to traditional adhesion factors in order to provide selective and specific neural-substrate adhesion.

CONCLUSIONS

The optimization of electrode-neuron couplings requires the development of adapted substrate functionalization methods for inhibiting inter-electrode neuronal localization and for promoting neuronal adhesion on electrode sites. Here, after selecting a suitable adhesion factor able to control neural placement on gold substrates with micrometer precision, we developed a functionalization technique able to lead neuronal adhesion onto model substrates based on silicon nitride and gold, which replicate the active area of a CMOS-MEA device providing thousands electrodes. The presented two-steps functionalization procedure consists in the introduction of a passivating layer of hydrophobic silane molecules (PTES) which constrain cell body exclusively onto gold surfaces that are subsequently modified with adhesion factor (AT). On model substrates we obtained well-ordered neural networks and a good interaction between neurons and gold regions even reaching the optimal coupling of a single neuron with single micrometric gold square. Moreover, this two-steps functionalization was transferred onto

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FIGURES

Figure 1. The top two panels show confocal immunofluorescent images at 7 DIV of hippocampal neurons plated on AT-Au substrates functionalized by dip coating technique (A), and patterned through the benchtop microdropper (B) at different magnification; inset of panel (B) shows the optical image of a spot array of AT deposited on gold by microdropper; scale bars 50 µm. The bottom four panels respectively illustrate confocal immunofluorescent images (C,D) and SEM images (E,F) at 7 DIV of hippocampal neurons plated on PTES-AT/Si3N4-Au functionalized substrates; scale bar 20 µm

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Figure 2. WCA measurements on gold and silicon nitride regions before and after functionalization steps. The change of the drop shape is correlated to the respective interactions: AT-Au (top three images), PTES-Si3N4 (bottom three images).

Figure 3. XPS spectra on Si3N4-Au substrates functionalized with PTES followed by AT (two-step functionalization); blue and black profiles respectively represent bare substrate and two-step functionalized substrate. AT-Au reaction is demonstrated by the analysis on Au 4f, S 2p and N 1s regions, respectively (A), (B) and (C) panels; the absence of the Si 2p peak on Au side (inset panel A) confirms that PTES molecule does not react with Au substrates. PTES immobilization on silicon nitride is confirmed by the analysis on Si 2p , panel (D). Panels (E) and (F) confirm that on Si3N4 side there is no trace of thiol S and of amine N peaks on the data acquired over the S 2p and N 1s energy regions.

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Figure 4. Spontaneous neuronal activity at 18 (left) and 25 (right) Days in vitro (DIV) recorded from a hippocampal culture grown on a chemically functionalized CMOS-MEA device; (A) raster plot representation of spiking events occurring during 10 minutes basal recording (X axis) on about 200 active electrodes (Y axis, each dot represents a detected spike). Electrical activity shows different temporal dynamic in the two recordings due to culture maturation; it can be appreciated a shift from tonic short synchronous events (left) to long lasting rhythmic bursts (right) as expected during culture maturation; (B) raw data plots of the electrical activity recorded from 3 electrodes during a synchronous burst event. Signals recorded at 25 DIV show stronger amplitudes and an increased synchronization than at 18 DIV and are comparable in term of amplitudes (200-500 uV) and dynamic with signals acquired on CMOS-MEA coated with standard adhesion factor (i.e. polylysine 0.1 mg/ml) as shown in the red inset on the right; (C) time lapse of the spatial propagation of a burst event recorded at 25 DIV. The activity recorded from the 64 x 64 electrode array is represented in a false color map where each pixel represents the maximal variation of the signal for each microelectrode over a time bin of 20 msec.

ASSOCIATED CONTENT Supporting Information. Confocal and optical images of neurons plated on AT functionalized Si3N4-Au substrate at 7 DIVs; optical image at 21 DIV of hippocampal neurons plated on PTES-AT functionalized substrate. Detail of XPS in Si 2p region, AFM images and RMS surface roughness of Si3N4-Au two-step functionalized substrate. Example of

spontaneous neuronal activity recorded from a CMOS-MEA device coated with standard adhesion molecules. This material is available free of charge via the Internet at http://pubs.acs.org.

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Page 10 of 12 Guidance Techniques to Create a Neuron Biochip. Optoelectron. Lett. 2008, 4 (5), 387–390.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

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Staii, C.; Viesselmann, C.; Ballweg, J.; Shi, L.; Liu, G. Y.; Williams, J. C.; Dent, E. W.; Coppersmith, S. N.; Eriksson, M. A. Positioning and Guidance of Neurons on Gold Surfaces by Directed Assembly of Proteins Using Atomic Force Microscopy. Biomaterials 2009, 30 (20), 3397–3404.

Present Addresses †

Université de Liège, Faculty of Science, Department of Chemistry, Bât. B6A Nanochemistry and Molecular System, Quartier Agora, allée du six Août 17B 4000 Liège, Belgium.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors would like to thank Marina Nanni for providing neural cells, Marco Scotto for the assistance with confocal microscopy analysis, Roberta Ruffilli for SEM imaging, Marco Leoncini and Eliana Rondanina for the substrates preparation.

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