Spatial Bias in Antibody Microarrays May Be an Underappreciated Source of Variability.

Antibody microarrays enable multiplexed protein detection with minimal reagent consumption, but they continue to be plagued by lack of reproducibility. Chemically functionalized glass slides are used as substrates, yet antibody binding spatial inhomogeneity across the slide has not been analyzed in antibody microarrays. Here, we characterize spatial bias across five commercial slides patterned with nine overlapping dense arrays (by combining three buffers and three different antibodies), and we measure signal variation for both antibody immobilization and the assay signal, generating 270 heatmaps. Spatial bias varied across models, and the coefficient of variation ranged from 4.6 to 50%, which was unexpectedly large. Next, we evaluated three layouts of spot replicates-local, random, and structured random-for their capacity to predict assay variation. Local replicates are widely used but systematically underestimate the whole-slide variation by up to seven times; structured random replicates gave the most accurate estimation. Our results highlight the risk and consequences of using local replicates: the underappreciation of spatial bias as a source of variability, poor assay reproducibility, and possible overconfidence in assay results. We recommend the detailed characterization of spatial bias for antibody microarrays and the description and use of distributed positive replicates for research and clinical applications.

Capillary microfluidics in microchannels: from microfluidic networks to capillaric circuits.

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.

Metal Binding Properties of the N-Terminus of the Functional Amyloid Orb2.

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.

Comparative study of the structure and interaction of the pore helices of the hERG and Kv1.5 potassium channels in model membranes.

The hERG channel is a voltage-gated potassium channel found in cardiomyocytes that contributes to the repolarization of the cell membrane following the cardiac action potential, an important step in the regulation of the cardiac cycle. The lipids surrounding K+ channels have been shown to play a key role in their regulation, with anionic lipids shown to alter gating properties. In this study, we investigate how anionic lipids interact with the pore helix of hERG and compare the results with those from Kv1.5, which possesses a pore helix more typical of K+ channels. Circular dichroism studies of the pore helix secondary structure reveal that the presence of the anionic lipid DMPS within the bilayer results in a slight unfolding of the pore helices from both hERG and Kv1.5, albeit to a lesser extent for Kv1.5. In the presence of anionic lipids, the two pore helices exhibit significantly different interactions with the lipid bilayer. We demonstrate that the pore helix from hERG causes significant perturbation to the order in lipid bicelles, which contrasts with only small changes observed for Kv1.5. These observations suggest that the atypical sequence of the pore helix of hERG may play a key role in determining how anionic lipids influence its gating.

Magnetically Oriented Bicelles with Monoalkylphosphocholines: Versatile Membrane Mimetics for Nuclear Magnetic Resonance Applications.

Bicelles (bilayered micelles) are model membranes used in the study of peptide structure and membrane interactions. They are traditionally made of long- and short-chain phospholipids, usually dimyristoylphosphatidylcholine (D14PC) and dihexanoyl-PC (D6PC). They are attractive membrane mimetics because their composition and planar surface are similar to the native membrane environment. In this work, to improve the solubilization of membrane proteins and allow their study in bicellar systems, D6PC was replaced by detergents from the monoalkylphosphocholine (MAPCHO) family, of which dodecylphosphocholine (12PC) is known for its ability to solubilize membrane proteins. More specifically 12PC, tetradecyl- (14PC), and hexadecyl-PC (16PC) have been employed. To verify the possibility of making bicelles with different hydrophobic thicknesses to better accommodate membrane proteins, D14PC was also replaced by phospholipids with different alkyl chain lengths: dilauroyl-PC (D12PC), dipalmitoyl-PC (D16PC), distearoyl-PC (D18PC), and diarachidoyl-PC (D20PC). Results obtained by 31P solid-state nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) at several lipid-to-detergent molar ratios (q) and temperatures indicate that these new MAPCHO bicelles can be formed under a variety of conditions. The quality of their alignment is similar to that of classical bicelles, and the low critical micelle concentration (CMC) of the surfactants and their miscibility with phospholipids are likely to be advantageous for the reconstitution of membrane proteins.

Lipid concentration and molar ratio boundaries for the use of isotropic bicelles.

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 (31)P 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 of cell-free expressed ion channels by direct incorporation in lipid bilayers.

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 hERGS5-S6 can be obtained by direct insertion in interdroplet lipid bilayers from microliters of a cell-free expression medium.

Choosing membrane mimetics for NMR structural studies of transmembrane proteins.

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.