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

Membranes

Mathias Winterhalter¹  Jayesh Arun Bafna¹  Richard Wagner¹  Michael J. Vellekoop²  Sander van den Driesche²  Tanzir Ahmed²  Roland Hemmler³  Karsten Gall³ 

[1] Department of Life Sciences and Chemistry, Jacobs University, D-28759 Bremen, Germany;Institute for Microsensors, -actuators and -systems (IMSAS), University of Bremen, D-28359 Bremen, Germany;Ionovation GmbH, D-49143 Bissendorf, Germany;

关键词: silicon nitride aperture;    Parylene-C;    Parylene-AF4;    bilayer lipid membrane (BLM);    outer membrane vesicle (OMV);   

DOI  :  10.3390/membranes12030309

来源: DOAJ

【 摘 要 】

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 µm 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).

【 授权许可】

Unknown