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 α-hemolysin (α-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.