Multiplexed Microfluidic Device for Parallel Electrophysiological Measurements on Independent Planar Lipid Bilayers

Abstract

Bilayer lipid membranes (BLMs) are excellent platforms to study ion channels in a functional environment, and for drug screening assays. However, conventional electrophysiological BLM approaches do not meet the requirements for high throughput experiments due to the large volumes utilized, and the extensive manual procedures involved. In that context, microfluidics is a promising format to develop new platforms for high throughput electrophysiological measurements on ion channels incorporated in BLMs. Previously, we reported a simple microfluidic device for experimentation on BLMs (Stimberg, Small, 2013), and here we describe a multiplexed device along the same line, which contains 3 BLMs, each being optically accessible and independently electrically addressable. Due to the larger footprint, bonding of the device turned out to be a challenge and required thorough optimization. Furthermore, a novel procedure was developed for BLM formation: it consists of 3 pipetting steps, and is therefore easily amenable to automation using pipetting robots. BLMs were prepared using DPhPC (25 mg/mL) in n-decane, and measurements were conducted in 1 M KCl 10 mM HEPES buffer (pH 7.0). First, BLMs were formed successfully in the different apertures of the device. Next, they exhibit good stability over time upon continuous application of a 100 mV voltage, as well as excellent electrical properties (RSEAL= 27.9±4.9 GΩ; CMspec = 0.77±0.02 μF/cm2 & IRMS = 500±50 fA for 100-μm diameter apertures (n=6)), enabling low noise single channel recordings. Finally, first experiments conducted on pore-forming Gramicidin demonstrated a well-defined single channel behavior with a chord conductance of ca. 33 pS at 100 mV. We will report the fabrication of the multiplexed device, together with its thorough characterization, membrane preparation, as well as multiplexed measurements on various pore-forming peptides and proteins (e.g., gramicidin, alpha-hemolysin, OmpF).