Monitoring Contractile Cardiomyocytes via Impedance Using Multipurpose Thin Film Ruthenium Oxide Electrodes

Abstract

A ruthenium oxide (RuOx) electrode was used to monitor contractile events of human pluripotent stem cells-derived cardiomyocytes (hPSC-CMs) through electrical impedance spectroscopy (EIS). Using RuOx electrodes presents an advantage over standard thin film Pt electrodes because the RuOx electrodes can also be used as electrochemical sensor for pH, O₂, and nitric oxide, providing multisensory functionality with the same electrode. First, the EIS signal was validated in an optically transparent well-plate setup using Pt wire electrodes. This way, visual data could be recorded simultaneously. Frequency analyses of both EIS and the visual data revealed almost identical frequency components. This suggests both the EIS and visual data captured the similar events of the beating of (an area of) hPSC-CMs. Similar EIS measurement was then performed using the RuOx electrode, which yielded comparable signal and periodicity. This mode of operation adds to the versatility of the RuOx electrode's use in in vitro studies.

Keywords: Fourier analysis; cardiomyocytes; electrical impedance spectroscopy; ruthenium oxide.

Figure 1

(a) Photograph of the chip- and Transwell insert-holder used in the experiment using RuOx electrode. Chip connection block equipped with pogo pins is used to connect the on-chip RuOx electrode to the impedance spectroscope. (b) Illustration of the cross-sectional view of the setup. Chip- and Transwell insert-holder (II) suspends the Transwell insert’s (III) membrane above the glass chip (I), with O-ring (IV) to seal the electrochemical cell. Cells were cultured on the top side of the membrane.

Figure 2

(a,b) SEM images of typical resulting RuOx nanorods. The nanorods grew on the precursor islands, with typical width between 15–25 nm and length between 110–150 nm.

Figure 3

(a) Frequency dependence of the system’s impedance in the presence of empty Transwell insert, measured in the hPSC-CMs medium at room temperature. (b) Expected equivalent circuit of the system with the hPSC-CMs in place and the (open) Warburg element as surface porosity model in the case of the RuOx nanorods. Abbreviations: C_dl, double layer capacitance; R_el, electrolyte resistance; C_mem, cell membrane capacitance; R_cell, cytoplasm resistance; C_par, parasitic capacitance; Z_w, Warburg element.

Figure 4

Visual data and their analyses recorded in the well-plate setup. (a) Still image of the recorded video of beating hPSC-CMs. Frames note example areas (35 by 25 pixels; corresponding to 85 by 60 μm) that were analysed. (b) Mean intensity over time of the two areas noted in (a), filtered by moving average (N = 50 frames, corresponding to 0.5 s integration time) for clarity. The black frame on the image corresponds to the black line in this graph, blue frame to the blue line. The graphs have different waveforms as well as a slight phase difference. (c) Frequency components of the recorded average intensity of the area in black frame in (a) (corresponds to the black graph in (b)).

Figure 5

(a) The magnitude of the recorded impedance of the contractile hPSC-CMs, recorded by Pt wires in the well-plate setup, plotted over time. (b) Frequency components of the recorded magnitude data shown in (a).

Figure 6

(a) The phase of the recorded impedance of the contractile hPSC-CMs, recorded by Pt wires in the well-plate setup, plotted over time. (b) Frequency components of the recorded phase data shown in (a).

Figure 7

(a) Overlaid impedance magnitude and phase of beating hPSC-CMs recorded between RuOx and Pt wire electrodes in the Teflon chip holder setup. (b) Frequency components of the magnitude and (c) the phase data.