Freestanding lipid bilayers are one of the most used model systems to mimic biological cell membranes. To form an unsupported bilayer, we employ two aqueous fingers in a microfluidic chip surrounded by an oily phase that contains lipids. Upon pushing two aqueous fingers forward, their interface becomes decorated with a lipid monolayer and eventually zip to form a bilayer when the monolayers have nanoscopic contact with each other. Using this straightforward approach, the quick and easy bilayer formation is facilitated by oil draining into the microfluidic device material consisting of polydimethylsiloxane. However, the oil drainage limits the lifetime of a bilayer to about 1 h. We demonstrate that this drainage can be managed, resulting in superior bilayer stability and an increased lifetime of several hours when using a pressure-controlled system. Applying different pressures to the aqueous fingers in the microfluidic chip, the formed bilayer can even be bent to a desired curvature. Extracting the contact angle and the resulting curvature of the bilayer region, for a given applied pressure difference, both the bilayer tension and the surface tension of each lipid monolayer can be derived from a single experiment using the Young Laplace pressure equation.
Transmembrane receptor proteins are located in the plasma membranes of biological cells where they exert important functions. Archaerhodopsin (Arch) proteins belong to a class of transmembrane receptor proteins called photoreceptors that react to light. Although the light sensitivity of proteins has been intensely investigated in recent decades, the electrophysiological properties of pore-forming Archaerhodopsin (Arch), as studied in vitro, have remained largely unknown. Here, we formed unsupported bilayers between two channels of a microfluidic chip which enabled the simultaneous optical and electrical assessment of the bilayer in real time. Using a cell-free expression system, we recombinantly produced a GFP (green fluorescent protein) labelled as a variant of Arch-3. The label enabled us to follow the synthesis of Arch-3 and its incorporation into the bilayer by fluorescence microscopy when excited by blue light. Applying a green laser for excitation, we studied the electrophysiological properties of Arch-3 in the bilayer. The current signal obtained during excitation revealed distinct steps upwards and downwards, which we interpreted as the opening or closing of Arch-3 pores. From these steps, we estimated the pore radius to be 0.3 nm. In the cell-free extract, proteins can be modified simply by changing the DNA. In the future, this will enable us to study the photoelectrical properties of modified transmembrane protein constructs with ease. Our work, thus, represents a first step in studying signaling cascades in conjunction with coupled receptor proteins.
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