Silicon Nitride-Based Micro-Apertures Coated with Parylene for the Investigation of Pore Proteins Fused in Free-Standing Lipid Bilayers

In this work, we present a microsystem setup for performing sensitive biological membrane translocation measurements. Thin free-standing synthetic bilayer lipid membranes (BLM) were constructed in microfabricated silicon nitride apertures (<100 mu m in diameter), conformal coated with Parylene (Parylene-C or Parylene-AF4). Within these BLMs, electrophysiological measurements were conducted to monitor the behavior of different pore proteins. Two approaches to integrate pore-forming proteins into the membrane were applied: direct reconstitution and reconstitution via outer membrane vesicles (OMVs) released from Gram-negative bacteria. The advantage of utilizing OMVs is that the pore proteins remain in their native lipid and lipopolysaccharide (LPS) environment, representing a more natural state compared to the usage of fused purified pore proteins. Multiple aperture chips can be easily assembled in the 3d-printed holder to conduct parallel membrane transport investigations. Moreover, well defined microfabricated apertures are achievable with very high reproducibility. The presented microsystem allows the investigation of fast gating events (down to 1 ms), pore blocking by an antibiotic, and gating events of small pores (amplitude of approx. 3 pA).