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Sensors and Actuators B 258 (2018) 1042–1050

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journa l homepage: www.e lsev ier .com/ locate /snb

onductive hydrogel/nanowire micropattern-based sensor for neuraltem cell differentiation

ong Min Lee a,1, Joo Yoon Moon b,1, Tae Hyun Kim a, Seung Won Lee a,hristian D. Ahrberg a, Bong Geun Chung a,∗

Department of Mechanical Engineering, Sogang University, Seoul, 04107, Republic of KoreaDepartment of Biomedical Engineering, Sogang University, Seoul, 04107, Republic of Korea

r t i c l e i n f o

rticle history:eceived 9 May 2017eceived in revised form3 November 2017ccepted 24 November 2017vailable online 28 November 2017

a b s t r a c t

One of the main challenges regarding treatment of neurodegenerative diseases in the central nervoussystem (CNS) lies in the inability of neurons to undergo mitosis. Stem cell therapy could provide a possiblesolution to treat the neurodegenerative diseases. Here, we developed a conductive polyethylene glycol(PEG) hydrogel/silver nanowire (AgNW) composite micropattern-based sensor to direct differentiationof neuronal stem cells (NSCs) and guidance of neurite outgrowth. The ridge micropatterns made from

eywords:icropatternanowirelectrical stimuli

the composite PEG hydrogel/AgNW materials were fabricated on a flexible polyethylene terephthalate(PET) film. Through the combination of these composite materials and the ridge micropatterns, the out-growth of neurites could be efficiently guided in a controlled manner. Therefore, this conductive PEGhydrogel/AgNW micropattern-based sensor could potentially allow future use of the flexible device asan implant for NSC therapy applications.

ensoreural stem cell differentiation

. Introduction

Despite of the significant improvements achieved in the lastecades, diseases affecting the nervous system like Alzheimer,arkinson, and Huntington’s disease are still not curable [1,2]. Like-ise damage to the peripheral nervous system (PNS) or central

ervous system (CNS) is not reversible due to the inability of neu-ons to undergo mitosis [3]. Although function can be restored withutographs in the PNS, these lead to a loss of function in the donorite, necessarily require several operations, and have a success ratef 80% [3]. Thus, researchers are developing new generations of

mplants with novel materials that should provide stability dur-ng nerve regeneration while being biodegradable [4]. In 1995,

cDonald et al. have shown that functional improvements can bebtained through transplantation of neuronal cells differentiatedrom mouse embryonic stem cells into a damaged spinal cord [5].s the rodent CNS is similar to the human one [6], this has sparked

he hope of curing physical damage of the CNS and PNS through

tem cell therapies. Moreover, stem cell therapy might be able torovide cures for neural degenerative diseases. Currently, the mainhallenges faced in stem cell therapy are directing stem cell differ-

∗ Corresponding author.E-mail address: bchung@sogang.ac.kr (B.G. Chung).

1 These authors contributed equally to this work.

ttps://doi.org/10.1016/j.snb.2017.11.151925-4005/© 2017 Elsevier B.V. All rights reserved.

© 2017 Elsevier B.V. All rights reserved.

entiation into the desired target phenotypes and spatial guidanceof cell growth in a homogeneous manner [7].

Stem cell differentiation can be influenced by a variety of factors,such as chemical transcription factors [8,9], chemogradients [10],and physical/mechanical factors [11]. In particular, the electricalstimuli can be used to guide stem cell differentiation towards a neu-ral fate [12]. The micro-electromechanical system (MEMS) providesa good method to apply the electrical fields [13]. Through electricalstimuli, it is not only possible to influence stem cell differentiationbut also possible to direct neurite outgrowth, as was previouslydemonstrated guiding neurites into a hollow silicone implant [4].In addition, faster growth rates and neurite migration towards thecathode can be observed when applying electrical stimuli [14,15].There are two different approaches for obtaining a well-defined andhomogenous electrical field in the growth substrate. One optionis the integration of conductive nanomaterials into the growthsubstrate, as was previously performed with gold nanoparticles[16]. The other option is to create the growth substrate from theconductive material itself. Previously, this was made by spinningnanofibers from conductive polymers [17], or using carbon nano-tubes (CNT) forming two-dimensional (2D) conductive scaffolds[18,19]. Three-dimensional (3D) graphene foam was also used asa conductive scaffold, as they reassemble in vitro conditions better

than 2D scaffolds [20]. Besides, the researchers have also varied thephysical and mechanical properties of the substrate. Hydrogels areparticularly interesting for this task, as their elastic modulus can

J.M. Lee et al. / Sensors and Actuators B 258 (2018) 1042–1050 1043

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ig. 1. Schematic of the production of PEG hydrogel/AgNW micropattern-based seEG hydrogel/AgNW micropattern is stained with red dye. Scale bar is 1 cm (B). FlDMS mold. For illustration purposes the hydrogel is stained with fluorescein dye.

e varied over several orders of magnitude [21], while providing aigh permeability to water, oxygen, and water soluble metabolites22,23]. Thus, polyethylene glycol (PEG) hydrogels have been usedor neural differentiation [24] and creation of 3D neuronal networks25]. Biodegradable gelatin-hydroxyphenylpropionic acid hydro-els have been suggested as an injectable implant for stem cellherapy [26]. Furthermore, neurite outgrowth can be guided byhysical features on the surface of the substrate. This was demon-trated using a grooved poly(dimethylsiloxane) (PDMS) structure27]. The growth rate of neurites and stem cell differentiation effi-iency could be increased by applying mechanical stress to thesetructures [28]. Feasibility of using patterned substrates as implantsas also demonstrated by Hsu et al. [29].

Here, we demonstrate a novel approach combining a micropat-erned PEG hydrogel substrate with silver nanowire (AgNW) toncrease the conductivity of the substrate. Electrical stimuli appliedo the substrate to enhance neural stem cell (NSC) differentiationnd guide neurite outgrowth. To increase the conductivity of theaterial and provide a homogenous electrical field, AgNWs are

ntegrated into the PEG hydrogel matrix. To further guide neuriteutgrowth, a series of parallel microridges are created from theybrid hydrogel material. This, to our knowledge, is the first reportf combining electrical stimuli and physical micropatterns contain-

ng AgNWs in one device for neurite guidance and NSC-derived

euronal differentiation. Therefore, we can achieve synergisticffects and obtain a more efficient guidance of neurites and a higherate of neurite outgrowth compared to using electrical stimuli or

icropatterns on their own.

A). Photograph of the PEG hydrogel/AgNW micropattern inside of the PDMS mold.cent microscopy image of PEG hydrogel/AgNW micropattern after removal of thear is 100 �m (C).

2. Material and methods

2.1. Fabrication of PEG hydrogel/AgNW micropatterns

PDMS stamps for the fabrication of the PEG hydrogel/AgNWmicropatterns were produced in a standard soft lithographyprocess, as previously described [24]. Briefly, photomasks weredesigned using Autocad (Autodesk, USA) and reproduced in SU8-50(Microchem Corp., USA) on silicon wafers (Wangxiang Silicon-PeakElectronics, China), and details for the fabrication process can befound in supplemental materials (Table S1). PDMS (10:1 Monomer:curing Agent, Microchem Corp., USA) was poured onto the sil-icon masters and cured in an oven at 85 ◦C for one hour. Thecured PDMS was carefully peeled from the silicon wafer and cutto size using a surgical blade, resulting in the PDMS stamps. Toproduce the PEG hydrogel/AgNW micropatterns, the PDMS stampswere placed onto a flexible PET film with a thickness of 100 �m(Filmbank, Korea) and a precursor solution was injected in thegap between the PDMS stamp and the PET film. The precursorconsisted of 10 �L silver nanowire solution (Nanopyxis Co. Ltd.,Korea), 100 �L PEG-dimethacrylate (1000 Da, Sigma Aldrich, USA),and 1 w/v% of the photoinitiator 2-hydroxy-2-methyl propiophe-none (Sigma-Aldrich, USA). The hydrogel was polymerized in aradical chain growth reaction by illuminating for 30 s using ultra-

violet light (320–500 nm, Omni Cures Series 1500 curing station,EXFO, Canada). Finally, the PDMS stamp was carefully peeled off,leaving the PEG hydrogel/AgNW micropatterns on the PET film.PEG hydrogel/AgNW micropatterns were coated with a solution

1044 J.M. Lee et al. / Sensors and Actuators B 258 (2018) 1042–1050

Fig. 2. SEM images of the PEG hydrogel (A), AgNW (B), and PEG hydrogel/AgNW composite material (C). X-ray element spectroscopy of PEG hydrogel/AgNW compositematerial (D).

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ig. 3. Stress-strain curve of compression test of different hydrogel materials (A) anetween PEG hydrogel/AgNW composite material and matrigel (**p < 0.01).

ontaining 100 �g/mL poly-l-ornithine (PLO, Sigma Sigma-Aldrich,SA) and 5 �g/mL laminin (Sigma-Aldrich, USA) for one day tochieve a better attachment of the neurospheres to the PET film.

.2. Characterization of composite hydrogel material

Surface morphology of the pure PEG hydrogel, AgNW, andEG hydrogel/AgNW composite material were analyzed using SEM

icroscopy (JSM-7100f, Jeol USA, USA). Further mechanical prop-

rties of the pure PEG hydrogel and PEG hydrogel/AgNW compositeaterial were tested. For this, disk of the materials were prepared

y pipetting the prepolymer solution into a PDMS mold (8 mm

ysis of compressive modulus (B). The compressive modulus is significantly different

diameter, 2 mm height) and were subsequently polymerized. Thecompressive modulus was tested using a CT3 Texture Analyzer(Brookfield Engineering Laboratory, USA) equipped with a 4500 gload cell (Brookfield Engineering Laboratory, USA). A probe diam-eter of 38 mm was used for compressing the sample disks at a rateof 0.05 mm/s. Stress-strain curves were recorded and compressivemodulus was determined from the slope of a linear regression inthe range of 5–15% strain. For comparison, gelatin methacrylate(GelMA, Sigma-Aldrich, USA), and matrigel (Gibco, USA) hydrogels

were also tested for their mechanical properties using the samemethods. Lastly, stress-strain curves of the PET film and the PEGhydrogel/AgNW micropatterns on PET film were analyzed.

J.M. Lee et al. / Sensors and Actuators B 258 (2018) 1042–1050 1045

F ropatc f simuc s (D).

2

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ig. 4. Simulations of electrical behavior of the composite PEG hydrogel/AgNW micurrent density in the micropattern with an applied voltage of 10 V (B). Analysis ourrent for the PEG hydrogel/AgNW composite material at different applied voltage

.3. Simulation of electrical behavior

Prior to experiments, the electrical behavior of the PEGydrogel/AgNW micropatterns was simulated using a Comsol Mul-iphysics 4.4 (Comsol Inc., USA) model. For the models, square

odulated voltages with a frequency of 139 �Hz and magnitude of, 10, and 20 V were chosen as used in experiments. The surface cur-ent density (�j)was then calculated using the following equation:

= � �E =�E�

here �E is the electric field in vector form, and � is the conduc-ivity of the material which can be expressed as the inverse ofhe resistivity (�). The electrical conductivity of water, and cop-er, which was used for the two electrodes, was taken the Comsolaterial library. For the other materials, no values were available

n the material library, so values were taken from literature for PET30], PEG [31], and AgNW [32]. As the conductivity of PEG hydro-el/AgNW composite materials is similar to bulk AgNWs [33], wesed the conductivity of AgNW.

.4. NSC culture and neural differentiation

NSCs cells were extracted from cortex tissues of E12 C57BL/6

ice (Daehan Biolink, Korea). The cells were incubated with Accu-

ase (Innovation Cell Technology, USA) at 37◦ C for 50 min beforerowing them as neurospheres on ultra-low attachment surfacesor 4 days in DMEM/F12 medium (Gibco, USA) containing 20 ng/mL

tern. Schematic of experimental setup for applying electrical stimuli (A). Simulatedlated average current for different materials at an applied voltage of 10 V (C) and

basic fibroblast growth factor (bFGF) (R&D Systems, USA), 20 ng/mLepidermal growth factor (EGF) (Invitrogen, USA), 1% N-2 Supple-ment, 2% B27 supplements, and 1% penicillin-streptomycin (Gibco,USA). The neurospheres were harvested and suspended in 100 �L ofthe same medium and afterwards seeded onto PEG hydrogel/AgNWmicropatterns on the flexible PET film. After incubation for one day,a further 3 mL of medium was added. The electrical stimuli werethen applied using a function generator (AFG1062, Tektronix, USA)and copper electrodes. Output voltages were increased to 5, 10, and20 V using a voltage amplifier (HSA4011, NF Co, Japan). All voltageswere modulated to give a single pulse every 2 h (139 �Hz) for a totalduration of 6 days. Control experiments were conducted on PETfilm submerged in culture medium, with application of identicalelectrical stimuli.

2.5. Immunochemistry

NSC differentiation was confirmed by an antibody stainingreaction. In the first step, the cells on the PEG hydrogel/AgNWmicropatterns were treated with 4% paraformaldehyde (YMSKorea, Korea) for 30 min and were subsequently permeabilizedusing 1% Triton X-100 (Sigma-Aldrich, USA) in phosphate-bufferedsaline (PBS, Sigma-Aldrich, USA). Non-specific protein binding wasreduced through incubation with bovine serum albumin (BSA,1 W/W%, Sigma-Aldrich, USA) in PBS at room temperature for

2 h. The cells were then incubated with anti-neural class III pri-mary antibodies, such as Tuj1 (Stem Cell Technology, Canada)or nestin (Abcam, UK) or MAP2 (Abcam, UK), at 4 ◦C overnight.They were rinsed with PBS buffer and were subsequently incu-

1046 J.M. Lee et al. / Sensors and Actuators B 258 (2018) 1042–1050

F rns wu

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3

3m

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ig. 5. Fluorescent microscopy images of NSCs on PEG hydrogel/AgNW micropattesed for Tuj1, red for nestin, and blue for DAPI.

ated overnight at 4◦ C with Alexa Flour 488-conjugated secondaryntibody (Tuj1, Invitrogen, USA) and Alexa Flour 594-conjugatedecondary antibody (Map2/nestin, Abcam, UK). Finally, nuclei weretained by a solution of 0.1 g/mL 4′,6-diamidino-2-phenylindoleDAPI, Thermo Fisher, USA) in PBS buffer for 30 min at roomemperature. After rinsing PEG hydrogel/AgNW micropatternsith PBS buffer, fluorescent images were taken using a confocal

aser-scanning microscope (LSM710, Carl Zeiss, Germany). Image JNational Institute of Health, USA) was used to measure the direc-ion of neurite outgrowth, which was classified into eight differentirections.

. Results and discussion

.1. Fabrication and characterization of PEG hydrogel/AgNWicropatterns

PEG hydrogel/AgNW micropatterns on a flexible PET film wereade from PDMS molds (Fig. 1A). 200 �m ridge microstruc-

ures were made in a controllable and reproducible mannerFig. 1B and C). Afterwards the PEG hydrogel/AgNW micropat-erns were submerged in the cell growth medium and a functionenerator-mediated voltage subsequently applied after the celleeding. Material properties were observed by scanning electronicroscopy (SEM) of the PEG hydrogel, AgNW, and the composite

0% PEG hydrogel/AgNW material (Fig. 2). The 10% PEG hydrogelisplayed a series of crests and valleys on its surface (Fig. 2A).

he AgNWs, as expected, showed long and thin wires (Fig. 2B).he PEG hydrogel/AgNW composite material displayed a surfaceorphology similar to the PEG hydrogel with ridges and valleys

f similar dimensions as the pure hydrogel (Fig. 2C). Within the

ith 100 �m (A), 200 �m (B), 300 �m (C), and 500 �m (D) width. A green dye was

hydrogel, individual AgNW can be observed in the bulk hydro-gel. As expected, the AgNWs are randomly distributed within thehydrogel. Randomly distributed AgNWs allow for the formation ofconductive networks, as previously shown [34]. However, aligningAgNWs leads to a higher conductivity at the same nanowire concen-tration [35], which could allow reducing the material costs of thedevice. Additionally, X-ray spectroscopy was used to analyze theelemental composition of the composite material (Fig. 2D). Unsur-prisingly, the two most abundant elements found were carbon andoxygen in nearly the ratio expected from the hydrogel. Additionally,almost 5 wt% silver was found by elemental X-ray spectroscopy,which was higher than the expected value of 2 wt%. As only a smallsample, containing AgNW, was chosen for X-ray spectroscopy, thecontent of AgNW was not representative for the bulk material.Hence, the measured content of silver nanowires was higher thanthe expected value. Lastly, small amounts of sodium and chloridewere found. These originated from salts in the hydrogel precursorin the synthesis of the hydrogel. Through the drying of the materialprior to SEM analysis, the concentration was increased comparedto the hydrated form of the hydrogels. We also characterized themechanical properties of the PEG hydrogel/AgNW materials andtheir properties compared to other hydrogels (Fig. 3). Although onewould expect an altered mechanical behavior of the hydrogel afterthe addition of AgNWs, the stress-strain curves for PEG and thePEG hydrogel/AgNW composite materials were similar and theircompressive moduli were not significantly different. It is due to theamount of AgNWs (∼0.2 wt%). Although the desired electrical prop-

erties are achieved, the amount of AgNW (∼0.2 wt%) is too smallto lead to a measureable difference in mechanical properties. Asa result, we confirmed that the addition of low concentrations ofAgNWs to PEG hydrogels did not significantly alter their mechani-

J.M. Lee et al. / Sensors and Actuators B 258 (2018) 1042–1050 1047

Fig. 6. Fluorescent microscopy images of NSCs after culture in control experiments (A–D) and PEG hydrogel/AgNW micropatterns (E-H). To facilitate NSC differentiation,various electric stimuli are applied. Cells are immunostained by TuJ1 (green) and cell nuclei are stained by DAPI (blue). Scale bars are 200 �m.

eurite

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Fig. 7. Analysis of average neurite length (A), average number of n

al properties. In contrast, the compressive modulus of the matrigelas significantly lower than that of PEG hydrogel/AgNW compositeaterials (Fig. 3B). Furthermore, we analyzed stress-strain curves

or tension tests between PEG hydrogel/AgNW micropatterns onET film and pure PET film (Fig. S1). As expected, it showed thathey were almost identical. The addition of the supporting PET filman lead to suitable mechanical properties. However, the PET filmeeds to be replaced by a biodegradable material to obtain a fullyegradable neural implant or graft.

.2. Simulation of electrical behavior

Before conducting experiments, the electrical behavior of PEGydrogel/AgNW micropatterns was simulated using Comsol Multi-hysics software (Fig. 4). Firstly, the current density within the PEGydrogel/AgNW micropatterns was simulated (Fig. 4). From theimulation, it can be seen that when applying a voltage the currentensity within the PEG hydrogel/AgNW structure is higher thanhe current density of the surrounding medium and the supportingET film. Simulations of the PEG hydrogel/AgNW microstructure

arallel to the applied voltage have shown that the current den-ity within the structures would be less homogeneous. Hence,o enhance homogeneity, it was decided to use the PEG hydro-el/AgNW microstructure perpendicular to the applied voltage. The

s (B) for NSCs cultured on the PEG hydrogel/AgNW micropattern.

model further suggests that the simulated current density withinthe individual ridges of the PEG hydrogel/AgNW is inhomogeneous.This effect is related to the mesh size of the Comsol model andnumerical instabilities during the simulation. In experiments, weexpect a homogeneous current distribution within the individ-ual ridges of the microstructure. Secondly, the average current atthe wall in the different materials was simulated (Fig. 4C). It wasobserved that PET and PEG showed lower currents than AgNWdue to the lower conductivity of the materials. For the PEG hydro-gel/AgNW material, a current similar to AgNWs can be expecteddue to similar conductivities [33], showing the feasibility of thematerial in the conduction of electrical stimuli. Furthermore, thedependency of the current inside the ridges on the applied volt-age was simulated (Fig. 4D). As expected from Ohm’s law, a linerrelationship between current and applied voltage was found.

3.3. NSC-derived neural differentiation

To optimize the dimension of PEG hydrogel/AgNW micropat-terns, we investigated various widths of PEG hydrogel/AgNW

micropatterns without applying a voltage (Fig. 5). Choosing a widthof the micropatterns smaller than the diameter of the neuro-spheres can lead to neurosphere growing on several microridgessimultaneously. Through the spatial proximity of neurospheres

1048 J.M. Lee et al. / Sensors and Actuators B 258 (2018) 1042–1050

F on in

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ig. 8. Rose diagrams indicating percentage of neurites growing in a certain directiicropatterns (B). Rose diagram comparing the percentage of neurites growing in a

op of the micropattern and 90◦ is growth towards the cathode, respectively. Grow

n neighboring ridges, the fusion of several neurospheres wasbserved (Fig. 5A). If a PEG hydrogel/AgNW micropattern widthider than the diameter of the neurospheres is used, the guid-

ng efficiency of neurites cultured in the micropatterns may beost. This is observable by the radial outgrowth of neurites (Fig. 5Cnd D). Therefore, we used PEG hydrogel/AgNW micropatternsith 200 �m width, which was similar to the diameter of neuro-

pheres (Fig. 5B), showing the best compromise between neuriteuiding and neurosphere seeding efficiency. To determine theifferentiation of NSCs into neurons in the PEG hydrogel/AgNWicropatterns, the neural markers (e.g., Tuj1, MAP2, and nestin)ere assessed by specific immunostaining with fluorescently

abeled antibodies. From the fluorescent microscopy images, itas observed that a large number of cells differentiated into

uj1-postive neuronal cells (Fig. 6). To gain further informationbout NSC differentiation behaviors, we used Map2 and nestiniomarker (Fig. S2). From the fluorescence microscopy images, webserved that a number of NSCs differentiated into MAP2-positiveature neuronal cells, while a small number of cells are nestin-

ositive neural progenitor cells. Furthermore, the outgrowth ofeurites mediated from the electrical stimuli was observed. How-ver, the strongest growth of neurites, both by number and length,as observed when applying intermediated voltages of 5 and

0 V (Fig. 6B,C,F and G). Longer neurites can be observed in theEG hydrogel/AgNW micropatterns (Fig. 6G) compared to con-rol experiments at the same voltage (Fig. 6C). Furthermore, whilehe direction of neurite growth appears to be random in controlxperiments, neurite outgrowth appears to follow the PEG hydro-el/AgNW micropatterns.

.4. Analysis of neurite outgrowth

The neurite outgrowth was measured with Image J and ana-yzed regarding neurite number and length (Fig. 7) and therowth direction (Fig. 8). In experiments conducted with the PEGydrogel/AgNW micropattern, the average length of neurites was

ncreasing with increasing applied voltages (Fig. 7A). Furthermore,

he number of neurites increased with the applied voltage (Fig. 7B).pplication of low voltages of 10 V significantly increases the lengthf neurites compared to control experiments, while the numberf neurites also increases compared to no voltage control exper-

control experiments (A) and in experiment conducted on the PEG hydrogel/AgNWin direction of control and micropattern experiments (C). 0◦ is growth towards the

classified into eight different directions, each occupying a radial angle of 45◦ .

iments, confirming results by Patel et al. [36]. However, whenapplying a higher voltage of 20 V, neither neurite length nor neuritenumber is significantly increased compared to control experimentswithout electrical stimuli. Similarly, Yamada et al. observed a lowerdifferentiation efficiency at voltages of 20 V compared to 15 V [12].The mechanism for this is still not fully understood [37]. A possiblereason is that low electric fields that present in wound healing,affect the cell steering mechanism, while higher electric fields(>2 V/cm) also stimulate overall cell motor activity [38]. In additionto the length and number of neurites, the direction of neurite out-growth was also analyzed (Fig. 8). In control experiments, neuritescould be observed growing in all directions, with a fairly even distri-bution (Fig. 8A). In literature a tendency of neurites to grow towardsthe cathode was observed [36]. However in our experiments thistendency was not observed. A possible reason for this might bethat in our experiments the frequency of stimulation (f = 139 �Hz)is four orders of magnitude lower than used in previous studies(f =1 Hz) [36], which observed growth towards the cathode. In con-trast, neurite growth orients along the ridges of the substrate whenan electrical field is applied (Fig. 8B). In experiments with no appliedvoltage, the direction of neurite outgrowth appears to be random,as observed in control experiments. Nevertheless, when a voltageof 5 or 10 V is applied, the outgrowth of neurites orients itself alongthe ridges of the PEG hydrogel/AgNW micropattern. In direct com-parison of the control experiment and the PEG hydrogel/AgNWmicropattern at 10 V, it can be seen that on the microstructure 60%of neurites follow the structure in their growth direction, whileonly 20% of neurites grow in the direction of the electrode for thecontrol experiments (Fig. 8C). This superior performance of the PEGhydrogel/AgNW micropattern could be attributed to two differentfactors. Firstly, the ridges of the material impose spatial limitationsto the growth of neurites, especially for neurites close to the edgeof a ridge. This effect would be more visible with longer neurites, asobserved in 10 V experiments, as they are more likely to encounteran edge of the ridges. Secondly, AgNW in the composite materialmight aid as a guide to the outgrowth of neurites.

4. Conclusions

We presented a conductive PEG hydrogel/AgNW compositemicropattern-based sensor that was used for NSC differentia-

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ion into neurons under the application of electrical stimuli.icrostructures were formed from the composite material with

he aid of a PDMS mold, through which neurite outgrowth coulde guided. Simulation of the electrical behavior of the compos-

te material and structure demonstrated the advantages of theEG hydrogel/AgNW composite material compared to conven-ional hydrogels. Therefore, our PEG hydrogel/AgNW composite

icropattern-based sensor on a flexible PET film could allow theotential future use as a neural implant device for NSC therapypplications.

cknowledgements

This research was supported by BioNano Health-Guardesearch Center funded by the Ministry of Science and ICTMSIT) of Korea as Global Frontier Project (grant number H-UARD 2014M3A6B2060503), Republic of Korea. This work was

upported by the National Research Foundation (NRF) of Korearant funded by the MSIT (Grant number 2016R1A6A1A03012845).his research was also supported by Basic Science Research Pro-ram through the NRF funded by the Ministry of Education (Grantumber 2016R1A6A3A11931838). Authors appreciate Prof. Sunoong in Korea University to kindly provide NSCs.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at https://doi.org/10.1016/j.snb.2017.11.151.

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Biographies

Jong Min Lee received his B.S. degree in Biochemical Engineering from Korea Poly-technic University, Korea in 2010, respectively. In 2016, he received his M.S. andPh.D. degree in Mechanical Engineering, both from Sogang University, Korea. Cur-rently, he is a research professor for the Department of Mechanical Engineering atSogang University, Seoul, Korea. His current research interests are development ofmicrofluidic sensing devices for healthcare and medical applications.

Joo Yoon Moon received his B.S. degree in Biomedical Engineering at Kyung Hee Uni-versity, Korea in 2015 and received his M.S. from Sogang University, Korea (2017).

His research interests are microfluidics and stem cells for diagnosis and treatmentof disease.

Tae Hyun Kim received the B.S. degree from Seoul National University of Science andTechnology in Mechanical System design engineering (2012). He received his M.Sdegree in Mechanical Engineering in 2015 at Sogang University, Korea. Currently,

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Research Fellow and an Instructor in Harvard Medical School, USA. Currently, he

050 J.M. Lee et al. / Sensors and A

e is a Ph.D. degree candidate at Department of Mechanical Engineering in Sogangniversity. His research interests are modeling and simulation of multi-physics inio-medical application.

eung Won Lee received his B.S. degree at Hanyang University with Materialngineering in 2000. In 2017, he received his M.S. at Department of Mechanicalngineering in Sogang University, Korea. Currently, he is a Ph.D. degree candidate atepartment of Mechanical Engineering in Sogang University. He is also Senior Man-ger for Platform Technology Lab, SAIT, in Samsung Electronics, Korea. His current

esearch interests are fabricating sensing device for electrical applications.

hristian D. Ahrberg obtained his Bachelor and Master Degree in Chemical andioengineering in 2012. He worked at Bayer Technology Services in Leverkusenefore starting his Ph.D. under supervision of Andreas Manz at the Mechatronics

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department of Saarland University in cooperation with KIST Europe, during which heworked on a portable real-time PCR device. Since graduating in 2016, he is currentlya postdoctoral researcher at the BioNano Technology Lab in Sogang University, Seoul,Korea.

Bong Geun Chung received the B.S. and M.S. degree from Hanyang University, Seoul,Korea. In 2007, he obtained Ph.D. degree from Materials Science Engineering in Uni-versity of California Irvine, USA. After Ph.D. degree, he worked as a Postdoctoral

is an Associate Professor at Department of Mechanical Engineering in Sogang Uni-versity, Seoul, Korea. His main research interests are development of functional 3DLab-on-a-Chips, Biosensors, and Nanomaterials to direct the stem cell and cancerfate.