Solute transport on the sub 100 ms scale across the lipid bilayer membrane of individual proteoliposomes

Abstract

Screening assays designed to probe ligand and drug-candidate regulation of membrane proteins responsible for ion-translocation across the cell membrane are wide spread, while efficient means to screen membrane-protein facilitated transport of uncharged solutes are sparse. We report on a microfluidic-based system to monitor transport of uncharged solutes across the membrane of multiple (>100) individually resolved surface-immobilized liposomes. This was accomplished by rapidly switching (<10 ms) the solution above dye-containing liposomes immobilized on the floor of a microfluidic channel. With liposomes encapsulating the pH-sensitive dye carboxyfluorescein (CF), internal changes in pH induced by transport of a weak acid (acetic acid) could be measured at time scales down to 25 ms. The applicability of the set up to study biological transport reactions was demonstrated by examining the osmotic water permeability of human aquaporin (AQP5) reconstituted in proteoliposomes. In this case, the rate of osmotic-induced volume changes of individual proteoliposomes was time resolved by imaging the self quenching of encapsulated calcein in response to an osmotic gradient. Single-liposome analysis of both pure and AQP5-containing liposomes revealed a relatively large heterogeneity in osmotic permeability. Still, in the case of AQP5-containing liposomes, the single liposome data suggest that the membrane-protein incorporation efficiency depends on liposome size, with higher incorporation efficiency for larger liposomes. The benefit of low sample consumption and automated liquid handling is discussed in terms of pharmaceutical screening applications.

Size-dependent, stochastic nature of lipid exchange between nano-vesicles and model membranes

“…The aim of the work is to decipher the dominant mechanism for the exchange, being either transfer of individual lipids in many steps or the transfer of many lipids in one or a few steps as an effect of stalk formation. To overcome these limitations, a range of assays, based on microscopy imaging of surface adhering vesicles, have been developed for a range of studies, including vesicles fusion, 25 membrane transport, 26,27 protein/peptidemembrane interaction [28][29][30] and membrane active enzymes. The close contact between membranes can be established by nonspecific interactions, 19 such as electrostatic charge on membrane surfaces, [20][21][22][23] or forces due to solute depletion in the inter-membrane hydration layer.…”

Controlled microfluidic switching in arbitrary time-sequences with low drag

“…In many single-molecule and single-cell studies it is of interest to use microfluidic systems to explore a molecule's or cell's response to changes in the surrounding chemical environment. For instance, microfluidic switching devices have been used to study ion translocation in ion channels in response to quick changes in ion concentration, [1][2][3] and neuron cell response to transmitters and drugs, 4,5 and they have enabled performance measurements of single motor proteins in response to controlled changes in chemical environment. 6 A limitation of existing techniques is the inability to switch three or more fluids in an arbitrary time sequence while keeping drag forces on surface-adhered molecules low.…”

Development of a Biomembrane Sensor Based on Reflectometry

“…However, the focus will be placed here on surface-based biosensor technologies that allow for the direct measurement of ligand binding, since compared to other techniques, they offer the possibility of rapidly screening multiple recognition events. Furthermore, they may be combined with microfluidic handling, which makes them compatible with small sample volumes and thus ideally suited for studies of substances that are rare or time-consuming and expensive to obtain [7].…”

“…However, the separation can not be absolute. In order for cells to maintain energy production, biosynthesis and communication with their environment, they are critically dependent on molecules, ions and signals being selectively transported across this barrier [7]. Membrane proteins control many of these processes.…”

“…One of the main advantages of surface-based bioanalytical sensor technologies is that they offer the possibility of rapidly screening multiple recognition events either sequentially or simultaneously. The emerging importance of these sensors in the field of life science stems from the fact that they can be combined with microfluidic handling, which makes them compatible with small sample volumes and multiplexing rendering them ideally suited for studies of substances that are rare or time-consuming and expensive to obtain [7]. Transport assay formats relying on immobilised vesicles have been developed based on ATR-FTIR, SPR, fluorescence and second harmonic signal generation (SHG) spectroscopy.…”