Nanopatterned Nafion microelectrode arrays for in vitro cardiac electrophysiology

Abstract

In this study, we report nanopatterned Nafion microelectrode arrays for in vitro cardiac electrophysiology. With the aim of defining sophisticated Nafion nanostructures with highly ionic conductivity, fabrication parameters such as Nafion concentration and curing temperature were optimized. By increasing curing temperature and Nafion concentration, we were able to control the replication fidelity of Nafion nanopatterns when copied from a PDMS master mold. We also found that cross-sectional morphology and ion current density of nanopatterned Nafion strongly depends on the fabrication parameters. To investigate this dependency, current-voltage analysis was conducted using organic electrochemical transistors (OECT) overlaid with patterned Nafion substrates. Nanopatterned Nafion was found to allow higher ion current densities than unpatterned surfaces. Furthermore, higher curing temperatures were found to render Nafion layers with higher ion/electrical transfer properties. To optimize nanopattern dimensions, electrical current flows, and film uniformity, a final configuration consisting of 5% nanopatterned Nafion cured at 65°C was chosen. Multielectrode arrays (MEAs) were then covered with optimized Nafion nanopatterns and used for electrophysiological analysis of two types of induced pluripotent stem cell-derived cardiomyocytes (iPSCs-CMs). These data highlight the suitability of nanopatterned Nafion, combined with MEAs, for enhancing the cellular environment of iPSC-CMs for use in electrophysiological analysis in vitro.

Graphical Abstract

1. Introduction

Most cell-based drug screening assays have historically relied on conventional tissue culture plasticware that lacks the aligned topographic architecture of the native microenvironment. Moreover, most current cell assays suffer from high equipment cost, laborious procedures, and a lack of a biomimetic culture environment. Thus, the pharmaceutical industry has suffered from a very high failure rate in the development of new drugs. Critically, unidirectional surface topography provides some advantages, such as enhanced cellular maturation and functionality in both cardiac and neuron cultures.^[1–4] Kim et. al. have developed an engineered nanotopography that promotes significant cardiomyocyte maturation and aligns the myocytes into a functional, electrically connected anisotropic monolayer.^[1] This nanotopographic culture surface enables the reliable and reproducible generation of sheets of heart muscle that recapitulate the highly aligned architecture and functional phenotype of the mature human myocardium.^[3] Moreover, physical guidance can affect neurite alignment and outgrowth of neuron cells.^[5] Similarly, many studies have demonstrated a capacity for physical cues to affect neuron development and function.^[6–9] Anisotropic topographies alternating lines of grooves and ridges with submicron lateral dimensions have been intensively investigated as one of the most effective systems for neuronal development, and have been shown to enhance differentiation, polarity, and viability. Hence, incorporation of anisotropic nano-features into electrophysiological cell-based assays has the potential to enhance output as the improved biomimicry of the culture environment can tailor the sensitivity of tissues to various test compounds, yielding a more predictive assay.

Since recording electric field potentials from excitable cells typically utilizes electrodes underneath the cultured cells, use of an ion permeable polymer to fabricate nanopatterned substrates was identified as the most suitable class of material. Recently, the Kim group demonstrated that the cation permeable polymer, Nafion, enables high quality field potential recordings from human induced pluripotent stem-cell derived cardiomyocytes (hiPSC-CMs) or hPSC-neurons cultured on commercially available microelectrode arrays (MEA) integrated with high fidelity nanotopographic surfaces.^[10,11] Electrical behaviors and activities of electroactive cells can be recorded by means of microelectrodes that offer non-invasive and label-free methods, which enables real-time analysis without cell death.^[12,13] Such devices can also stimulate the cell by applying continuous or intermittent electrical pulses. Since microelectrodes were introduced for electrophysiological cell and tissue analysis, the electrode-based technology has been continually improved and widely adopted in both academic and industrial laboratories. Particularly, industrial laboratories have shown great interest in this technology as they need to screen large numbers of compounds in their native environment at the tissue, cellular, and sub-cellular level.^[14] Among the MEA systems, organic electrochemical transistors (OECTs) have been widely investigated for various biosensing applications because of their ability to measure ionic and electronic charges through the semiconductor channels.^[15–19] OECTs show effective use of ion injection from electrolyte into semiconductor channel and modulate the charge carrier conductivity of organic semiconductor layers by controlling gate voltages. This operating principal can be applied for monitoring electrical activity of excitable cells that generate ion displacement across a cell membrane that contacts semiconducting channel.^[15] It is therefore possible to make real-time recordings of the density of generated ions from the cells to the organic semiconductor layer. Khodagholy et. al. showed the use of a flexible OECT device on the brain to record in vivo electrophysiological signals with superior signal-to-noise ratios.^{[20] [21]}

Here, we introduce anisotropic Nafion nanopatterns as a semiconductor channel layer on an OECT device to establish ion-transport pathways for recording electrical signals through the Nafion-coated electrodes. The highly ion permeable Nafion nanopatterns were then applied onto an MEA system to investigate the relationship between Nafion ion conductivity and electrophysiological signal clarity when recording from cardiomyocytes. The resulting data will be of critical importance for the future design of patterned electrodes for studying electrically excitable cell behaviors. In order to optimize and enhance Nafion fidelities and ion conductivity, Nafion nanofabrication processes was varied by changing fabrication parameters, such as curing temperature and concentration. The different Nafion nanostructures and their electrical properties were studied by scanning electron microscopy (SEM), atomic force microscopy (AFM), x-ray diffraction (XRD) and current-voltage (I-V) curve analysis to investigate how electrical properties and nanopattern size affect cellular electrophysiology. Incorporation of anisotropic nanopatterns of highly ion permeable Nafion into extant electrode systems can enhance cell culture environment during the real-time measurement of electrical signals from cardiac cells.

2. Results and discussion

2.1. Fabrication of Nafion nanopattern with high structural fidelity

We obtimized Nafion nanopatterns with high structiral fidelity due to the its shrinkage phenomenon after drying solution. Figure 1 shows the Nafion nanofeatures prepared with different fabrication conditions. Nafion nanopatterns were fabricated using a PDMS mold (Figure 1A).^[10] Here, we found that the formation of Nafion nanostructures was greatly influenced by the curing temperature and its concentration. The dimension of the Nafion nanofeatures became closer to the master mold size (800nm in ridge and groove, 600nm in depth) when the Nafion was cured at higher temperatures and higher concentrations of Nafion was used (Figure 1B, C). For example, when a 20% Nafion concentration was used, 532 ± 23 nm in ridge, 1008 ± 19 nm in groove and 447 ± 16 nm in depth was obtained at 105°C curing temperature, while 270 ±15 nm in ridge, 1291 ±17 nm in groove and 276 ± 10 nm in depth was obtained at Room Temperature (R.T.). Furthermore, the graphs (Figure 1B) show that curing temperature affects the Nafion nanostructure dimensions more dramatically than Nafion concentration change. Hence, curing temperature is the target parameter to optimize in order to obtain high fidelity nanostructure copies from the PDMS master mold. This is also greatly governed by the PDMS swelling phenomenon (Figure S1). Since the evaporating and drying time is longer at R.T than 105°C, the Nafion solution can have sufficient time to swell the PDMS master mold at R.T., thereby considerably deforming the resulting PDMS nanopattern structures. However, as the PDMS does not go through a siginificant volume change at the higher temperature (because the Nafion cures faster), the copy fidelity between the PDMS mold and the resulting Nafion nanopattern is increased.

Figure 1. Fabrication and characterization of nanopatterned Nafion layer.

(A) Fabrication scheme of Nafion nanostructures (n = at least 15 samples in ridges and grooves, n = at least 5 samples in depth). (B) Ridge, groove and depth of Nafion nanostructures produced at different conditions (temperature & concentration). (C) Representative SEM images of anisotropic Nafion nanopatterns

There are challenges associated with curing Nafion at high temperatures. For example, we tried to cure at 145°C but the PDMS mold was bent during the curing in the oven (data not shown). Also, we tried to use other materials as master molds, such as polyurethane acrylate (PUA), perfluoropolyether (PFPE) and Norland optical adhesive (NOA), but these materials showed some problems, including dissolving in Nafion, poor film uniformity, and sticking to Nafion, respectively. To make perfect nanopattern dimensions in Nafion (800 nm in ridges and grooves), we also investigated highly concentrated Nafion solution (upto 20%) by adding soluble Nafion powders (POWDion™ ~200 Mesh, Ion power, Inc.). More than 20% Nafion was hard to handle, due to its high viscosity. Height and depth profiles of Nafion nanopatterns (Nafion 20% concentration), characterized by AFM, are shown in Figure S2. In short, at a low concentration and low curing temperature, lower fidelity of the Nafion nano-size ridges were observed, but the ridges became wider, and closer to the master mold dimensions, as the Nafion concentration and curing temperature increased.

2.2. Analysis of current-voltage characteristics and ion current pathways using OECT

In order to measure Nafion’s electrical properties, Nafion layer was combined with OECT device. Figure 2A illustrates the entire scheme of OECT fabrication and integration with nanopatterned Nafion. An OECT was fabricated on a glass substrate. Au (200nm in thickness) was patterned using a lift-off process to create source, drain, and gate electrodes. A PDMS chamber was set-up around these three electrodes in order to confine Nafion and electrolyte within the channel. Nanopatterned Nafion was used as a conductive channel between source and drain. To make the anisotropic nanostructures on the OECT device, a nanopatterned PDMS block was prepared from a PUA master mold with anisotropic 800 nm (ridge and groove) and 600 nm (depth) dimensions. Carson et. al. showed that 800 nm nanogroove size improved cardiomyocyte organization and structural development.^[22] The Nafion solution was placed inside the PDMS chamber and pressed using a PDMS master mold. After curing it in an oven, the PDMS master mold was carefully removed from the OECT device. The completed device is shown in Figure 2B.

Figure 2. Electrical properties of Nafion substrates using OECT.

(A) Fabrication scheme for production of OECT device integrated with a nanopatterned Nafion interface. (B) Image of fully fabricated OECT device. (C) Schematic illustrating hypothetical electrical current flows with and without the nanopatterned Nafion layer. (D-G) I-V curves recorded when electrodes were coated with 5%/20% and unpatterned/nanopatterned Nafion (n = 3~5 per each condition). (H-K) 2D Simulation modelling of ion current density and flow pathways. (H, I) Unpatterned Nafion layers and (J, K) nanopatterned Nafion layers are deposited onto the electrodes. (I, K) Images are magnified from dotted area highlighted in H and J, respectively.

An OECT that uses Nafion as a channel between the source and drain electrodes is useful, not only to analyze electrical properties of Nafion, but also to measure its ionic permeability and predict the electrical current flow pathway by switching gate voltages. ^[23,24] Figure 2C shows hypothetical ion current pathways between the source and drain electrodes through PBS and Nafion layers. PBS is an electrolyte solution and Nafion works as a cationic transport layer by taking in the saline solution. ^[25] When Nafion is not used, ion currents must flow only through the PBS solution (Figure 2C, left illustration, black dot arrow), but there is another path for ion currents when a Nafion layer is applied between the source and drain electrodes (Figure 2C, middle and right illustration, red and blue dot arrow). Since the resistance of the Nafion layer between the source and drain electrodes is generally much higher than in PBS, most of the ion currents still conduct through the PBS solution after passing through the Nafion layer vertically. (Figure 2C, middle and right illustration, black and red dot arrow). If the resistance of the Nafion layer was lower than the PBS solution, most of the ion currents would flow through the middle of the Nafion layer. (Figure 2C, middle and right illustration, blue dot arrow). To test this hypothesis, the effects of flat and nanopatterned Nafion layers were investigated using I-V analysis. The gate voltage was varied from 0V to 1.2V, with steps of 0.3V. When the gate voltage was less than 0.9V, the drain current did not seem to depend on how the Nafion layer was prepared (Figure S3). However, large variations in the drain current were observed when the gate voltage exceeded 0.9V. Once the gate voltage reached 1.2V, bubbles were generated on both the electrodes and generated I-V curves were no longer accurate. Therefore, the gate voltage was fixed at 0.9V for further experiments. First, we aimed to investigate how nanopatterned Nafion layers affected electrical current flow in the OECT device. Figure 2D–G shows the I-V curve characteristics of Nafion layers fabricated using two different concentrations (5% and 20 %) and three curing temperatures (Room Temp (RT), 65°C, and 105°C). When flat Nafion layers were used for the channel layer between the source and drain electrodes, the Nafion curing temperature did not significantly affect the drain current (Figure 2D, F). However, drain current was changed substantially when nanopatterned Nafion was used (Figure 2E, G), i.e., the drain current increased rapidly as the drain voltage increased. This suggests that nanopatterns distinctly affect ion movement and pathway. It can be considered that most of the ion currents move directly up to the surface of nanopatterned Nafion. In order to further examine whether ion currents move in the middle of the Nafion layer or on top of the nanopatterned layer (Figure 2C, middle and right illustration, blue/red dot arrow), three different channel distances between source and drain electrodes were used: 20, 50 and 100 μm. If the ion currents flow predominantly through the middle of the Nafion layer (Figure 2C, middle and right illustration, blue dot arrow), the drain current should show a great dependence on the channel distance, i.e., current decreases considerably as channel length increases. As expected, the measured drain current was slightly affected by the electrode distance when no Nafion was used (Figure S4A, no Nafion layer). Similarly, nanopatterned Nafion layers did not show significant changes in drain current, regardless of the electrode distance (Figure S4B, C). This indicates that most ion currents measured in the OECT device vertically passed through the Nafion layer and conducted through the PBS solution and/or the surface of nanopatterned Nafion layer (Figure 2C, middle and right illustration, black/red dot arrow).

A simulation study of ion current pathways through Nafion was also conducted using COMSOL Multiphysics® Modeling Software, as shown in Figure 2 H–K. These results are consistent with the I-V curve data obtained from the OECT measurements. Interestingly, the majority of the ion current flows through the PBS or the surface of Nafion layer (not middle of the Nafion layer). Also, the number of streamlines, indicative of current density, on nanopatterned Nafion was roughly 2-times higher than that recorded for flat Nafion substrates. Therefore, it is likely that Nafion not only changes the electrical current pathway, compared with bare electrodes, but also that nanopatterned Nafion shows higher electrical conductivity than unpatterned surfaces.

For the detailed quantitative analysis, mean conductance was calculated in which the drain voltage ranged from −0.5 ~ −0.4V as shown in Figure 3. This result is summarized in Table 1. All nanopatterned layers exhibited higher drain currents than flat samples, as expected. We attribute the higher conductance observed in patterned samples to the large interfacial area between PBS and the electrode. The dependence of the conductance on the curing temperature is more significant for the nanopatterned Nafion layer compared to the flat Nafion layer as the nanopatterned Nafion layer experiences more dramatic volume changes during the curing process.

Figure 3. Analysis of transconductance of unpatterned and patterned Nafion layers.

Electrical transconductance was calculated by the I-V curve slope range from −0.5 to −0.4 in drain voltage. Higher transconductance indicates that greater current changes were obtained at a given drain voltage. (A) R.T., (B) 65°C, (C) 105°C curing temperature. (n = 3~5 per each condition) In presented data, ***P<0.001, **P<0.01; Data were analyzed by the one-way ANOVA

Table 1.

Ion conductance of Nafion measured at room temperature (R.T.), 65°C, and 105°C, respectively.

	Conductance of Flat Nafion (μS)			Conductance of patterned Nafion (μS)
	R.T.	65°C	105°C	R.T.	65°C	105°C
5% Nafion	28.1 ± 6.5	31.9 ± 10.0	32.3 ± 6.4	34.4 ± 5.9	111.4 ± 37.4	160.5 ± 73.7
20% Nafion	50.6 ± 3.0	25.1 ± 0.5	66.5 ± 54.4	109.0 ± 22.6	60.4 ± 13.8	177.5 ± 83.1

2.3. Effects of Nafion concentration and annealing temperature on substrate morphologies.

In order to correlate Nafion ion permeability and morphology, samples with different conditions (concentration (5% vs. 20%), curing temperature (R.T., 65°C, and 105°C)) were characterized with SEM imaging, AFM surface topography, and X-ray diffraction (Figure 4). These data demonstrate that the morphology of Nafion strongly depends on the Nafion concentration and curing temperature. Cured Nafion substrates appeared more crystalized and tightly packed when a low Nafion concentration and low curing temperatures were used. We assumed that these morphological changes lead to different porosity in the Nafion layer, resulting in different ion permeability. As you can see from the AFM images, root mean square (RMS) values of surface roughness were deceased with increasing curing temperatures. At room temperature, RMS were 0.73 nm but dropped to 0.62 and 0.47 nm at 65°C and 105°C, respectively. These results indicate that changes in Nafion morphologies, due to varying curing temperatures and Nafion concentrations, are closely related to the ion permeability of the resulting Nafion layer, and that this is likely to also affect the recording sensitivity of Nafion-coated electrodes used to study cellular electrophysiological function.

Figure 4. Effects of Nafion concentration and annealing temperature on nanopatterned layer morphologies.

SEM and AFM analysis for cross-sectional view and top surface topography of Nafion layers produced using different fabrication conditions (concentration & curing temperature). Root mean square (RMS) of surface roughness was calculated from 3 repeated experiments. (A) 5%, (B) 20 % Nafion, (C) X-ray diffraction spectra for different Nafion layers.

To assay for crystallinity in Nafion layers prepared under different condition, X-ray diffraction spectra studies show only two intrinsic peats at 2θ = 16~18° and 2θ = 39~40°, as illustrated in Figure 4C. These two peaks at 16~18° and 39~40° are attributed to crystallin/amorphous regions and only amorphous regions, respectively. ^[26,27] Improved crystallinity of Nafion was achieved at 5% concentration because no peak occurred at 39~40° but, slightly broad regions at around 39~40° were observed in the only 20% Nafion samples. These results support our SEM/AFM analysis and suggest that Nafion morphologies in low concentration are more crystalized and tightly packed, leading to better ion conductivity than high concentration Nafion substrates. Hence, this research will be useful to elucidate the effect of Nafion porosity and morphology on ion conduction through Nafion substrates.

2.4. In vitro cardiac electrophysiology recording using nanopatterned Nafion MEA

MEAs coated with flat or patterned Nafion were used in conjunction with commercially-sourced CDI (Cellular Dynamics, Inc.) hiPSC-CMs and in-house differentiated hiPSCs-CMs to investigate which Nafion substrate was most effective for facilitating cellular electrophysiological recordings from overlying patterned cells. Considering nanopattern dimensions and electrical/ion current flows, we first chose 5% Nafion cured at R.T. or 65°C to compare the effect of curing temperature on field potential recordings. 65°C was then set as a curing temperature and Nafion concentration was examined for the same metrics. Due to the high viscosity of 20% Nafion, a 100 g weight was put on the PDMS stamp during 20% Nafion solvent drying. (see Figure S5) Final tested Nafion thicknesses for recording electrophysiology were roughly 1.1 ± 0.53 μm and 1.2 ± 0.42 μm in 5% and 20% Nafion, respectively.

Figure 5 and Figure S6 summarizes the electrophysiological responses of CDI hiPSCs-CMs on Nafion-coated electrodes for 14 and 21 days. Electrodes coated with Nafion cured at 65°C recorded higher spike amplitudes and spike slopes than R.T. cured Nafion electrodes. Also, 5% Nafion showed a higher average spike amplitude and spike slope than 20% Nafion, while no differences were observed in beat period or field potential duration corrected for beat rate (FPDc). Based on these results, they illustrate why electrodes coated with 5% Nafion cured at 65°C show better performance than 20% Nafion coated electrodes. 5% Nafion layers cured at 65°C exhibited the best conductance (Figure 2 and 3), suggesting efficient cation-transfer from overlying cardiac cells to the electrode via the Nafion layer. 20% Nafion has a denser molecular structure in its back bone than 5% Nafion, which may have impeded cation-transfer from excitable cells to the recording electrodes. Although 20% nanopatterned Nafion layers cured at R.T. show similar conductance measurements to those recorded from 5% nanopatterned Nafion layers (see Figure 3 A and B), hiPSCs-CMs exhibit poor long-term survival on 20% Nafion layers, possibly due to the incomplete removal of organic solvents at R.T. that cause subsequent cytotoxicity.

Figure 5. Electrophysiological responses of hiPSC-derived cardiomyocytes on Nafion-coated electrodes.

(A-D) Commercially differentiated CMs from CDI recorded at day 21. Cells were assessed for differences in: (A) spike amplitude, (B) depolarizing spike slope, (C) beat period, and (D) field potential duration corrected for beat period using Fridericia’s formula (FPDc). (n = 25, 21, 6, 6, 4, and 5 for RT_5% UP, RT_5% NP, 65°C _5% UP, 65°C _5% NP, 65°C _20% UP and 65°C _20% NP, respectively) ****P<0.0001 ***P<0.001, **P<0.01, *P<0.05; Data were analyzed by the one-way ANOVA

In-house differentiated hiPSCs-CMs were cultured on patterned MEAs to investigate whether the substrate variables had any impact on electrophysiology in these cells and to compare the device performance across multiple cell sources (Figure S7). Similar results to those obtained with CDI cells were observed. The more ion-permeable 5% Nafion layer enabled recording of larger spike amplitudes and spike slopes than electrodes coated with 20% Nafion, while no differences were observed in beat period or FPDc. Spike amplitude and spike slope signals were lower than those recorded from hiPSCs-CMs from CDI, possibly due to the unmatured nature of the in-house differentiated cells or some effect of the dystrophin mutation present in these cardiomyocytes. We plotted representative field potential waveforms for both in-house differentiated and commercially-sourced cardiomyocytes in Figure S8. These data reveal that 5% and 20% Nafion layers, prepared at 65°C, facilitate recording of spontaneous baseline beat rate. However, spike amplitude and spike slope recordings were enhanced when measured through a 5% Nafion layer.

Overall, the higher ion/electrical transfer properties of 5% Nafion layers cured at 65°C appeared to enhance the electrophysiology signals recorded from overlying hiPSC-CMs. Spike amplitude and spike slope recorded from cells on these surfaces exhibited no loss of signal compared with R.T cured Nafion layer. Moreover, 5% Nafion layers enabled better spike amplitude and spike slope recordings than 20% Nafion layers, even though 20% Nafion shows better nanopattern fidelity. According to previous work, nanopatterns promote cardiomyocyte organization and structural development.^[22] However, no correlation between cardiomyocyte organization and field potential measurements were obtained through this study. Regardless, integration of nanotopographic cues into MEAs using Nafion to facilitate structural organization in cultured cardiomyocytes prior to electrophysiological recordings represents a useful screening platform with which to enhance cell culture environments during the real-time functional recordings.

3. Conclusions

Nafion was layered and nanopatterned over microelectrodes by simple imprinting methods and due to its ion permeable properties, especially for cations, the electrophysiology of overlying excitable cells was able to be recorded by underlying, Nafion-coated electrodes. In order to better understand the ion current pathways through Nafion-coated electrodes and investigate how fabrication parameters affect ion flow pathways, Nafion layers with different process conditions were studied using OECT devices for electrical conduction characterization. We showed that Nafion nanopattern fidelity was affected by Nafion concentration and curing temperatures. With increasing concentration and curing temperatures, dimensions of Nafion nanopatterns became closer to initial mold dimensions. Also, ion permeable properties of nanopatterned Nafion layers were characterized and compared by OECT transconductance. 5% Nafion showed better electrical properties than the 20% Nafion layers. Moreover, nanostructures on the Nafion surface were found to enhance ion current conduction more than flat Nafion layers. 5% nanopatterned Nafion layer, cured at 65°C, was chosen for cellular electrophysiological responses and recording sensitivity of Nafion-coated electrodes, based on nanopattern dimension, electrical current flow, and film uniformity data. Two types of hiPSCs-CMs were then utilized for electrophysiological recordings from Nafion-coated MEAs. Nanopatterned and ion permeable polymer-coated electrodes can be utilized for enhancing the cellular environment of excitable cells within electrode-based assays. The data presented here will be invaluable for informing substrate fabrication parameters when using these surfaces in combination with electrode-sensing arrays.

4. Experimental Section

Fabrication of the nanopatterned Nafion layer:

Figure 1A shows how Nafion nanostructures were prepared from a master mold. Nanopatterned PUA molds were prepared from a silicon (Si) master mold bearing anisotropic 800 nm (ridge and groove) and 600 nm (depth) dimensions. The PUA solution was dropped onto the Si mold and pressed using a polyethylene terephthalate (PET) film. The solution was then homogeneously spread to make an even surface. Under 2 min UV irradiation, the PUA was cured and then peeled off of the Si master mold. Using this secondary PUA mold, the PDMS mold was then prepared. PDMS mixtures with 10:1 weight ratio were poured onto PUA master mold, degassed, and cured in an oven. The Nafion solution was dropped on PET substrate and pressed with a patterned PDMS mold. After cure in an oven, the PDMS master mold was carefully removed from PET substrate.

Fabrication of OECT device to measure the Nafion’s electrical properties:

Figure 2A shows the entire scheme of OECT fabrication with Nafion nanopatterns. Each electrode unit includes gate, source, and drain. First, a negative photoresist was patterned via photolithography to define the electrode patterns and then, 20 nm of Cr and 200 nm of Au were deposited in sequence by a thermal evaporator. The photoresist was lifted-off with acetone overnight, and SU-8 was then spin-coated and patterned to make an opening for the contact with Nafion and electrodes while providing electrical insulation in other areas. In order to confine the Nafion and electrolyte solution on the OECT device, a PDMS sheet with 3 mm in thickness was prepared by mixing PDMS prepolymer and curing agent (10:1 weight ratio). Two reagents were mixed, degassed to remove bubbles, and placed in an oven to cure overnight. PDMS chambers were prepared using a 6 mm hole puncher. A PDMS chamber was then attached and set-up around the drain, source and gate electrode area. To make the anisotropic Nafion nanostructures onto on an OECT device, the Nafion solution was dropped inside a PDMS chamber and pressed with a patterned PDMS mold including weights to make an identical Nafion layer thickness (see Figure S5). After curing in an oven, the PDMS master mold was carefully removed from the OECT device. An example of an OECT integrated with a nanopatterned Nafion is shown in Figure 2B.

Electrical characterization of nanopatterned Nafion layer:

A Keithley 4200 semiconductor characterization system was used to characterize the OECT devices. Before the experiments, the PDMS chamber was filled with PBS to swell the Nafion layer. The gate and drain voltages were then applied with respect to the source electrode, i.e., source electrode was grounded. The current between the source and drain electrode (Ids) was measured while drain voltage was swept between −0.5 and – 0.2V at various gate voltages. The gate voltage (Vgs) was applied in the 0–0.9V range, with step heights increasing progressively by 0.3V.

Simulation study of electrical flow pathway on Nafion combined electrodes:

A 2D simulation model via COMSOL Multiphysics® Modeling Software consisted of PBS, glass substrate, Au electrodes (thickness: 200 nm), Nafion layer (thickness: 2 μm) to analyze the electric current pathway on flat and nanopatterned Nafion layer. The material property of Au electrodes and glass substrate was set from the COMSOL material library. However, there is no material information about Nafion in the COMSOL library. Therefore, we referred to the references^[28] and company’s provided information (Biosolve Chem.). The conductivities (σ) of Nafion and PBS were set at 10 and 2 S/m, respectively. The dimensions of the nanopatterns were modeled as 800 nm ridges, 800 nm grooves and 600 nm depth. The 0.9V was applied to the left-side electrode, and right-side electrode was used as the drain electrode. The gap between electrodes was 50 μm.

CDI iCell cardiac cell culture on Nafion nanopatterned MEA:

commercial hPSC-CMs (iCell) were purchased from CDI and stored according to the manufacturer’s protocol. Cultures were maintained in a conventional cell culture condition throughout the culture period. A 5 μg/ mL fibronectin in PBS containing Ca²⁺ and Mg²⁺ was first applied for 3 hours to all MEA substrates for cardiomyocyte culture. A 6 μL droplet of fibronectin solution was applied and positioned to cover all the recording electrode area. Distilled H₂O was plated around each MEA well to humidify the plate and prevent solution evaporation. Following overnight incubation, the fibronectin was aspirated and 16,000~17,000 cells, resuspended in 6 μL of medium, were transferred to the fibronectin-coated nanopatterned electrodes. MEAs were then incubated at 37°C, 5% CO₂ for 5 hours until complete cell attachment. At this point, additional medium was added to the MEA well to support the cultured cells. Cultures were maintained and electrophysiology was recorded for 7, 14 and 21 days, and medium was replaced every 2–3 days during this period.

Cardiac differentiation and culture on Nafion nanopatterned MEA:

1006–1 human induced pluripotent stem cell (hiPSC) colonies^[29] were differentiated into beating cardiomyocytes using an established cardiac differentiation protocol.^[30] Briefly, colonies were maintained on Matrigel (1:60) coated tissue-culture plates in mTeSR medium until colonies reached 80% confluency. Two days before induction of cardiac directed differentiation programs, colonies were dissociated and re-plated into a monolayer at 250k cells/cm². Human iPSC-monolayers were induced with 5 μM Chiron (CHIR99021) in RPMI/B-27 medium without insulin on Day 0 and cultured for 24 hours (37°C, 5% CO₂) before replacing medium again with RPMI/B-27 without insulin and without Chiron. Medium was changed on Day 3 with RPMI/B-27 without insulin and 2 μM WNTC59, and replaced again on Day 5 without addition of WNTC59. On Day 7 and onward, cells were cultured with RPMI/B-27 medium containing insulin. Beating monolayers of hiPSC-cardiomyocytes (hiPSC-CMs) were observed as early as Day 8, but cultures were maintained until Day 12 before dissociating into single cells for cardiac purification. Human iPSC cardiac-differentiated populations were subjected to a lactate-rich glucose-poor selection medium at D14 for 3 days to enrich the population to 99%+ cardiac troponin T-positive CMs as confirmed by flow cytometry.^[31] Before plating with purified hiPSC-CMs, commercial multi-well MEA plates (Axion Biosystems) were patterned with the ion permeable polymer Nafion via solvent-mediated capillary force lithography. The plate was placed under a UV-C light source for 30 min to sterilize, then incubated for 24 hours at 37°C with Matrigel (Corning, Cat. No. 354277). Human iPSC-CMs were then plated at 16,000~17,000 cells/cm² in 20 μL droplets onto only the recessed portion of the well to ensure even coverage over all electrodes for optimal recording of the electrical properties of the cardiac monolayer. Human iPSC-CMs were allowed to adhere to the MEA electrode surface for 24 hours (37°C, 5% CO₂) before adding 200 μL of RPMI/B-27 with insulin for further culture. MEA recordings were taken 7 days after plating or until a beating monolayer were observed.

MEA electrophysiological analysis:

Electrophysiological analysis of spontaneously beating cardiomyocytes was collected for 2 minutes using the AxIS software (Axion Biosystems). After raw data collection, the signal was filtered using a Butterworth band-pass filter and a 90 μV spike detection threshold. Field potential duration was automatically determined using a polynomial fit T-wave detected algorithm.

OECTs with Nafion nanointerfaces are presented not only to give in vivo-like fibrous cellular environments, but also integrated with microelectrode arrays to better understand the mechanism of recording cellular electrophysiology through ion permeable polymer layers. Optimization of nanostructure fidelity and ion permeability under different fabrication parameters are conducted. Moreover, nanopatterned Nafion-coated electrode arrays are used to evaluate electrophysiological recordings of iPSCs-CMs.