Jakešová M et al. (2019), Optoelectronic control of single cells using or...

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Sci Adv 2019 Apr 05;54:eaav5265. doi: 10.1126/sciadv.aav5265.

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Optoelectronic control of single cells using organic photocapacitors.

Jakešová M , Silverå Ejneby M , Đerek V , Schmidt T , Gryszel M , Brask J , Schindl R , Simon DT , Berggren M , Elinder F , Głowacki ED .

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Optical control of the electrophysiology of single cells can be a powerful tool for biomedical research and technology. Here, we report organic electrolytic photocapacitors (OEPCs), devices that function as extracellular capacitive electrodes for stimulating cells. OEPCs consist of transparent conductor layers covered with a donor-acceptor bilayer of organic photoconductors. This device produces an open-circuit voltage in a physiological solution of 330 mV upon illumination using light in a tissue transparency window of 630 to 660 nm. We have performed electrophysiological recordings on Xenopus laevis oocytes, finding rapid (time constants, 50 μs to 5 ms) photoinduced transient changes in the range of 20 to 110 mV. We measure photoinduced opening of potassium channels, conclusively proving that the OEPC effectively depolarizes the cell membrane. Our results demonstrate that the OEPC can be a versatile nongenetic technique for optical manipulation of electrophysiology and currently represents one of the simplest and most stable and efficient optical stimulation solutions.

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GO keywords: potassium channel inhibitor activity

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	Fig. 1. The OEPC at the single-cell level.(A) The mechanism of action of OEPC devices relies on photoinduced charge transfer between H2Pc (P-layer) as the electron donor and PTCDI (N-layer) as the electron acceptor. The conducting ITO layer plays a critical role in storing positive charge and serving as the return electrode in solution. (B) EPR measurements are used to characterize the photocharging dynamics of OEPC devices. Photovoltage is measured between the ITO and an Ag/AgCl reference electrode in solution, while the corresponding charging current is registered by measuring a voltage drop across a resistor. The insets show a photograph of an OEPC device on ITO-coated PET foil and a schematic of the EPR measurement configuration. (C) Cross-section of the OEPC device architecture, with an illustration of the capacitive coupling mechanism with an adjacent oocyte in the physiological electrolyte. Illustration shows the positive (+) and negative (−) charge density at the moment when photoinduced currents in the electrolyte have fully capacitively charged the oocyte. Single-cell electrophysiology experiments were carried out using one or two intracellular electrodes. The transient potential measured intracellularly by the voltage electrode is defined as VT in this paper. The actual membrane potential across a given region of the cell membrane is defined as VM. The sign of the induced VM is represented in the illustration by the blue (positive) and red (negative) color of the cell membrane. In the vertical dimension, the thickness of the ITO/P/N structure is expanded for clarity. The thickness of the device layers is 110/30/30 nm, respectively. For voltage-clamp experiments where longer voltage transients are necessary, the capacitance of the device is boosted by adding a poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) layer onto the ITO. (D) Photograph of the oocyte measurement setup with bottom LED illumination, with labeled PN region and ITO. The white arrow indicates the oocyte cell. Photo credit: E. D. Głowacki, Linköping University.
	Fig. 2. Stability and nanomorphology of OEPC devices.(A) Peak photovoltage measured using the EPR of a series of devices subjected to a light stress test over 178 days. Three different sterilization conditions were applied, with autoclave yielding the most stable devices and UV resulting in faster performance decay. (B) Peak photocurrent of the same set of samples measured over the stress/stability test. (C) SEM micrograph of the edge of the organic PN layer on the ITO bottom electrode. This interface was stable for a month of stress but then began to experience delamination. (D) SEM of an as-fabricated organic PN layer and of a device (E) after 178 days of light pulse stress.
	Fig. 3. Photoinduced transient voltages and their effect on oocyte membrane potential.(A) Photoinduced transient potential change measured intracellularly in an oocyte, showing the biphasic trace produced by a 10-ms illumination pulse. (B) VT for 1-ms pulse length. (C) Peak cathodic VT dependence on light intensity averages for 12 oocytes (pulse length, 250 μs). (D) Model of electrical potential during OEPC charging, showing both VT and arrows indicating ionic current direction and magnitude. The cell membrane is colored with a different color scale representing the calculated induced membrane potential, ∆VM. A resting membrane potential of −33 mV is assumed for all calculations. The bottom of the cell near the OEPC is depolarized, while the membrane on the top of the oocyte experiences a small hyperpolarization. A zoom of the cleft region showing the strongly depolarized region is given in the inset. A distance of 3 μm between the OEPC surface and the cell membrane is assumed for this calculation. Because of symmetry, only half of the oocyte is shown. (E) The calculated VT for a 1-ms pulse in the upper intracellular region of the oocyte (red trace) corresponds to the experimentally measured VT values (blue trace). The inset shows a three-dimensional (3D) projection of VM in different regions of the oocyte at the time point (50 μs after the light pulse is turned on) where the cathodic VT is maximum. The red region at the bottom of the oocyte represents the strongly depolarized part of the membrane near the OEPC surface. (F) 2D projection showing the time evolution of the induced membrane potential over the course of a 1-ms light pulse (turned on at t = 0.5 ms, turned off at t = 1.5 ms) and subsequent discharging (i.e., anodic peak).
	Fig. 4. Photostimulation effects on KVShaker-type channels in oocytes.(A) Voltage measurements on uninjected oocytes using a 5-ms light pulse in the case where the bare ITO is the back electrode and the ITO is modified with PEDOT:PSS. Because of the higher capacitance given by PEDOT:PSS, the photocapacitive charging currents are larger and are exhibited over longer time, thus applying higher transductive extracellular potentials. Maximizing VT in a time scale of ≥2 ms is critical for observing effective stimulation of KV channels. We use VT@5 ms here as a convenient reference. (B) Voltage-clamp measurements at various voltage steps in uninjected oocytes, showing the voltage-independent capacitive response of the cell to the light pulse. (C) Voltage-clamp measurements of an oocyte transfected with the Shaker KV channel, demonstrating the photoinduced increase in outward K+ current. (D) G(V) characteristic for WT Shaker KV channels, for dark conditions (black trace) and during the light pulse (red trace). The green trace is for measurement after addition of the KV channel blocker 4-AP. (E) G(V) characteristics comparing dark conditions versus light pulses for 3R Shaker mutant channels. The onsets are shifted relative to WT. However, the photoinduced shift behavior is the same. (F) Correlation between the observed voltage shift in the light versus dark G(V) and the transient voltage at the end of a 5-ms light pulse measured in the same cell. The higher transient voltage VT corresponds to greater observed shifts in the G(V) characteristics.

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