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Inkjet-Printed Bio-Functional Thermo-Plasmonic Interfaces for Patterned Neuromodulation Hongki Kang, Gu-Haeng Lee, Hyunjun Jung, Jee Woong Lee, and Yoonkey Nam ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b06617 • Publication Date (Web): 05 Feb 2018 Downloaded from http://pubs.acs.org on February 5, 2018

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Inkjet-Printed Bio-Functional Thermo-Plasmonic Interfaces for Patterned Neuromodulation

Hongki Kang, Gu-Haeng Lee, Hyunjun Jung, Jee Woong Lee and Yoonkey Nam* Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Republic of Korea

KEYWORDS (5-7 keywords) Thermo-plasmonics, inkjet printing, nanoparticle assembly, polyelectrolyte layer-by-layer coating, contact line pinning, neuromodulation, microelectrode array

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ABSTRACT

Localized heat generation by thermo-plasmonic effect of metal nanoparticles has great potential in biomedical engineering research. Precise patterning of the nanoparticles using inkjet printing can enable the application of thermo-plasmonic effect in a well-controlled way (shape and intensity). However, universally applicable inkjet printing process that allows good control in patterning and assembly of nanoparticles with good biocompatibility is missing. Here we developed an inkjet printing based bio-functional thermo-plasmonic interfaces that can modulate biological activities. We found that inkjet printing of plasmonic nanoparticles on polyelectrolyte layer-by-layer substrate coating enables high quality biocompatible thermo-plasmonic interfaces across various substrates (rigid/flexible, hydrophobic/hydrophilic) by induced contact line pinning and electrostatically assisted nanoparticle assembly. We experimentally confirmed that the generated heat from the inkjet-printed thermo-plasmonic patterns can be applied in micron resolution over large area. Lastly, we demonstrated that the patterned thermo-plasmonic effect from the inkjet-printed gold nanorods can selectively modulate neuronal network activities. This inkjet printing process therefore can be a universal method for bio-functional thermo-plasmonic interfaces in various bioengineering applications.


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Localized heat generation by nanoparticles has emerged as a promising thermal interface platform in nanomedicine. Most common application is the treatment of cancer cells by localized heating, (e.g., photothermal therapy1–3 or hyperthermia4). More advanced and promising application is the modulation of brain activity. Brain cells, often controlled by electrical stimulation, can be genetically modified to be thermo-senstive5 or have been reported to be thermo-responsive6–10 so that neural activity could be precisely controlled by heat. Among various ways to deliver heat, thermo-plasmonic nanoparticles—metal nanoparticles generating heat in nanoscale from significantly enhanced light absorption at particular wavelength by localized surface plasmons—have gained strong attention due to their versatile functionalities and matured synthesis methods.11 Plasmonic nanoparticles, tuned for absorbing near infrared (NIR) light, are especially a good candidate as heat generation can be remotely controlled by optical interfaces, which eliminates many complicated problems with wired technologies. In order to apply the thermo-plasmonic effect in more advanced and controlled manner to various biological systems (i.e. patterned thermo-biological functions), suitable precision nanoparticle patterning methods must be developed. Large-area applicability with cellular level spatial resolution, precise nanoparticle density control, uniform assembly of the nanoparticles and applicability to various substrates are all desirable specifications of the patterning methods. There have been various top-down or bottom-up approaches to pattern plasmonic nanoparticles or metal films to create plasmonic interfaces12: e-beam13,14, nanosphere15, colloidal16 and dippen17 lithography techniques; soft lithography including micro-contact printing variants18,19; and inkjet printing20–22. Among those techniques, inkjet printing is the most versatile tool as it is easy to scale-up the patterning area while maintaining high spatial resolution with minimal cost and


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effort. Inkjet printing can have single cell resolution with easily customizable pattern design.23–26 It can effortlessly vary the areal density of the printed particles, allowing precise control of nanoparticle effects in shape and intensity. These benefits are in line with the technology trends in bio-printing.27 However, previous works utilizing inkjet printing for thermo-plasmonic interface was limited in terms of spatial resolution, printing scale, substrate choices, printed image quality, and bio-functionalities.21,22 Precision nanoparticle inkjet printing especially requires good control on dewetting of the droplets and on the assembly of nanoparticles during solvent drying.28–31 Therefore, biocompatible nanoparticle inks and printing substrate surface treatment need to be developed to universally fabricate high quality thermo-plasmonic interfaces with good controllability, high-resolution and bio-functionality for various bioengineering applications. Here, we developed a universally applicable inkjet printing process for bio-functional thermo-plasmonic interfaces. We found that biocompatible polyelectrolyte layer-by-layer (LbL) coating successfully transforms any non-printable substrate (e.g. freely moving contact lines or hydrophobic surface) into nanoparticle printable substrates with excellent pattern fidelity by inducing contact line pinning of the nanoparticle ink while achieving uniform assembly. Using the optimized plasmonic nanoparticle inkjet printing process, we show that thermo-plasmonic effect can be efficiently patterned on various substrates in micron scale resolution with varying intensity over large area. As an experimental validation of bio-functionality, we demonstrate that the heat patterns generated by the inkjet-printed thermo-plasmonic nanoparticles can selectively modulate neuronal network activities. In Figure 1a and b, schematic illustrations of the inkjetprinted thermo-plasmonic interface for patterned neuromodulation and the overall inkjet printing process developed in this work are presented.


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RESULTS Precise patterning of inkjet-printed gold nanorods enabled by polyelectrolyte LbL coating In order to achieve high quality inkjet printing process with gold nanorod ink, we attempted to functionalize both printing substrate and GNR surfaces. As shown in Figure 1a, printing substrates were coated with LbL coating and the GNR inks were immobilized via electrostatic interaction between the ink and substrate surfaces. These functionalization processes were important to obtain the contact line pinning (CLP) and high contact angle. To precisely control the shape of the printed patterns, CLP is required so that the printed droplet holds its initial contact line boundary during drying of solvent or merging with nearby droplets as described in Figure 1b.32,33 When the GNR ink droplet was printed on bare surfaces of four different model substrates (glass, PECVD silicon nitride, silicon oxide/nitride/oxide or fluorinated ethylene–propylene membrane), CLP did not occur regardless of the surface hydrophobicity of the substrates, and the droplet dewetted the substrate as the solvent dried, eventually leaving much smaller footprint than the initially landed droplet contact area (Figure 1c). When droplets were triggered to merge, the droplets were dislocated to become a new shape, and such surface dewetting behaviors did not allow forming more complex patterns such as lines and films (Figure 1d, top). When LbL coating was applied to these substrates, however, we observed that contact line pinning was induced on all the substrates regardless of which polarity the coating terminates the surface. Then, the printed droplets always pinned and maintained the original footprints even when the droplets merged. Thus, it became possible to precisely control the shape of patterned nanoparticles. Figure 1d shows that it was possible to print line or ‘L’ shapes with CLP. It has been understood that the chemical or physical inhomogeneity induces the contact line pinning behavior.34 We speculated that the induced CLP by the polyelectrolyte


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coating could be due to the increased surface roughness35 and also possibly due to the attraction of polar solvent such as water to the polar LbL substrates. While printed pattern fidelity was significantly improved by the induced CLP regardless of the LbL polarity, positively charged LbL coating was more advantageous in obtaining the high printing resolution. Equilibrium contact angle (θeq) on the printing substrate defines spatial resolution of printed features as it determines the diameter of landed droplets in spherical cap shape. In general, it is well known that higher equilibrium contact angle leads to higher resolution, but higher than 90º is not desired for printing more complicated shapes than circles (e.g. lines).26,36–38 Based on our equilibrium contact angle measurements over various printing substrates (as shown in Figure 1e), we observed that positively charged polyelectrolyte, poly(allylamine hydrochloride), terminated LbL coating (PAH+) showed more desirable contact angle values for inkjet-printed pattern generation. For originally hydrophobic substrates such as Nitride 2 surface, PAH+ showed θeq smaller than 90º, which avoids the instability of printed lines.26,36–38 For other substrates, θeq values nearly unchanged (or at most slightly decreased) from the bare substrates, thus not deteriorating the printing resolution much in the presence of the LbL coating. On the other hand, negatively charged polyelectrolyte, poly(4-styrenesulfonic acid) coating (PSS-) always showed the smallest θeq, resulting in poorest printed droplet resolution. Thermo-plasmonic effect control by inkjet printing We observed that the electrostatic force between gold nanorod (GNR) and LbL coating improved the uniform assembly of the nanorods after drying, which was critical for obtaining reproducible localized surface plasmon resonance (LSPR) absorption peak and uniformly


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distributed thermo-plasmonic effect in nanoscale. Owing to the Marangoni flow implemented in our co-solvent ink system (see Methods section for details), nanorods were expected to circulate within the droplet as the solvent dries. With the presence of circulatory flow and pinned contact line boundary, the nanorods must be favorably attracted to the substrate surface. As shown in Figure 2a, when negatively charged GNRs were printed on positively terminated LbL coating surface (PAH+), the dried GNR patterns showed uniform nanorod assembly without coffee-ring effect within the printed pattern boundary. On the other hand, when the nanorods and the LbL coated substrates had the same surface charge polarities, the nanorods were not uniformly distributed as shown in Figure 2b (either significant coffee ring (left) or incomplete inner surface coverage (right)) as the nanorods were repelled from the substrate surface. As confirmed with SEM images (Figure 2c – 2e), the electrostatically attracted gold nanorods were assembled uniformly from the center to the edge of the printed dot without significant agglomeration or coffee-ring. In addition, the negatively charged printed GNRs on the PAH+ surface showed good surface immobilization after washing with water whereas GNRs on bare substrate were significantly detached from the surface after wash (see Supporting information Figure S1). Printing negatively charged gold nanorods on positively charged PAH+ coated substrates, therefore, enabled both precise nanorod ink patterning in high resolution and uniform nanorod assembly with more efficient thermo-plasmonic effect (47% higher than the identically printed bare substrate as shown in Supporting information Figure S1b). Next, we investigated how we can precisely control the intensity of thermo-plasmonic effect using inkjet printing. With inkjet printing, GNR areal density and thus the intensity of thermo-plasmonic effect could be easily varied by either changing the number of droplets or the concentration of GNR ink. We printed arrays of GNR patterns on the PAH+ LbL coated


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substrates with high surface coverage for efficient thermo-plasmonic effect (overall size of 5 mm by 9 mm, Figure 2f) with different particle areal densities by varying the number and the concentration of printed droplets. The light absorbance of the printed substrates in NIR range monotonically increased with increased GNR ink areal density (Figure 2g), and the temperature changes were linearly proportional to the printed GNR ink areal densities under the same laser intensity (Figure 2h). These data confirmed that the inkjet printing could precisely control the amount of thermo-plasmonic effect in large scale. Inkjet-printed micro thermo-plasmonic (µTP) heater We examined whether the inkjet-printed micro patterns of GNRs could be used as microthermo-plasmonic (µTP) heater for locally confining the heat transiently. When NIR light was illuminated to cover an inkjet-printed GNR pattern (Figure 3a), the GNR pattern acted as a micron-scale localized thermo-plasmonic heater. The 1-dimensional and 2-dimensional temperature change profiles of the µTP heater in response to the illuminated light on/off are shown in Figure 3b and 3c. First, the µTP heat source was quickly established as described from step #1 to #2. Within a few tens of milliseconds after the laser was illuminated, Gaussian profile temperature change with the effective diameter same as the size of the GNR pattern was observed. The intensity of the Gaussian temperature profile quickly grew and reached a steadystate condition in less than 300 msec (step #3). The steady-state heat source was approximately three times wider than the diameter of the printed GNR pattern. After reaching the steady-state, while maintaining the Gaussian spatial temperature profile, the µTP heater started to slowly heat up the surrounding solution equally. From step #3 to #4, the temperature increased with the same amount throughout the entire image area. After the light was turned off (from step #5 to #6), the micro heat source quickly disappeared within a few tens of milliseconds. The heated surrounding


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solution slowly cooled down afterwards (step #6 to #7). The heat generation recording is available online as Supporting information movie 1. This observation confirmed that the µTP heat source was locally confined on the printed GNR pattern. With very short time duration of light exposure (i.e. tens of msec), the micro heat source was only as big as the printed GNR pattern.11 Even with much longer exposure time (i.e. > 300 msec), the micro heat source was still only three times wider than the printed GNR pattern. When multiple µTP heater patterns were located with distance, these µTP heaters could operate individually as shown in Figure 3d and 3e. The 50-µm diameter inkjet-printed µTP heaters in Figure 3d operated as 150-µm wide micro heaters after reaching steady-state condition. When the µTP heaters were placed even closer, the heat sources merged and acted nearly as a single source (see supporting information Figure S2). Using the Gaussian spatial profile and transient behavior of a single µTP heat source, the behavior of multiple sources could be precisely estimated by linear superposition of the single heat source data (see Supporting information movie 2). Printed large-area thermo-plasmonic image Another benefit of using inkjet printing over other patterning techniques is varying the printed nanorod areal density over large area with easily modifiable digital input image. In this way, we can selectively generate heat with different degree of intensity over large area with a simple light illumination. As a demonstration of this capability, we inkjet-printed multi-level thermo-plasmonic GNR images on PAH+ LbL coated substrates using digital bitmap input image files as shown in Figure 4. An exemplary thermo-plasmonic image on a glass coverslip (Glass 1) with high spatial resolution (564 dots per inch, pattern size: 45-µm) confirmed that the multi-levels of GNR density (the number of density levels: 3) in the thermo-plasmonic image are clearly distinguishable under dark-field microscopy as shown in Figure 4a. Another example is a


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larger thermo-plasmonic portrait image (GNR density level: 5, input image in Figure 4b) on a 20-µm thick flexible fluorinated ethylene–propylene substrate (FEP membrane) (4.8 cm big as shown in Figure 4c). The FEP membrane was hydrophobic (equilibrium contact angle of 97 ° for the gold nanorod ink without CLP), but through PAH+ LbL coating, the equilibrium contact angle was decreased to 47 ° with CLP induced. Due to the optical characteristics of GNRs, the printed thermo-plasmonic image was quite transparent under visible light range. Upon NIR laser illumination on the printed GNR image (Figure 4c) on the flexible FEP membrane, an identical thermal image was generated as monitored by an infrared camera (Figure 4d and 4e). Though the resolution of the captured thermal image was poorer than the printed GNR image due to the low spatial resolution of the IR camera (170 µm at best), it could be readily confirmed visually that the printed thermo-plasmonic image could generate precisely defined thermal image pattern over large area. Selective neuromodulation using printed thermo-plasmonic patterns In order to validate the bio-functionality of the printed thermo-plasmonic interface, we used in vitro neuronal cultures to attempt to selectively modulate the electrical activity of the cells by patterned photo-thermal effect. It has been reported that thermal effect could either excite or inhibit neural activity in vivo or in vitro.

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For in vitro, GNR patterns can be

directly printed on cell culture substrates in order to selectively apply heat. Therefore, we demonstrated selective neuronal activity modulation on microelectrode array (MEA) chips with thermo-plasmonic patterns inkjet-printed using the LbL coating. As shown in Figure 5a, µTP heaters were inkjet-printed on a part of PAH+ LbL coated MEA channel areas (top right corner; 500-µm square). The GNR printed MEAs showed good


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extracellular field potential recording capabilities (see supporting information Figure S4). After 12-hour of poly-D-lysine coating on the printed MEA chip, hippocampal neurons were seeded and cultivated for up to 5 weeks. There was no cellular preference in terms of adhesion and neurite growth between the GNR printed domain and non-printed domain (see Figure 5a, supporting information Figure S5 and S6), which also indicates that the biocompatibility of the printing process, qualitatively. Upon the wide-field illumination of NIR laser on the cultured neuronal network (11 DIV), we observed the inhibition of neuronal activity only around the GNR printed domain. When NIR laser with 138 mW/mm2 power density was illuminated, the spiking activities within or near the GNR area were nearly 100% suppressed (Figure 5b). On the other hand, spiking activity distant from the GNR printed domain was not affected (Figure 5c). In the case of control condition (NIR power density of 26 mW/mm2), there was no significant activity change from baseline level near or far from GNR patterned domain. The localized suppression of neural activity was consistent with other studies that induced the selective neural suppression using optical instrumentation.40 Next, we further designed the thermo-plasmonic interfaces such that we can apply the partial thermal stimulation to a highly synchronized neural network through GNR µTP heater patterns while we monitor and analyze the network synchrony response behavior at the same time. A neuronal network was prepared by cultivating hippocampal neurons for 15 days on an identically prepared LbL coated MEA chip with a GNR pattern (400 µm wide, 2 mm long) printed only at the center of the cell growth and recording area such that only the partial area of the entire channel recording area (2 mm × 2 mm) can be thermally stimulated. The central GNR printing patterns divide the entire recording area into two sub-domains (left and right) where heat is not directly generated (Figure 6a). We then applied wide-field NIR light illumination when the


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cultured neuronal network showed network-wide synchronized bursting activity as shown in the spike raster plot (control, 26 mW/mm2) in Figure 6b. When the illuminated light was weak (26 mW/mm2, control), there was no significant change in the synchronized activity. However, when NIR light power was increased to fully suppress the individual electrode activities (138 mW/mm2, as confirmed in the previous experiment), the neuronal activities in central GNR domain was almost completely reduced (mean ± SEM: -93.74 ± 2.32 %, n=6) (Figure 6b, c). On the two non-GNR domains (left and right), we also observed that many of the active channels showed reduced spike rates (left: -52.98± 30.07 %, n=7; right: -60.11 ± 13.77 %, n=8) upon NIR illumination possibly due to diffused heat from the central GNR domain or due to the completely suppressed central GNR domain neurons that could cause the suppression of the activities of the non-GNR domain neurons (Figure 6c). However, we clearly observed decent number of highly active channels within the non-GNR domains upon the illumination. Some channels in the nonGNR domains even showed increased spike rates upon the illumination. These changes in turn resulted in temporary transition in synchronization of overall network. During the NIR illumination, the network occasionally showed either partially or fully synchronized activities whereas fully synchronized strong burst activities were observed during NIR off. (Figure 6b, d). Figure 6e shows cross-correlation coefficient matrices and connectivity maps that describe the degree of synchrony between active electrodes in 138 mW/mm2 condition. From these graphs, clear transition of network synchrony was observed as we apply NIR light. When the NIR is off, the entire network is strongly correlated. However, when the NIR is turned on, only a subset of the electrodes maintained the correlation. We also quantified the degree of overall network synchrony by the mean values of the correlation coefficients.41 As displayed in Figure 6f and Figure S7, the overall network synchrony was reduced as the NIR stimulation is on, and the


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transition in synchrony was temporary and reversible. Unlike the control experiment showing no change, we confirmed that the overall synchrony transition upon light was statistically significant (Figure 6g). These results confirmed that the inkjet-printed µTP heaters allow us to precisely apply thermal stimulation to only a part of highly synchronized neural network without affecting the network analysis capability in the multi-channel microelectrode array system. The observed changes in the network characteristics upon light illumination was temporary as the network activity was quickly recovered to fully synchronized mode as soon as the light stimulation was removed.

DISCUSSION The successful implementation of the inkjet printing of thermo-plasmonic patterns was possible by simple but versatile layer-by-layer coating of biocompatible polyelectrolytes. This LbL coating method provides significant advantages over other approaches that had been reported for enhancing printability such as thick polymer spin coating or surface roughening by etching or grinding42–44. The LbL coating is applicable to larger substrates in any form, and compatible for batch process at low cost. The coated layer thickness is only a few tens of nanometer thick at most.45 Thus, structural characteristics of the underlying surface (e.g. micromachined surfaces) can be maintained. In addition, the LbL coating is also suitable for biological system studies. It has been shown that the electrostatic polarity of the LbL coated layer has been successfully used for various types of cell culture and immobilization of nanoparticles in biological media as we also demonstrated in our work.35,46–48 While other thin surface coating methods such as self-assembled monolayers are often used for cell


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immobilization or functionalized nanoparticle assembly, the self-assembled monolayer leads to chemically and physically homogeneous surface, ending up with very small contact angle hysteresis. The small contact angle hysteresis (in other words, no contact line pinning) is not suitable for printing based patterning.26,49 Therefore, the LbL coating we introduced for precision nanoparticle patterning along with excellent biocompatibility has a great potential in biofunctional nanoparticle based bioengineering research. Our thermo-plasmonic patterns can provide multi-site differential focal stimulation which is one of the important requirements for neuromodulation technology. As we have shown in Figure 2 and 3, our patterning platform can generate focal heat patterns in tens to hundreds of micrometer scale by combining single or multiple micro thermo-plasmonic heaters. As our largescale thermal patterning implies (Figure 4), one can also generate differential heat level in space by controlling the GNR areal density, which will induce different inhibition effect. More complex inhibitory stimulus patterns such as thermal gradient patterns or hollow geometries at multiple locations can be easily designed through similar process. According to our cell culture experiments (Figure 5 and 6), patterned thermo-plasmonic stimulation can not only induce localized inhibition, but also modulate the connectivity. It might be possible to find optimal patterns for large-scale neural networks, which would be conducive to the treatment of epileptic neural activity. As a translational approach for in vivo or clinical applications, our platform can be easily integrated with transparent and implantable neural interface devices. Because the patterns of GNRs can be printed on flexible substrates as shown in Figure 4, we can fabricate the patterned thermo-plasmonic platform onto flexible, transparent microelectrodes such as graphene electrodes50 or implantable, transparent substrates such as elastomeric silicone substrates51 or


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fiber optic cables for optogenetics.52 With these integration, we can precisely apply patterned heat stimulation to the implanted region while simultaneously recording neural signals or optogenetically modulating neural activities. In addition to the multi-functionality, we can provide easy customization. By the nature of printing, customized thermal stimulation patterns for each subjects or patients can be easily applied. We, therefore, believe that the patterned neuromodulation using our platform can open up various opportunities in current neural engineering research. CONCLUSIONS We developed inkjet printing process for high quality bio-functional thermo-plasmonic interfaces using biocompatible gold nanorods and polyelectrolyte layer-by-layer coating. The induced contact line pinning of aqueous gold nanorod ink by LbL coating on various printing substrates, and uniform nanorod assembly without coffee-ring effect using the electrostatic attraction led to maximized thermo-plasmonic effect of printed GNR patterns with excellent pattern fidelity. With this printing process, we show that thermo-plasmonic functions can be freely patterned on any substrate. Depending on applications, we can generate locally confined micron-scale heat, or can collectively generate thermal pattern with gradient over large area. We then demonstrated that hippocampal neuronal network can be cultured on the bio-functional thermo-plasmonic interface, and inkjet-printed µTP GNR heaters can be used to selectively suppress the neuronal activities without damaging the cultured neuronal network. This could be used to either turn off the hyperactive area or selectively isolate malfunctioning neurons from the entire network by disconnecting synchronized network activity. While only thermo-plasmonic effect of the inkjet-printed gold nanorods is demonstrated in a biological system, the simplicity of this process with good cell culture capability35,46–48 allows that this printing process can be a


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universal method for functional nanoparticle patterning in various bioengineering applications. We therefore believe it can also open up other functional interfaces using nanoparticles.

METHODS Gold nanorod ink synthesis Gold nanorod printing ink was synthesized based on a seed mediated method.53 Firstly, 5 ml of 0.5 mM Tetrachloroauric(III) acid (HAuCl4) (520918, Aldrich), 5 ml of 0.2 M cetyltrimethylammonium bromide (CTAB) (H6269, Sigma), 600 µl of ice-cold 0.01 M sodium borohydride (NaBH4) (71321, Fluka) were mixed in deionized water during ultra-sonication for 4 min at 26˚C. The seed solution was left at room temperature for 2 hours. 12 µl of seed solution was mixed with 5 ml of 0.2 M CTAB, 5 ml of 1 mM HAuCl4, 250 µl of 4 mM silver nitrate (AgNO3) (209139, Sigma-Aldrich), and 70 µl of 78.84 mM L-Ascorbic acid (A5960, Sigma) to grow the seed into rod shape. After that, the solution was kept at room temperature until the longitudinal absorption peak was observed around 800 nm. The GNRs were then washed with deionized water using centrifuge. For negatively charged nanorod inks, the GNR surface was coated with methoxyl polyethylene glycol thiol (mPEG-SH) (PG1-TH-5k, Nanocs) for at least 12 hours at room temperature. For positively charged nanorod inks, the GNR surface was coated with amine polyethylene glycol thiol (NH2-PEG-SH) (PG2-AMTH-5k, Nanocs) for at least 12 hours at room temperature. After the PEG coating, GNR solution was concentrated using centrifuge. Final ink concentration was characterized as the maximum peak absorbance value in


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optical density (OD) as high as 288 OD. GNR ink used throughout this study showed maximum absorbance peak around 785 nm in water and zeta potential of -24.5 mV for negatively charged nanorods and +24.7 mV for positively charged ones (Zetasizer Nano ZS, Malvern). See the supporting information Figure S8 for more details. For stable piezoelectric inkjet jetting of the aqueous nanoparticle solution, ink rheology must be optimized (e.g. capillary number and Weber number).26,54 Without adding any kind of surfactants which could affect biocompatibility, we only added ethylene glycol as a co-solvent to increase viscosity to avoid satellite droplet formation. By adding at least 50% of ethylene glycol (324558, Sigma-Aldrich), ink viscosity was increased over 5 cPs, and other dimensionless fluid quantities were tuned to move toward experimentally defined stable printing zone in other works26,54 (see Supporting information Figure S9 for more details). In addition to the ink jettability, ethylene glycol with higher boiling point than water but lower surface tension is also known to induce Marangoni flow to help solvent recirculation to suppress coffee-ring effect. Inkjet printer system and printing process An inkjet printing system (UJ200MF, Unijet) with piezoelectric inkjet nozzles (MJ-AT01 series, MicroFab) of different orifice sizes (30 – 50 µm) was used in this work. Ink volume was ranged from 5 pL to 22 pL with minimum jetting velocity higher than 1.5 m/s. Printing GNR ink was filtered (5 µm pore size) before loading in order to avoid nozzle clogging. All printing was done in a clean room facility at temperature below 40 °C. GNR ink was printed directly on the LbL coated surface without any pre-treatment. An input image file of a portrait of Siberian tiger (also known as Korean tiger) that we used in this work was purchased from Getty Images (http://www.gettyimages.com/) with


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royalty-free license (Rüdiger Katterwe / EyeEm / Getty Images) and was used after modification for color-scale simplification and file-type conversion to bitmap type. Absorbance analysis, printed ink areal density Absorbance of the synthesized GNR ink and the printed substrates was measured using a spectrometer covering both visible and near-IR range (350 – 1000 nm; USB4000-VIS-NIR-ES with halogen light source HL-2000, Ocean Optics). Pure solvents without GNRs or LbL coated substrates without GNRs were used as reference. Printed GNR ink areal density was characterized using the absorbance of the printed ink (optical density, OD) as the mass loading of the GNRs in the solution was difficult to control or characterize. For example, when 10 pL of 180 OD ink was printed within 60 µm × 60 µm area, the areal density was 0.5 OD×pL/µm2. Layer-by-layer polyelectrolyte coating and substrate surface analysis We used the same polyelectrolyte layer-by-layer (LbL) coating method we briefly introduced with preliminary data.55 10 mg/mL of positive polyelectrolyte, poly(allylamine hydrochloride) (PAH) (283215, Aldrich), and negative polyelectrolyte poly(4-styrenesulfonic acid) (PSS) (561258, Aldrich), were dissolved in 10 mM NaCl aqueous solution, respectively. Printing substrates were dipped into each solution for 5 min alternatively with deionized water washing in-between. At least 5 bilayers were deposited before the final layer deposition. The coated layer thickness was expected to be only a few tens of nanometer thick at most.45 Thus, structural characteristics of the underlying surface could be maintained. Contact angles on all the substrates were measured using a sessile drop method (EasyDrop FM40; Kruss, Germany). In order to determine if the contact line was pinned, the


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contact angle and contact area of the sessile droplets were repeatedly recorded as the droplets dried. Printing substrates In order to validate the applicability of the printing process with LbL coating, various substrates were tested. The following are the list of substrates used in this study. Glass 1: microscope glass coverslip (0101030, 18 mm × 18 mm, Marienfeld-Superior), Glass 2: sodalime glass wafer (Namkang, Seongnam, Korea), Nitride 1: 500-nm thick plasma-enhanced chemical vapor deposition (PECVD) silicon nitride deposited on sodalime glass wafer, Nitride 2: 500-nm thick low pressure chemical vapor deposition (LPCVD) silicon nitride on glass (60MEA200/30IR-ITO-gr, Multi Channel Systems), ONO: PECVD silicon dioxide 100 nm / PECVD silicon nitride 150 nm / PECVD silicon dioxide 100 nm on sodalime glass wafer. Nitride 1, 2 and ONO substrates were prepared from National Nanofabrication Center (Daejeon, Korea). Gold: 100-nm thick gold film was e-beam deposited on a sodalime glass wafer with 20nm thick titanium layer as adhesion layer. FEP membrane: 20-µm thick transparent and hydrophobic fluorinated ethylene–propylene flexible substrate often used as selective gas permeable cell culture chamber lids (O2 and CO2 permeable, but water impermeable).56 Dark field microscopy Inkjet-printed gold nanorod patterns on various substrates were mainly characterized using dark field microscopy (U-DCD darkfield condenser, Olympus) in an inverted microscope (IX71, Olympus). Compared to other metal nanoparticle inks often used for conductive electrodes, the nanoparticle concentration required for thermo-plasmonics was much lower to identify under typical bright field microscopy. Dark-field microscopy images of printed GNRs


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were used to analyze printing results with better contrast. Cell images were obtained using phase-contrast microscopy (IX71, Olympus). Near infrared laser system Two different NIR laser systems were used in this work. One was 785-nm continuous wave laser (450 mW, B&W Tek) that directly illuminated samples (maximum power density up to 24 mW/mm2 with 5-mm diameter illumination areas). The other was 808-nm continuous wave mode laser (4 W, Laserlab, Anyang, Korea) integrated with a custom-built digital micro-mirror device (DMD, DLP3000 light crafter module, Texas Instruments) based light illumination system.57 Though the DMD-based system was built originally for patterned light illumination application, in this work, it was only used for non-patterned, whole illumination mode when higher NIR power density more than 24 mW/mm2 was needed. For 808-nm laser based illumination conditions, actual laser output power and the illumination area are listed herein (with the DMD system unless noted otherwise): (4.23 mW/mm2: about 4 W, about 35-cm diameter, no DMD; 26 mW/mm2: 54.5 mW, 1.65-mm diameter; 138 mW/mm2: 295 mW, 1.65mm; 1.16 W/mm2: 658 mW, 850-µm diameter; 1.38 W/mm2: 125 mW, 340-µm diameter) Temperature measurement For the large-scale printing test, temperature change measurement (i.e. millimeters or larger) was carried out with handheld infrared cameras (E40 and A655SC, FLIR), and the data were analyzed using MATLAB (Mathworks). Maximum spatial resolution was at best several hundreds of µm. For the micro-scale temperature change measurement (used in Figure 4), fluorescence

thermal

imaging

was

used.57,58

A

fluorescence

dye

(tris

(2,2’-

bipyridine)ruthenium(II), Sigma-Aldrich, #652407) that shows highly linear relationship


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between temperature and fluorescence intensity over the wide range of temperature was used to indirectly measure high spatial resolution temperature change (2.4 µm per pixel with 4X objective lens). The scanning frame rate of fluorescence imaging was 17 Hz (57 msec per frame). Microelectrode array and Cell culture Microelectrode array (MEA) chips with ITO conductive lines and 30-µm TiN electrodes with 200-µm spacing (60MEA200/30IR-ITO-w/o, Multi Channel Systems) were used for cultivating neuronal cultures and recording extracellular neural spikes. After the cleaning of asreceived MEAs with acetone, IPA and deionized water in sonication for 5 min each, aforementioned polyelectrolyte LbL coating was applied to MEAs with the positive PAH coating as the top layer. Then, the GNR micro-patterns were inkjet-printed only on desired areas of the MEAs. The TiN microelectrodes modified by LbL coating retained good extracellular recording characteristics in terms of RMS noise levels (RMS noise: 7.5 ± 0.2 µV, n=59; see more details in Supporting information Figure S4). As a cell culture media chamber, a glass ring was attached onto the MEAs using polydimethylsiloxane (Sylgard 184, Dow Corning) cured at room temperature for 48 hours. The cell seeding and growth area was 6 mm in diameter centered with 8x8 microelectrode layout. To promote adhesion of cultured neurons The GNR printed MEA chips were then coated with poly-D-lysine (0.1 mg/mL in Trizma buffer of pH 8.5, Sigma Aldrich) for 12 hour in a humidified incubator (37 ºC, 5% CO2). Before the cell seeding, the MEA chamber was rinsed with sterilized ultrapure water multiple times, sterilized by 70% of ethanol for 1 min and dried.


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Hippocampal neurons were dissected from embryonic day 18 Sprague–Dawley rat brains. The dissected tissue was rinsed and dissociated in Hank’s balanced salt solution (HBSS, WelGENE, Korea) by multiple pipetting. Cells collected through centrifugation at 1000 rpm, and resuspended in a plating medium (Neurobasal medium with 2% of B27 (Invitrogen), 2 mM of glutamax (Invitrogen), 12.5 µM of glutamate (Sigma Aldrich) and 1% of penicillinstreptomycine (Invitrogen)). Cells were plated at density of 1000 cells/mm2 and maintained in a humidified incubator (37 ºC, 5% CO2). 50% of medium was replaced twice a week with fresh medium which is the same as the plating medium except glutamate. All experiments were performed in accordance with the guidance of the Institutional Animal Care and Use Committee (IACUC) of Korea Advanced Institute of Science and Technology (KAIST), and all experimental protocols were approved by IACUC of KAIST. Neural recording and analysis Extracellular spikes were recorded using a custom-built multichannel voltage amplifier system (gain: 1000 V/V, bandwidth: 150 Hz to 4 kHz). The amplified analog voltage signals were digitized by MC card (12 bit, 25 kS/s, Multi Channel Systems), and spikes were detected using a threshold method (-6 × rms noise) in MC Rack program. Spike train analysis was performed using a neurophysiological data analysis software (Neuroexplorer, Nex Technologies). Light-induced artefacts were removed from the recorded spike data set. For network analysis, cross-correlation matrix was obtained by following the literature.41,59 Briefly, spike rate histogram was obtained with bin size 10 ms and gaussian smoothing by size 5 in Neuroexploer. Cross-correlation (xcorr ( ) normalized by ‘coeff’, MATLAB) was calculated with the smoothed rate histograms and maximum values were taken as the correlation coefficient for the matrix. To quantify the synchrony, the mean value of the correlation matrix was used.


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Figure 1. Inkjet-printed bio-functional thermo-plasmonic interface for patterned neuromodulation using LbL coating, and the effect of contact line pinning (CLP) induced by LbL coating on the pattern fidelity of the printed thermo-plasmonic gold nanorods. a, Schematic illustration of inkjet-printed thermo-plasmonic interface for patterned neuromodulation on in vitro cultured hippocampal neuronal network. Printed gold nanorods define thermo-plasmonic regions where heat is generated upon NIR illumination. Action potential (AP) spikes of the neurons on the thermo-plasmonic region were turned off upon NIR illumination via photothermal inhibition. Electrostatic force between oppositely charged gold nanorods and LbL coating is formed to help immobilization and uniform assembly of the nanorods. b, Gold nanorod ink is inkjet printed on the LbL coated substrates in desired patterns, and the printed GNR patterns after drying lead to patterned heat (left). The effect of CLP induced by LbL coating on the GNR ink droplet drying is described (right). c, CLP observed only on the LbL coated substrates. Water droplets are deposited using micropipette (2.5 µL) on Nitride 2 substrates. All the other substrates used in this work also showed the induced CLP only after the LbL coatings. d, Inkjet-printed gold nanorod ink droplet array (scale bar: 100 µm). As more printings are added, droplets get bigger, touch each other and merge. When CLP exists (bottom: PAH+ coated nitride 1), droplets maintain original position and shape before/after the solvent drying. On the other hand, droplets on bare substrate without CLP (top) relocate and reshape in merging, and shrink as solvent dries (much smaller coverage area eventually). e, Equilibrium contact angle of water on various surface conditions (different substrate materials: see more details in Methods, different coating conditions: uncoated (bare), PAH (LbL coating with positively charged polyelectrolyte finish), PSS (LbL coating with negative charge finish) (n=5, error bar: s.e.m.).


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Figure 2. Inkjet printed gold nanorod patterns on LbL coated nitride substrate (PAH on nitride 2) and their photothermal effect. a, Dark field microscope image of densely printed gold nanorod patterns (scale bar: 200 µm). b, Identically charged GNR and LbL substrates (scale bar: 50 µm). Positively charged GNR on PAH+ coated glass coverslip (Glass 1, left). Negatively charged GNR on PSS- coated Nitride 1 (right). c, Scanning electron microscope image of the inkjet printed nanorod patterns showing uniform nanorod coverage within the patterns (scale bar: 100 µm). d,e, Magnified SEM images of a GNR pattern as described in (c): (d) in the center, and (e) on the edge; assembly and density of the nanorods are quite similar in both regions, indicating good uniformity and insignificant coffee-ring effect (scale bar: 500 nm). f, Printed array of GNR dots with different GNR ink areal densities on PAH+ LbL coated nitride on sodalime glass wafer (scale bar: 5 mm). Magnified dark field microscope images for different densities are presented (scale bar: 50 µm). g, Absorbance spectrum of the GNR printed samples in (f). The enhanced absorbance value at 785 nm (the wavelength of NIR laser used for these samples) is indicated. h, Temperature changes of the samples in (f) with respect to different NIR laser power density and GNR ink areal density.


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Figure 3. Heat generation of inkjet-printed micro thermo-plasmonic (µTP) heaters. a, Experimental setup: inkjet-printed GNR µTP pattern array on PAH+ coated glass coverslip using 50-µm inkjet nozzle with 350-µm drop spacing. 808-nm NIR laser with 1.38 W/mm2 power density is illuminated only on a single dot pattern. Image spatial resolution: 2.4 µm/px. b,c, 2D and 3D profile of temperature change around a µTP heater in (a). Gaussian profile heat source formed within 300 msec after NIR laser turned on (step #3); overall temp increases through heat dissipation from the heater (step #3 to #4); heat source quickly disappears as the laser is turned off (step #5, #6). d,e, Smaller µTP heaters (dark field image) and their heat profiles at maximum temperature change (45-µm diameter, 250-µm drop spacing, 30-µm inkjet nozzle used, ~5 pL droplet volume, NIR laser power density: 1.16 W/mm2). A single large NIR illumination covered the four µTP heaters altogether, and micro heat patterns were only generated from the inkjetprinted GNR patterns.


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Figure 4. Large-area inkjet-printed thermo-plasmonic images on various PAH+ LbL coated substrates. a, Dark field microscope image of an inkjet-printed GNR image (right, 45µm single pattern size with 45-µm center-to-center drop spacing; 564 dots per inch printing resolution) on a glass coverslip using the input bitmap image (left). Four different GNR levels: 0 (none), 1 (single layer), 2 (bi-layer) and 3 (three layers). Tightly spaced printed dots show how high the percentage of printed GNR coverage area can be without losing its individual characteristics (i.e. microshape, GNR areal density). b, Original input image file (a portrait of a Siberian tiger, 5-level grey levels) for 4.8-cm tall GNR image in (c) (Reproduced with permission, Rüdiger Katterwe / EyeEm / Getty Images). c, Inkjet-printed thermo-plasmonic GNR image on the flexible FEP membrane (5-level grey scale; 600 pixel long; 40-µm drop size; 80-µm center-to-center drop spacing; 318 dots per inch printing resolution; magnified dark field and phase contrast images can be found in Supporting information Figure S3). d, Thermal image (temperature change extracted from baseline) recorded by an infrared camera upon the illumination of 808-nm NIR light with 4.23 mW/mm2 power density on the printed thermoplasmonic image in (c). Recorded thermal image movie after temperature change extraction is provided as Supporting information movie 3. e, Magnified thermal image in (d) and corresponding input image in (b). Darker spot in the input image looks brighter in the thermal image as higher density of GNRs leads to higher temperature change.


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Figure 5. Selective photothermal neuromodulation using inkjet-printed gold nanorod thermo-plasmonic heaters. a, GNR printed MEA (dark field image): 6×6 array of 50-µm GNR dots with 100-µm center-to-center spacing on top right corner of the MEA channel area, and hippocampal neurons cultured on the GNR printed MEA (1,000 cells/mm2, at 10 DIV, phase contrast image). All the 59 microelectrodes are illuminated with 808-nm NIR laser with circular illumination area as described in the phase contrast image. b, Dependence of distance from the printed GNR patterns on extracellular spike rate changes of active electrodes upon the NIR laser illumination (power density: 26 mW/mm2 and 138 mW/mm2) (10 sec rest, 10 sec illumination, 10 sec rest, repeat 20 times; electrodes with spike rates over 0.1 Hz/sec were only considered; inset represents spike rate graphs and raster plots of selected electrodes; 11 DIV). c, Statistical analysis of the effect of laser power and distance from the GNR area on photothermal neural activity suppression (Two-tailed paired t-test; n=20 for all cases; *** represents p < 0.0001; p values for other cases specified in the graph). Inset represents how spike activity suppression looks like in raw recording data.


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Figure 6. Neuromodulation of synchronized hippocampal neuronal network using inkjetprinted gold nanorod thermo-plasmonic heaters. a, Darkfield microscopy image of the cultured neuronal network (15 DIV) on an MEA chip. The chip is divided into three domains: GNR domain (center); non-GNR domains (left, right). Photo-thermally generated heat is formed only in the GNR domain. b, Spike raster plots of all the active electrodes (n=22 for control, n=21 for 138 mW/mm2) under NIR light stimulation with different light intensities (repeated 2 min illumination with 2 min light off; 5 times repeat; the light illumination time window is specified with color bars below each plot). c, Average spike rate of active channels by domains and light illumination conditions (mean ± s.e.m.). d, Representative raw recording data for NIR off and NIR on conditions. e, Network analysis. Cross-correlation matrices (top) (active electrodes: >10 spikes per 10 minutes; bin: 10 msec; rate histogram smoothing with Gaussian, size 5) under two conditions: NIR on and NIR off during 138 mW/mm2 illumination. Connectivity map (bottom) based on the cross-correlation coefficients (threshold coefficient: 0.6). Darker lines represent stronger correlation. f, Mean correlation coefficient of the overall active channels by NIR illumination conditions. g, Statistical analysis of the mean correlation coefficient (two-tailed unpaired t-test; n=5; *** represents p < 0.0001).

ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. Additional information (PDF) Heat generation of single µTP heater (AVI) Measured and simulated temperature change profile of multiple µTP heaters (AVI)


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Large-area thermo-plasmonic image heat generation movie (AVI)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was supported by the National Research Foundation of Korea (NRF) grant (NRF2015R1A2A1A09003605) funded by the Ministry of Science, ICT & Future Planning.

ACKNOWLEDGMENT This work was supported by the National Research Foundation of Korea (NRF) grant (NRF2015R1A2A1A09003605) funded by the Ministry of Science, ICT & Future Planning. Authors thank Professor Kyung Cheol Choi and Professor Seunghyup Yoo and for kindly allowing us to use their inkjet printer. Authors thank Nari Hong for providing technical assistance on neuronal network connectivity analysis.

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Figure 1 662x340mm (300 x 300 DPI)


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Figure 3 283x284mm (300 x 300 DPI)


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