Self-Assembly of Mammalian-Cell Membranes on Bioelectronic Devices with Functional Transmembrane Proteins.

Transmembrane proteins (TMPs) regulate processes occurring at the cell surface and are essential gatekeepers of information flow across the membrane. TMPs are difficult to study, given the complex environment of the membrane and its influence on protein conformation, mobility, biomolecule interaction, and activity. For the first time, we create mammalian biomembranes supported on a transparent, electrically conducting polymer surface, which enables dual electrical and optical monitoring of TMP function in its native membrane environment. Mammalian plasma membrane vesicles containing ATP-gated P2X2 ion channels self-assemble on a biocompatible polymer cushion that transduces the changes in ion flux during ATP exposure. This platform maintains the complexity of the native plasma membrane, the fluidity of its constituents, and protein orientation critical to ion channel function. We demonstrate the dual-modality readout using microscopy to characterize protein mobility by single-particle tracking and sensing of ATP gating of P2X2 using electrical impedance spectroscopy. This measurement of TMP activity important for pain sensing, neurological activity, and sensory activity raises new possibilities for drug screening and biosensing applications.

Optical and Electronic Ion Channel Monitoring from Native Human Membranes.

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.