Aquaporin embedded suspended lipid bilayer on anodized alumina nanoporous substrates for studying stability and functionality: towards the development of a miniaturized water purifier

Abstract

Porous substrates are crucial for suspended lipid bilayers, yet the influence of pore dimensions on bilayer stability and function remains underexplored. Understanding how these complementary platforms affect bilayer properties is essential for developing reliable biomimetic systems, and studying transmembrane proteins. We fabricated pores at different scales (micro- and nanoscale) by employing an aluminium-based substrate, using manual puncturing for micropores and anodization for nanopores. This approach was designed to address the intricate role of porous substrates in modulating the bilayer suspension, stability and function of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) lipid bilayers. Micropores provide optical access for real-time imaging of bilayer coverage and defects, while nanopores offer stronger mechanical stability, as confirmed by impedance spectroscopy with giga-seal resistance (∼2 GΩ). Using this stable platform, aquaporin, a water-channel protein, was reconstituted into lipid bilayers using an n-octyl-β-D glucopyranoside (NOG) detergent-mediated method. Dynamic light scattering, zeta potential, and impedance analyses confirmed successful insertion, and forward osmosis assays demonstrated functional water transport. The study represents a comparative framework, demonstrating the trade-off between accessibility and stability and providing design principles for future bilayer-based biomimetic and protein reconstitution platforms.