Abstract
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).
Highlights
Pores located in the cell membrane of biological species play an important role in controlling cellular activities
To resolve the above-mentioned issues, we have presented a Parylene-coated silicon/silicon nitride chip where a thin bilayer lipid membranes (BLM)
Microfabricated silicon nitride apertures on a silicon substrate were conformal coated with Parylene
Summary
Pores located in the cell membrane of biological species play an important role in controlling cellular activities. As a promising alternative, stroking an air bubble covered with lipid in the liquid reservoir near the aperture, a BLM can be formed This method requires the tip of a pipette precoated with lipid solution (lipids dissolved in n-octane). The lipid bilayer membrane with fused pore proteins realized in a Parylene-coated aperture functions as a biological sensing unit. Such systems are very suitable for membrane translocation measurements such as antibiotic candidate screening and DNA sequence analysis [47,48]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have