Faradaic Pixels for Precise Hydrogen Peroxide Delivery to Control M-Type Voltage-Gated Potassium Channels

Abstract

H₂ O₂ plays a significant role in a range of physiological processes where it performs vital tasks in redox signaling. The sensitivity of many biological pathways to H₂ O₂ opens up a unique direction in the development of bioelectronics devices to control levels of reactive-oxygen species (ROS). Here a microfabricated ROS modulation device that relies on controlled faradaic reactions is presented. A concentric pixel arrangement of a peroxide-evolving cathode surrounded by an anode ring which decomposes the peroxide, resulting in localized peroxide delivery is reported. The conducting polymer (poly(3,4-ethylenedioxythiophene) (PEDOT), is exploited as the cathode. PEDOT selectively catalyzes the oxygen reduction reaction resulting in the production of hydrogen peroxide (H₂ O₂ ). Using electrochemical and optical assays, combined with modeling, the performance of the devices is benchmarked. The concentric pixels generate tunable gradients of peroxide and oxygen concentrations. The faradaic devices are prototyped by modulating human H₂ O₂ -sensitive Kv7.2/7.3 (M-type) channels expressed in a single-cell model (Xenopus laevis oocytes). The Kv7 ion channel family is responsible for regulating neuronal excitability in the heart, brain, and smooth muscles, making it an ideal platform for faradaic ROS stimulation. The results demonstrate the potential of PEDOT to act as an H₂ O₂ delivery system, paving the way to ROS-based organic bioelectronics.

Keywords: Xenopus laevis oocytes; electrochemistry; organic bioelectronics; potassium channels; reactive oxygen species.

Figure 1

Faradaic pixels electrically modulate hydrogen peroxide and oxygen concentrations. a) Schematic and photo of the device, featuring a circular PEDOT:PSS cathode in the center, surrounded by a palladium anode. The cathode produces H₂O₂ via the 2‐electron oxygen reduction reaction, while the anode completes the DC electrochemical circuit by the anodic reactions of water oxidation and peroxide oxidation. b) Cyclic voltammetry recordings of PEDOT:PSS versus bare Titanium in pH 7.4 electrolyte with different O₂ contents: air saturated, 0% (purged with N₂), and 100% (purged with O₂). The volume of solution that was placed on top of the active area (6.15 mm²) was 30 µL. Larger volumes were initially purged with either O₂ or N₂ and subsequently an aliquot of 30 µL used for recordings. c) Cyclic voltammetry (4 cycles) measurement of the palladium counter electrode before and after addition of 10 mm H₂O₂. d) Fluorescent images of the electrolyte droplet containing Amplex UltraRed reagent placed on top of the PEDOT:PSS pixel. The PEDOT:PSS pixel device was operated for 10 min and changes in the fluorescent signal of the Amplex UltraRed reagent were recorded over time. Increase in fluorescence intensity (greyscale) was evaluated by plotting the intensity over time. e) Mean quantities (nmol, left axis) of H₂O₂ produced by the device obtained via the HRP‐TMB assay after running the PEDOT faradaic pixel device galvanostatically at 10 µA cm⁻² for 5, 10, 15 and 20 min (± SD, n = 3–5, number of measured samples in total = 5). Faradaic efficiency is calculated at each time point, and is plotted on the right axis. Threoretical values indicate a situation with 100% faradaic yield. f) Digital camera imaging was used to calculate the distance, d = 250, 500, 750 µm, of the amperometric sensor from the surface of the pixel. Distance was calibrated using the known thickness of the microscope slide as a standard. g,h) mean H₂O₂ and O₂ concentration traces acquired via local amperometric recordings using H₂O₂ and O₂ sensors 250, 500, and 750 µm above PEDOT:PSS film surface for a run time of 10 min. i,j) mean H₂O₂ and O₂ concentration traces measured 250 µm above the device surface during alternating on/off time periods with a duration of 5 min (on)/5.35 min (off), respectively (± SD, n = 3–6, number of measured samples in total: 4).

Figure 2

Finite element modeling illuminates the interplay of O₂ and H₂O₂ gradients. a) 2D slice of the 3D model, used during the simulations of H₂O₂ and O₂ diffusion with given boundary conditions and regions of production/consumption of diffused species. The geometry reproduces that of the electrophysiology experiments on oocytes (brown sphere in the center). b) 3D concentration profiles of H₂O₂ at different times from 0 to 1200 s (Current is turned on during the first 600 s, then switched off). c–e) Calculated [H₂O₂] (top row) and [O₂] (bottom row) at a different height from the center of the PEDOT pixel: 1 µm, 0.5 mm, 1 mm. The calculations closely follow the microamperometry experimental setup. Each calculation shows the effect of a different critical parameter: c) temperature, d) initial O₂ concentration, and e) applied current. At a distance of 1 µm, O₂ concentration drops to values below the measurable threshold, indicated by the flattening of the black lines.

Figure 3

Faradaic delivery of H₂O₂ modifies currents in H₂O₂‐sensitive Kv7.2/7.3 ion channels. Panels a–c): Effect of H₂O₂ delivered via perfusion on the Kv7.2/7.3 M‐channel. a) Representative current traces for one oocyte before (control, light orange) and after perfusing H₂O₂ with concentrations of 5, 50, and 300 µm (indicated by darker color gradient, as in (c) at following test voltages +40 mV (steady‐state), −30 mV (tail). Inset: voltage protocol showing 3 s‐long test pulses applied throughout all TEVC experiments. b) Relative change in steady‐state current at +40 mV (I _ss,test/I _ss,ctrl, Table 1) for 5, 50, and 300 µm H₂O₂ (mean ± SEM, n = 3). Concentration‐response curve fitted using Equation (8); A = 2.41 ± 0.07, C ₅₀ = 37.39 ± 6.86 µm. c) Representative normalized I _tail for one oocyte, curve fitted with Equation (9). Panels d–f): Activation of Kv7.2/7.3 channels upon electrochemical H₂O₂ delivery with PEDOT faradaic pixel devices. d) Representative steady‐state (+40 mV) and tail current (−30 mV) current traces measured in a single oocyte placed on top of the PEDOT cathode before (control) and after exposure to 2.19, 4.53, 6.53, and 9.17 nmol H₂O₂ (indicated by darker color gradient, as in (f). e) Relative change in steady‐state current ratios at +40 mV (I _ss,test/I _ss,ctrl, Table 1) (mean ± SEM, n = 11). Concentration‐response curve fitted using Equation (8), although a saturation in the curve is not reached. f) Normalized I _tail plotted against preceding test voltage, data fitted with Equation (9) (mean ± SEM, n = 11, in total 10 different PEDOT faradaic pixel devices were tested).

Figure 4

Increased potassium currents are primarily caused by delivered H₂O₂ acting on a cysteine triplet in Kv7.2. Panels (b–e) are fitted with sigmoidal Boltzmann function (Equation (9)). Panel a) concentration‐response plots for electrochemical H₂O₂ delivery, showing the results for steady‐state currents at +40 mV in Kv7.2/C150A/C151A/C152A and a time‐matched control with device switched off for Kv7.2/7.3 (mean ± SEM, n = 3 (Kv7.2/C150A/C151A/C152A), n = 3 (Kv7.2/7.3 time‐match)). Panels b,c) comparison between TEVC recordings of Kv7.2/7.3 after delivering 9.17 nmol of H₂O₂ via the PEDOT cathode (panel b, ± SEM, n = 11; includes same data as in panel 3(f) and during a 45 min‐long time‐match control without H₂O₂ where the cathode is not operated (panel c, mean ± SEM, n = 3). Panel d) TEVC recordings of H₂O₂‐insensitive Kv7.2/C150A/C151A/C152A after perfusing 500 µm H₂O₂ for 10 and 20 min (mean ± SEM, n = 3). Panel e) exposure of Kv7.2/C150A/C151A/C152A to 9.17 nmol H₂O₂ via PEDOT cathode (mean ± SEM, n = 3). Panel f) statistical analysis and comparison of relative steady‐state I _ss,test/I _ss,ctrl obtained for Kv7.2/7.3 and Kv7.2/C150A/C151A/C152A upon delivery of 9.17 nmol H₂O₂ by the faradaic pixel device. Middle bar “Time‐match Kv7.2/7.3” shows relative steady‐state I _ss,test/I _ss,ctrl of oocytes recorded without operating the PEDOT cathode in time‐match experiments, (mean ± SEM, n = 3–11, asterisk: p < 0.0001(****), p < 0.005 (***)).