Characterizing Moderately Short Antimicrobial Tryptophan/Arginine-Rich Peptides

Abstract

Lifetime and stability of lipid bilayer membranes are essential parameters in ion channel reconstitution experiments. Reports on nano-BLMs across porous silicon nitride and alumina membranes with nanometer-sized apertures show a dramatically enhanced bilayer lifetime. In addition, the particular porous structure of the membranes enables the formation of a nano-BLM array, where failure of one membrane will not affect the membranes suspended across the other nanopores. However, a tradeoff between membrane thickness and mechanical stability is necessary when manufacturing these devices, with a three-dimensional multi-layer structure, substantially raising the cost of top-down device fabrication. In our study, the use of biogenic nanopores as substrates to host nano-BLMs was explored. Diatoms are unicellular marine algae in porous silica shells, called frustules. These shells have a tiered structure, with pores of minimum diameter 40 nm. The hierarchical porous structure of these shells makes them exceptionally mechanically stable. Frustules obtained from the marine algae Coscinodiscus wailesii were supported on a silicon platform with a micron-sized through-wafer aperture that allows fluidic access to either side of the frustule. Nano-BLMs were formed across the frustule pores using bubble-collapse method and were monitored by measuring the seal resistance and capacitance. Untreated frustules are hydrophilic, preventing the organic solvent from wetting the surface, resulting in a low BLM formation probability. Our studies indicate a formation probability of 1 out of 10 attempts. To render the surface hydrophobic, frustules were functionalized usingsilane chemistry, significantly improving bilayer formation probability. To study the effect of pore size on the incorporation probability and functionality of membrane proteins, the self-insertion of outer membrane protein OmpF of E.Coli in the nano-BLMs was investigated using single channel recordings. Blocking of the channel in the presence of the antibiotic ampicillin indicates the full functionality of the channel protein.