Microfluidics offer economy of reagents, rapid liquid delivery, and potential for automation of many reactions, but often require peripheral equipment for flow control. Capillary microfluidics can deliver liquids in a pre-programmed manner without peripheral equipment by exploiting surface tension effects encoded by the geometry and surface chemistry of a microchannel. Here, we review the history and progress of microchannel-based capillary microfluidics spanning over three decades. To both reflect recent experimental and conceptual progress, and distinguish from paper-based capillary microfluidics, we adopt the more recent terminology of capillaric circuits (CCs). We identify three distinct waves of development driven by microfabrication technologies starting with early implementations in industry using machining and lamination, followed by development in the context of micro total analysis systems (μTAS) and lab-on-a-chip devices using cleanroom microfabrication, and finally a third wave that arose with advances in rapid prototyping technologies. We discuss the basic physical laws governing capillary flow, deconstruct CCs into basic circuit elements including capillary pumps, stop valves, trigger valves, retention valves, and so on, and describe their operating principle and limitations. We discuss applications of CCs starting with the most common usage in automating liquid delivery steps for immunoassays, and highlight emerging applications such as DNA analysis. Finally, we highlight recent developments in rapid prototyping of CCs and the benefits offered including speed, low cost, and greater degrees of freedom in CC design. The combination of better analytical models and lower entry barriers (thanks to advances in rapid manufacturing) make CCs both a fertile research area and an increasingly capable technology for user-friendly and high-performance laboratory and diagnostic tests.

The native environment of membrane proteins is complex and scientists have felt the need to simplify it to reduce the number of varying parameters. However, experimental problems can also arise from oversimplification which contributes to why membrane proteins are under-represented in the protein structure databank and why they were difficult to study by nuclear magnetic resonance (NMR) spectroscopy. Technological progress now allows dealing with more complex models and, in the context of NMR studies, an incredibly large number of membrane mimetics options are available. This review provides a guide to the selection of the appropriate model membrane system for membrane protein study by NMR, depending on the protein and on the type of information that is looked for. Beside bilayers (of various shapes, sizes and lamellarity), bicelles (aligned or isotropic) and detergent micelles, this review will also describe the most recent membrane mimetics such as amphipols, nanodiscs and reverse micelles. Solution and solid-state NMR will be covered as well as more exotic techniques such as DNP and MAOSS.

Bicelles are model membranes generally made of long-chain dimyristoylphosphatidylcholine (DMPC) and short-chain dihexanoyl-PC (DHPC). They are extensively used in the study of membrane interactions and structure determination of membrane-associated peptides, since their composition and morphology mimic the widespread PC-rich natural eukaryotic membranes. At low DMPC/DHPC (q) molar ratios, fast-tumbling bicelles are formed in which the DMPC bilayer is stabilized by DHPC molecules in the high-curvature rim region. Experimental constraints imposed by techniques such as circular dichroism, dynamic light scattering, or microscopy may require the use of bicelles at high dilutions. Studies have shown that such conditions induce the formation of small aggregates and alter the lipid-to-detergent ratio of the bicelle assemblies. The objectives of this work were to determine the exact composition of those DMPC/DHPC isotropic bicelles and study the lipid miscibility. This was done using 31P nuclear magnetic resonance (NMR) and exploring a wide range of lipid concentrations (2–400 mM) and q ratios (0.15–2). Our data demonstrate how dilution modifies the actual DMPC/DHPC molar ratio in the bicelles. Care must be taken for samples with a total lipid concentration ≤250 mM and especially at q ∼ 1.5–2, since moderate dilutions could lead to the formation of large and slow-tumbling lipid structures that could hinder the use of solution NMR methods, circular dichroism or dynamic light scattering studies. Our results, supported by infrared spectroscopy and molecular dynamics simulations, also show that phospholipids in bicelles are largely segregated only when q > 1. Boundaries are presented within which control of the bicelles’ q ratio is possible. This work, thus, intends to guide the choice of q ratio and total phospholipid concentration when using isotropic bicelles.

Single-channel electrophysiology with lipid bilayer systems requires ion channel expression, purification from cell culture, and reconstitution in proteoliposomes for delivery to a planar bilayer. Here we demonstrate that single-channel current measurements of the potassium channels KcsA and hERG S5-S6 can be obtained by direct insertion in interdroplet lipid bilayers from microliters of a cell-free expression medium.Electrophysiology is the gold standard for the functional characterization of ion channel proteins, including screening of new drugs that target this pharmaceutically important class of membrane proteins. 1,2 The patch clamp technique involves intimate contact of an electrode-containing glass pipette with the membrane of a cell in which the channel of interest is overexpressed, and is used most widely for ensemble current measurements of channel populations. The alternative 'bilayer lipid membrane' (BLM) approach, in which puried ion channels are introduced by proteoliposome fusion into a pure-lipid membrane that separates two electrode-containing aqueous compartments, is typically employed for current measurements of single channels. 3,4 Automated instruments for mediumthroughput patch clamp electrophysiology have entered the market in the last decade, 5,6 whereas systems for automated and/or parallel BLM electrophysiology are under development by several research groups and companies. [7][8][9] Although there are recent developments toward patch clamping of giant proteoliposomes, 10 patch clamping typically relies on eukaryotic cells that are able to overexpress the ion channel of interest with concomitant cell membrane incorporation. 2 However, this is not possible when the expressed channel aggregates or when membrane incorporation is toxic to the cell. Proteoliposome formation, for patch clamping or for the BLM method, also requires ion channel overexpression, but, at the cell culture stage, not necessarily as solubilized or membrane-incorporated protein, and is not restricted to eukaryotic cells. 3 This offers more options to obtain the desired ion channel but at the cost of having to purify it from cell culture, with subsequent reconstitution from detergent solution into liposomes, which is a laborious process that requires a relatively high protein yield.In recent years, considerable progress has been made with the cell-free expression of membrane proteins, 11 which bypasses any cytotoxicity problems and facilitates protein purication. Bacterial lysates that contain the ribosomal machinery and are supplemented with amino acids, a metabolic energy supply and a protein-encoding plasmid, have been shown to express a large variety of membrane proteins, either as precipitates or solubilized in detergent micelles. 12-14 Interestingly, when liposomes are added to the cell-free reaction mixture, spontaneous reconstitution has been demonstrated for a variety of cell-free expressed membrane proteins, including stearyl-CoA desaturase, glucan synthase, ATP synthase, DesK thermosensor, endothelin rece...

The cytoplasmic polyadenylation element binding protein (CPEB) homologue Orb2 is a functional amyloid that plays a key regulatory role for long-term memory in Drosophila. Orb2 has a glutamine, histidine-rich (Q/H-rich) domain that resembles the Q/H-rich, metal binding domain of the Hpn-like protein (Hpnl) found in Helicobacter pylori. In the present study, we used chromatography and isothermal titration calorimetry (ITC) to show that the Q/H-rich domain of Orb2 binds Ni2+ and other transition metals ions with μM affinity. Using site directed mutagenesis, we show that several histidine residues are important for binding. In particular, the H61Y mutation, which was previously shown to affect the aggregation of Orb2 in cell culture, completely inhibited metal binding of Orb2. Finally, we used thioflavin T fluorescence and electron microscopy images to show that Ni2+ binding induces the aggregating of Orb2 into structures that are distinct from the amyloid fibrils formed in the absence of Ni2+. These data suggest that transition metal binding might be important for the function of Orb2 and potentially long-term memory in Drosophila.