Multi-sized microelectrode array coupled with micro-electroporation for effective recording of intracellular action potential

Figures

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  Fig. 1. Fabrication and characterization of a MEA platform for intracellular AP recording in Cardiomyocytes. a Schematic of a MEA with multiple sizes, labelled with five sizes of microelectrodes. b By culturing cardiomyocytes of suitable density, their coupling to different sizes of planar microelectrode formation was ensured. c Cardiomyocytes were coupled to a microelectrode. Schematic representation of the instantaneous conversion of the extracellular FP of the microelectrodes into intracellular AP after micro-electroporation. Multi-sized microelectrode devices were connected to the recording instrument via PCBs, and software was used to record electrophysiological signals and control micro-electroporation voltages. d Cardiomyocytes presented intracellular and extracellular electrical signals with different waveforms before and after micro-electroporation due to the different coupling of cardiomyocytes at different-sized microelectrodes. e CAD design drawing of the photomask of a single Mix-MEA. f The main preparation process of MEA. (i) Cleaned of glass substrate. (ii) Spin-coated and cured photoresist. (iii) Developed photoresist. (iv) Obtained conductive layer by sputtering Cr/Au.…

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  Fig. 2. Simulation model of intracellular AP recordings and cell-electrode interface for simulated micro-electroporation. a, b Schematic diagrams of equivalent circuit models for extracellular recorded from unperforated cells and intracellular recorded from perforated cells, respectively. The passive circuits were based on RC (resistor-capacitor parallel) elements, including non-junctional RC (Rnj, Cnj), conjunctional RC (Rj, Cj), seal resistor (Rseal), microelectrode RC (Re, Ce), and amplifier RC (Cstary). c (i) Recorded typical original AP, (ii) simulated extracellular FP and (iii) simulated intracellular AP based on original AP. d Schematic diagram of an equivalent circuit model of intracellular AP recorded by one microelectrode perforating two cardiomyocytes simultaneously. e (i) and (ii) were the original APs of neighboring cardiomyocytes, respectively; there was a time difference (Δt) in the APs of neighboring cells due to the propagation of the electrical signals, and the appearance of two spikes in the simulated AP in (iii) was due to the superposition of the intracellular signals of the two cardiomyocytes that had a time difference. f Microscope images of cardiomyocytes coupled…

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  Fig. 3. MSMEA continuously recorded extracellular FP and intracellular AP in cardiomyocytes. a–e Microscopic images of 20, 50, 100, 200, and 400 μm-MEAs. f Extracellular FP and intracellular AP of typical cardiomyocytes were recorded continuously at 20 μm-MEA. g At ~36 s after micro-electroporation, the AP amplitude had decayed to 70% of its maximum value. At ~68 s after micro-electroporation, the waveforms of the recorded signals appeared characteristic of FP waveforms. At ~142 s after micro-electroporation, the recorded signals reverted to the amplitude and shape of extracellular FPs. h Extracellular FP and intracellular AP of typical cardiomyocytes were recorded continuously at 50 μm-MEA. i At ~25 s after micro-electroporation, the AP amplitude decayed to 70% of its maximum value. At ~70 s after micro-electroporation, the waveforms of the recorded signals appeared characteristic of FP waveforms. At ~229 s after micro-electroporation, the recorded signals reverted to the amplitude and shape of extracellular FP. j Extracellular FP and intracellular AP of typical cardiomyocytes were recorded continuously at 100 μm-MEA. k At ~18 s after micro-electroporation, the AP amplitude had decayed…

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  Fig. 4. Waveforms and amplitudes of myoelectric signals recorded by MSMEA. a, b Overlay of 28 extracellular FP and 28 intracellular AP waveforms for 20 μm-microelectrode, respectively.c, d Amplitude heatmaps of extracellular FP and intracellular AP were recorded by 20 μm-microelectrode. e, f Overlay of 32 extracellular FP and 32 intracellular AP waveforms for 50 μm-microelectrode, respectively. g, h Amplitude heatmaps of extracellular FP and intracellular AP were recorded at 50 μm-microelectrode. i, j Overlay of 28 extracellular FP and 27 intracellular AP waveforms for 100 μm-microelectrode, respectively. k, l Amplitude heatmaps of extracellular FP and intracellular AP were recorded at 100 μm-microelectrode. m, n Overlay of 20 extracellular FP and 20 intracellular AP waveforms for 200 μm-microelectrode, respectively. o, p Amplitude heatmaps of extracellular FP and intracellular AP were recorded at 200 μm-microelectrode. q, r Overlay of 29 extracellular FP and 29 intracellular AP waveforms for 400 μm-microelectrode, respectively. s, t Amplitude heatmaps of extracellular FP and intracellular AP were recorded at the 400 μm-microelectrode. u–x Statistical plots of (u) extracellular FP SNR…

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  Fig. 5. Mix-MEA was used for cardiomyocyte extracellular FP and intracellular AP recording, and parametric analysis. a Microelectrodes were numbered from smallest to largest microelectrode size (20 μm (1–8), 50 μm (9–16), 100 μm (17–24), 200 μm (25–28), and 500 μm (29–32)), and the numbering positions were arranged in rows. b–f Different-sized microelectrodes in Mix-MEA were used for extracellular FP and intracellular AP recorded in cardiomyocytes. Upper part: extracellular FP recorded before micro-electroporation. Lower part: intracellular AP recorded after micro-electroporation. g Amplitude heatmaps of extracellular FP and intracellular AP recorded by Mix-MEA. The positions of the amplitude heatmaps were arranged according to the numbered positions in Fig. (a). h Intracellular AP waveforms of cardiomyocytes were recorded continuously by Mix-MEA. i Overlay of single contraction-relaxation processes of intracellular AP recorded by Mix-MEA, containing 32 channels. j To explore the real-time potentials of cardiomyocytes recorded by Mix-MEA, the AP waveforms of individual systolic-diastolic processes were divided into 24-time segments. k Magnitude heatmaps for 24 time points in Fig. (j).…

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  Fig. 6. Analysis of extracellular FP and intracellular AP in cardiomyocytes recorded by Mix-MEA. a–h Statistical plots of (a) intracellular AP Vpp, (b) ratio of intracellular AP Vpp to extracellular FP Vpp, (c) extracellular FP SNR and intracellular AP SNR, (d) intracellular AP duration, (e) extracellular AP and intracellular AP beat frequency, (f) APD50 duration, (g) single cell AP signal ratio and (h) AP yield. i–m Radar plots of parameters under different microelectrode sizes (20, 50, 100, 200, and 400 μm) in MSMEA and Mix-MEA. n, o Heatmaps of parameters under different microelectrode sizes (20, 50, 100, 200, and 400 μm) in MSMEA and Mix-MEA. Statistical plots were analyzed for a sample size of n = 10 or 4 data per group and were expressed as mean ± sem. Significant differences were analyzed by one-way ANOVA, *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; NS, not statistically significant