Optical and Electronic Ion Channel Monitoring from Native Human Membranes

Abstract

Transmembrane proteins represent a major target for modulating cell activity, both in terms of therapeutics drugs and for pathogen interactions. Work on screening such therapeutics or identifying toxins has been severely limited by the lack of available methods that would give high content information on functionality (ideally multimodal) and that are suitable for high-throughput. Here, we have demonstrated a platform that is capable of multimodal (optical and electronic) screening of ligand gated ion-channel activity in human-derived membranes. The TREK-1 ion-channel was expressed within supported lipid bilayers, formed via vesicle fusion of blebs obtained from the HEK cell line overexpressing TREK-1. The resulting reconstituted native membranes were confirmed via fluorescence recovery after photobleaching to form mobile bilayers on top of films of the polymeric electroactive transducer poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS). PEDOT:PSS electrodes were then used for quantitative electrochemical impedance spectroscopy measurements of ligand-mediated TREK-1 interactions with two compounds, spadin and arachidonic acid, known to suppress and activate TREK-1 channels, respectively. PEDOT:PSS-based organic electrochemical transistors were then used for combined optical and electronic measurements of TREK-1 functionality. The technology demonstrated here is highly promising for future high-throughput screening of transmembrane protein modulators owing to the robust nature of the membrane integrated device and the highly quantitative electrical signals obtained. This is in contrast with live-cell-based electrophysiology assays (e.g., patch clamp) which compare poorly in terms of cost, usability, and compatibility with optical transduction.

The Role of Relative Capacitances in Impedance Sensing with Organic Electrochemical Transistors

“…One common manifestation of such an ion‐permeable layer is a supported lipid bilayer (SLB), which has been used in combination with OECTs. [ 23,24 ] To capture the transistor response with an SLB formed along the gate, we used a common SLB equivalent circuit comprising a capacitor, C b (for “bilayer”), in parallel with a resistor, R b , connected in series with the gate capacitor. [ 32,33 ] With the bilayer present, the fractional potential drop over the channel capacitor becomes, …”

“…Similar impedance sensing modes have been used to study the integrity of tight junction cell layers and black lipid membranes, as well as for the characterization of supported lipid bilayer (SLB) formed on the OECT channel. [ 16,21–24 ] The device geometry influences the transistor performance mainly through the transconductance, g m . [ 25,26 ] The transconductance is an important figure‐of‐merit for the OECT, in general and for sensing in particular, since it describes the current modulation versus the applied gate potential ( g m = ∂ I D /∂ V G ) and thereby determines the amplification that the transistor can deliver.…”

Current‐Driven Organic Electrochemical Transistors for Monitoring Cell Layer Integrity with Enhanced Sensitivity

“…[64] Utilizing the OECT supported by imaging methods is state-of-the-art and provides valuable insights to understand monitored results on biosensing. [69,71,98] The group of Owens evolved a fully automated electrical wound-healing assay utilizing OECTs. [69] Taking advantage of the transparent nature of PEDOT:PSS, the healing process has been simultaneously monitored electrically and optically in this project.…”

Rational ion transport management mediated through membrane structures

“…They are therefore considered promising bioelectronics that can be used to stimulate nerves and record real-time signals. [317][318][319][320][321][322][323] This corresponding process involves both the normal ion transport from the electrolyte solutions to the CP layers and horizontal electron transport between the source and drain electrodes along the CP layers simultaneously. The representative CP layer is made of poly(3,4-ethyle-nedioxythiophene) doped with poly(styrenesulfonate) (PEDOT:PSS) because of its high electrical conductivity and good biocompatibility.…”