Giga-Ohm Sealed 3D Artificial Cell Membranes in Microfluidic Chips for Advanced Electrophysiology Study

Abstract

Electrophysiological investigations of ion channels and nanopores require high-resolution, stable, and reproducible recording platforms. Conventional patch-clamp and bilayer lipid membrane (BLM) techniques suffer from limitations in scalability, stability, and compatibility with multiplexed recordings. To overcome these challenges, we develop a silicon-based microwell array chip integrated with Ag/AgCl microelectrodes, enabling multiplexed, high-resolution ion flux recordings. The platform incorporates a PDMS microchannel for controlled solution exchange and supports the formation of 3D freestanding lipid bilayers (3DFLBs) within 100 microwells. We employ electrochemical chlorination to achieve uniform Ag/AgCl electrode formation, ensuring stable and reproducible electrochemical performance across all 100 electrodes. To facilitate 3DFLBs formation under physiological conditions, we introduce a hydraulic pressure-assisted electroformation, which precisely regulates inter-lipid membrane fusion. By applying controlled hydraulic pressure, we achieve highly stable 3DFLBs with tunable size, ensuring biologic relevance while maintaining a completely solvent-free environment. The 3DFLBs exhibit giga-ohm electrical sealing, verified through fluorescent dye exclusion and current-voltage (I-V) measurements, confirming their suitability for single ion channel electrophysiology. We further demonstrate single ion channel recordings and biosensing applications using alpha-hemolysin (alpha-HL) nanopores, achieving real-time ion flux detection and molecular sensing using Alexa Fluor 488. These findings establish our Ag/AgCl-integrated 3DFLB platform as a robust alternative to conventional electrophysiological techniques, offering superior stability, scalability, and physiologic relevance for ion channel research, drug discovery, and synthetic membrane applications.