Phosphate-buffered saline was prepared using sodium chloride (NaCl), sodium phosphate monobasic monohydrate (H₂NaPO₄⋅H₂O), and sodium phosphate dibasic heptahydrate (HNa₂PO₄⋅7H₂O) (all from Sigma-Aldrich). Ingredients were dissolved in distilled water in proportion 0.13 M NaCl, 0.022 M H₂NaPO₄⋅H₂O, and 0.081 M HNa₂PO₄⋅7H₂O, yielding PBS with pH = 7.2.

Potassium hexacyanoferrate(II) trihydrate (K₄[Fe(CN)₆]⋅ 3H₂O) and potassium hexacyanoferrate (III) (K₃[Fe(CN)₆]) (both from Fluka)^TM were used to prepare the second buffer (K₃[Fe(CN)₆]/K₄[Fe(CN)₆]), both in 25 mM concentration.

Screen-printing pastes for conductive layer of neurostimulation electrodes were composed of polymethyl methacrylate (PMMA; BASF GmbH) polymer matrix dissolved in butyl diglycol acetate (OKB, Sigma-Aldrich)^® as a vehicle with silver micro-flakes as conducting filler. Silver printing paste was purchased from Novelinks (Poland).

Carbon-based screen-printing pastes for active layer of electrodes were likewise prepared using dissolved PMMA granulate (8 wt.%) in the OKB. GNPs with mean diameter 15 μm and thickness below 10 nm (XG Sciences, Inc., United States) were used to prepare the 12.5 wt.% GNP paste by adding them into the vehicle solution of PMMA. Prior to addition, GNPs were sonicated in acetone for 20 min. After 10 min of sonication, dispersing agent (DA) was added to one part of the suspension in an amount of 2 wt.% of GNP mass. As a DA, AKM-0531 (NOF, Corp., Japan) was used. After sonication, GNP suspension was dried at 90°C to evaporate acetone. DA was used to further enhance deagglomeration, chiefly for two reasons: agglomerated GNPs lead to poorer printability (Dybowska-Sarapuk et al., 2015) of the final paste and layers printed with such paste exhibit much lower electrical conductivity (Dybowska-Sarapuk et al., 2018). Dry GNPs were then added to PMMA solution and hand-mixed in agate mortar for 10 min. Finally, paste was rolled in the three-roll-mill with silicon carbide (SiC) rolls and 5 μm gap between rolls to obtain homogenous compositions and exclude the possibility of agglomeration of the GNPs. Thus, GNP and GNP/DA pastes were obtained used for fabrication of screen-printed SP-GNP and SP-GNP/DA electrodes.

For all printing steps, 77 T polyester screens were used, fabricated by Maroka (Poland).

SP-GNP and SP-GNP/DA electrodes were fabricated on 25.4 μm thick polyimide foil Kapton^® 100HN (DuPont Poland). Firstly, the holes were laser-drilled in the foil for further printing of vias between electric contacts and GNP electrodes. Secondly, on one side of the foil, silver paths were printed, and then on the other side, using the same method of screen printing, GNP electrodes were deposited using GNP and GNP/DA pastes. Finally, the side with silver paths was isolated with heat-curable 8155 paste (DuPont Poland).

To assess the hypothesis stated during the described experiments (Dispersing Agent Electrochemistry and Silver Contacts Electrochemistry sections), another version of SP-GNP/DA^∗ electrodes was also prepared, with GNP/DA paste employed as well to print contacts between electrode layers and conducting routes. Thus, the silver layer was absent beneath the part designed to contact tissue directly.

The GCE/GNP and GCE/GNP/DA working electrodes were fabricated on a glassy-carbon electrode (GCE) by firstly polishing and rinsing GCE in distilled water, and in the next step, it was rinsed in ultrasound. Next, sonicated GNP and GNP/DA suspensions in acetone were drop-casted (2 μl) on the GCE surface and dried at 40°C for 30 min.

Methods employed to investigate electrochemical properties of fabricated electrodes were CV and SWV performed in both types of buffer (K₃[Fe(CN)₆]/K₄[Fe(CN)₆] and dimethyl sulfoxide [DMSO]). EIS of SP-GNP and SP-GNP/DA electrodes was also performed in K₃[Fe(CN)₆]/K₄[Fe(CN)₆]. In every electrochemical experiment described, Ag/AgCl(s) 1 M KCl reference electrode was used.

Before every series of measurements, a previously unused electrode was prepared by washing with distilled water and drying at 120°C for 15 min. Gold auxiliary electrode was heated in fire and then washed in acetone, followed by rinsing with water.

Since in the living tissue the oxygen level can vary, the electrodes’ performance was examined in two PBS samples. One of those was kept in an open vessel during measurements to allow oxygen dissolution in the PBS. The second sample was subjected to an argon purging, by diffusing Ar in the solution for 30 min and then keeping the gas flow over the surface of the sample throughout the measurements. As it is visible on the overlaid CV plots (Figure 1), there are few differences between the electrode processes in both high and low O₂ concentrations in the sample. This may be explained by the lack of redox activity of PBS components in the applied potential window (−1 to 0.9 V). Thus, a wider window (−1 to 1.6 V) was also used, as shown on the inset in Figure 1. For the potentials above 1.3 V, current corresponding to O₂ evolution was observed. Thus, it may be concluded that 1.3 V is the upper limit for applied potential when stimulating tissue with fabricated electrodes for aqueous solutions. In both cases, wide oxidation regions are present above 0.4 V, which may be interpreted as oxidation of functional groups on GNP surface (Lee et al., 2017) or oxidation of Ag, which is contained in the bottom layer of SP-GNP/DA electrodes. The cathodic peak, observed below 0 V, may correspond to three overlapping processes: oxidized functional groups reduction, hydrogen adsorption, and H₂ evolution. Besides that, one anodic peak was observed in 0–0.4 V range, which was subjected to further examination as described further. Finally, since neither deoxidation nor wider potential window altered the obtained results significantly, in the following experiments, −1 to 0.9 V range and PBS solution without gas-purging were used.

To determine the cause of 0–0.4 V oxidation peak, SWV measurements were performed, revealing the multi-process origin of the peak. To investigate the possible occurrence of intermediate processes, between SWV measurements, a series of 30 CV scans was carried out. Results of the former are shown on the main plot in Figure 2 and of the latter on the inset, presenting the last CV plot in each series. After each CV series, EIS (Figure 3) was also performed to investigate electrode surface accessibility, indicated by charge transfer resistance, R_CT. It was then observed that between the first and second SWV measurements, i.e., after 30 CV scans, the most pronounced change was observed, whereas after another 30 CV scans, an alleged three-step redox process reached equilibrium to some measure. In that case, two sub-peaks present in the lower potential region of the peak were almost identical, with only the third one increasing its current. In addition, a significant decrease in R_CT was observed corresponding to the first scanning series (ΔR_CT = 183.5 Ω), whereas it only slightly decreased after the second one (ΔR_CT = 17.3 Ω). It was then presumed that the compound(s) undergoing the observed redox processes might be covering the electrode’s surface with its second and third forms hindering the access to the electrode to a lesser extent. Nevertheless, such alleged species presence on the electrode for direct neural stimulation was recognized as a potential risk and so demanded explanation and elimination.

It was hypothesized that the species undergoing the redox process in 0–0.4 V range was the DA employed for printing paste. Since it helps deagglomerate GNPs by coulombic forces, its oxidation might have caused the observed impedance decrease. To further investigate that, GCE/GNP and GCE/GNP-DA were used for CV measurements in −1 to 0.9 V range in PBS. Since in the obtained results no significant oxidation peaks were observed that could be linked to DA reactions (data not shown), it was then concluded that this compound was not the source of 0–0.4 V peaks in previous measurements. From this, it may also be inferred that DA employed in the fabrication of electrodes would not undergo any potentially dangerous process during neural stimulation.

Since the hypothesis of DA being the source of 0–0.4 V oxidation peaks was rejected, another presumption was made, namely, that the GNP printed layer is not entirely impermeable. Thus, silver-containing conductive routes lying beneath the GNP layer would be exposed to the solution. To assess this hypothesis, SP-GNP/DA^∗ was employed. SWV measurements conducted with those yielded no oxidation peaks (Figure 4, left). Analogical measurements with SP electrode with no GNP layer and thus having the Ag layer directly exposed to the solution yielded pronounced redox activity peaks (Figure 4, right). Those were more distinctly separable than the ones observed previously (Figure 2), which can be explained by the SP-Ag electrode’s surface being more easily accessible. Further supporting the hypothesis of Ag causing 0–0.4 V peaks are the findings by Choi and Luo (2011), describing complicated redox reactions occurring on the bulk silver electrode within this potential range, which involve Ag oxide as well as chloride formation.