Amphiphobic Septa Enhance the Mechanical Stability of Free-Standing Bilayer Lipid Membranes

Abstract

Artificial bilayer lipid membranes (BLMs) provide well-defined systems for investigating the fundamental properties of membrane proteins, including ion channels, and for screening the effect of drugs that act on them. However, the application of this technique is limited due to the low stability and low reconstitution efficiency of the process. We previously reported on improving the stability of BLM based on the fabrication of microapertures having a tapered edge in SiO/SiN septa and efficient ion channel incorporation based on vesicle fusion accelerated by a centrifugal force. Although the BLM stability and incorporation probability were dramatically improved when these approaches were used, some BLMs were ruptured when subjected to a centrifugal force. To further improve the BLM stability, we investigated the effect of modifying the surface of the SiO/SiN septa on the stability of BLM suspended in the septa. The modified surfaces were characterized in terms of hydrophobicity, lipophobicity, and surface roughness. Diffusion coefficients of the lipid monolayers formed on the modified surfaces were also determined. Highly fluidic lipid monolayers were formed on the amphiphobic substrates that had been modified with long-chain perfluorocarbons. Free-standing BLMs formed in amphiphobic septa showed a much higher mechanical stability, including tolerance to water movement and applied centrifugal forces with and without proteoliposomes, than those formed in the septa that had been modified with a short alkyl chain. These results demonstrate that highly stable BLMs are formed when the surface of the septa has amphiphobic properties. Because highly fluidic lipid monolayers that are formed on the septa seamlessly connect with BLMs in a free-standing region, the high fluidity of the lipids contributes to decreasing potential damage to BLMs when mechanical stresses are applied. This approach to improve the BLM stability increases the experimental efficiency of the BLM systems and will contribute to the development of high-throughput platforms for functional assays of ion channel proteins.

3D printed microfluidic devices for lipid bilayer recordings

“…It is known that the large capacitance and small resistance of a planar lipid bilayer impair the current signals of ion channels 35 and suppression of the electrical noise is indispensable to characterize ion channels. In the present study, the RMS electrical noise was suppressed to 0.56 ± 0.10 pA by optimizing dimensions of the separator, which was comparable to previous studies where the RMS noise ranged from 0.4 pA 33 to 0.9 pA. 23,26 Assuming a normal distribution of the current data, the peak-to-peak noise, which determines whether the target signals are resolved by the system, would be 3.3 pA (which is estimated to be 6 times of the RMS noise). A part of ion channels, such as the large-conductance calcium-activated potassium channel (BK) 8,36 or the transient receptor potential vanilloid 4 channel (TRPV4), 37 exhibit conductance over 80 pS, which represents the gating current larger than 4 pA at 50 mV bias.…”

“…29,31 When the separator met the above-mentioned conditions of high hydrophobicity and low surface roughness, the success rate of lipid bilayer formation reached 70-93%. In a previous study, the success rate ranged from 47% to 86%, 26 and our 3D printed device achieved a similarly good success rate.…”

“…4, the DCM devices fabricated by microArch or DigitalWax succeeded in forming a lipid bilayer when the device surfaces were hydrophobic, suggesting the results were in line with the previous ones. 26,27 In contrast, the device fabricated by AGILISTA showed a different trend. Lipid bilayer formation failed even though the surface of the device was hydrophobic with the Novec coating.…”

“…Regarding hydrophobicity, previous works also applied a hydrophobic separator to successfully form a lipid bilayer. 26 Since hydrophobic surfaces in air are oleophilic in water, 27 the hydrophobic separator will be covered with oil in aqueous solutions after the reagents are loaded to the DCM devices. In this context, a hydrophobic surface allows the separator to keep aqueous solution separate by oil at the aperture and to facilitate the orientation of lipid molecules on the surface, both of which are inevitable for forming a lipid bilayer.…”

Advances in Artificial Cell Membrane Systems as a Platform for Reconstituting Ion Channels

“…As another approach to stabilize BLMs, we next investigated the effect of surface modification of the Si chips on BLM stability. [46] The BLM in a free-standing region is seamlessly surrounded by lipid monolayers formed on chemically modified Si chips. A hydrophobic modification of the chip surface is necessary to align the fatty acid tails of phospholipids on the chip surface.…”