Micro‐ and Nano‐Technologies for Lipid Bilayer‐Based Ion‐Channel Functional Assays

Ion channel proteins provide gated pores that allow ions to passively flow across cell membranes. Owing to their crucial roles in regulating transmembrane ion flow, ion channel proteins have attracted the attention of pharmaceutical investigators as drug targets for use in the studies of both therapeutics and side effects. In this review, we discuss the current technologies that are used in the formation of ion channel‐integrated bilayer lipid membranes (BLMs) in microfabricated devices as a potential platform for next‐generation drug screening systems. Advances in BLM fabrication methodology have allowed the preparation of BLMs in sophisticated formats, such as microfluidic, automated, and/or array systems, which can be combined with channel current recordings. A much more critical step is the integration of the target channels into BLMs. Current technologies for the functional reconstitution of ion channel proteins are presented and discussed. Finally, the remaining issues of the BLM‐based methods for recording ion channel activities and their potential applications as drug screening systems are discussed.     

Synthesis of 3‐[4‐Acyl‐2‐(1‐methoxy‐1‐methylethyl)morpholin‐3‐yl]‐benzonitriles as Novel Potassium Channel Openers

Potassium channel openers (KCO's) have been demonstrated to possess potent relaxant‐activity on smooth muscle. Tissue‐selective KCO's may find use in the treatment of a variety of diseases, such as hypertension, asthma, and urinary incontinence. We have previously reported a series of 1,9‐dioxa‐4‐aza‐phenanthrene‐6‐carbonitriles, including compounds 2 & 3 , as bladder‐selective KCO's. As a continuation of our efforts, we have designed 3‐[4‐acyl‐2‐(1‐methoxy‐1‐methylethyl)morpholin‐3‐yl]‐benzonitriles as ring‐opened analogs of compounds 2 & 3 . In this report, we describe the efficient construction of the novel 2,3‐disubstituted morpholine structure, as represented by the synthesis of compounds 4‐7 . Compounds 4‐7 showed potent and selective relaxant‐activity on rat bladder detrusor strip preparation. In this series, the most potent derivatives are Boc‐substituted analogs 4 & 6 (IC₅₀ = 3.9 and 2.9 μM, respectively).     

Rapid mixing with high‐throughput in a semi‐active semi‐passive micromixer

In this paper, we investigate a novel alternating current electrothermal (ACET) micromixer driven by a high efficiency ACET micropump. The micromixer consists of thin film asymmetric pairs of electrodes on the microgrooved channel floor and array of electrode pairs fabricated on the top wall. By connecting electrodes with AC voltage, ACET forces are induced. Asymmetric microgrooved electrodes force the fluids along the channel, while lateral vortex pairs are generated by symmetric electrode pairs located on the top wall. Waviness of the floor increases contact area between two confluent streams within a narrow confinement. An active mixer operates as a semi active semi passive mixer. Effects of various parameters are investigated in details in order to arrive at an optimal configuration that provides for efficient mixing as well as appreciable transport. It is found that using a specific design, uniform and homogeneous mixing quality with mixing efficiency of 97.25% and flow rate of per unit width of the channel can be achieved.     

Ultrathin polymer gate buffer layer for air‐stable,low‐voltage,n‐channel organic thin‐film transistors

An ultrathin poly(methyl methacrylate) (PMMA) buffer layer was developed to improve the performance of n‐channel organic thin‐film transistors (OTFTs). The 8 nm‐thick PMMA film, prepared by spin‐coating, provided a very smooth surface and a uniform coverage on SiO₂ surface reproducibly, which was confirmed by X‐ray reflectivity (XR) measurement. Then, we fabricated N,N′‐ditridecyl‐3,4,9,10‐perylenetetracarboxylic diimide (PTCDI‐C13) thin‐film transistors with and without this 8 nm‐thick PMMA insulating layer on SiO₂ gate insulators and achieved one‐order increase of field‐effect mobility (up to 0.11 cm²/(Vs) in a vacuum), one‐half decrease of threshold voltage, and reduction of current hysteresis with the PMMA layer. Only TFTs with the PMMA layer displayed n‐channel operation in air and showed field‐effect mobility of 0.10 cm²/(Vs). We consider that electrical characteristics of n‐channel OTFTs were considerably improved because the ultrathin PMMA film could effectively passivate the SiO₂ insulator surface and decrease interfacial electron traps. This result suggests the importance of the ultrathin PMMA layer for controlling the interfacial state at the semiconductor/insulator interface and the device characteristics of OTFTs. Copyright © 2009 John Wiley & Sons, Ltd.     

Structure–Activity Studies of Cysteine‐Rich α‐Conotoxins that Inhibit High‐Voltage‐Activated Calcium Channels via GABAB Receptor Activation Reveal a Minimal Functional Motif

α‐Conotoxins are disulfide‐rich peptides that target nicotinic acetylcholine receptors. Recently we identified several α‐conotoxins that also modulate voltage‐gated calcium channels by acting as G protein‐coupled GABA_B receptor (GABA_BR) agonists. These α‐conotoxins are promising drug leads for the treatment of chronic pain. To elucidate the diversity of α‐conotoxins that act through this mechanism, we synthesized and characterized a set of peptides with homology to α‐conotoxins known to inhibit high voltage‐activated calcium channels via GABA_BR activation. Remarkably, all disulfide isomers of the active α‐conotoxins Pu1.2 and Pn1.2, and the previously studied Vc1.1 showed similar levels of biological activity. Structure determination by NMR spectroscopy helped us identify a simplified biologically active eight residue peptide motif containing a single disulfide bond that is an excellent lead molecule for developing a new generation of analgesic peptide drugs.     

Electrophysiological Characterization of Transport Across Outer‐Membrane Channels from Gram‐Negative Bacteria in Presence of Lipopolysaccharides

Multi‐drug resistance in Gram‐negative bacteria is often associated with low permeability of the outer membrane. To investigate the role of membrane channels in the uptake of antibiotics, we present an approach using fusion of native outer membrane vesicles (OMVs) into a planar lipid bilayer, allowing characterization of membrane protein channels in their native environment. Two major membrane channels from E. coli, OmpF and OmpC, were overexpressed from the host and the corresponding OMVs were collected. Each OMV fusion surprisingly revealed only single or few channel activities. The asymmetry of the OMVs translates after fusion into the lipid membrane with the lipopolysaccharides (LPS) dominantly present at the side of OMV addition. Compared to the conventional reconstitution method, the channels fused from OMVs containing LPS have similar conductance but a much broader distribution and significantly lower permeation. We suggest using outer membrane vesicles for functional and structural studies of membrane channels in the native membrane.     

4‐Naphthylmethyl Proline Forms a Channel Structure

Proline and proline derivatives are useful tools to control the structural properties of peptides and proteins, and thereby modulate numerous processes. Here, we show that proline derivatives can have unique structural properties in the solid state by presenting the crystal structure of zwitterionic (2S,4S)/(2S,4R)‐4‐[(naphthalen‐2‐yl)methyl]proline (H‐Nap‐OH). This amphiphilic proline derivative forms a columnar structure around large hydrophilic and small hydrophobic channels with diameters of 9 Å and 4 Å, respectively. We show that this architecture, which is unprecedented for amino acids, results from the combination of a hydrogen bond network between the ammonium and carboxylate moieties and π–π as well as CH–π interactions between the aromatic moieties.     

Ion Channel Behavior of a Supported Bilayer Lipid Membrane Composed of 5,5－Ditetradecyl－2－ （2－trimethyl－ammonioethyl）－l, 3－dioxane Bromide Modified Glassy Carbon Electrode

A synthetic cationic surfactant, 5,5-ditetradecyl-2-(2-trimethyl-ammonioethyl)-l,3-dioxane bromide (DTDB), was used to construct a supported bilayer lipid membrane (s-BLM) coatedon an underlying glassy carbon electrode (GCE). Electrochemical impedance spectroscopy (EIS), small-angle X-ray diffraction (SAXD) and cyclic voltammetry were used to characterize the s-BLM. Both EIS and SAXD data indicated that the synthetic lipid exists as a well-oriented bilayer in the membrane.The voltammetric study showed that the lipid membrane can open ion channels in the presence of ClO4^- stimulant with Ru(bpy)3^2 as marker ions and give distinct channel currents.The channels can be dosed and open up again many times by removing or introducing ClO4^- anions.     

Simple,low‐cost fabrication of semi‐circular channel using the surface tension of solder paste and its application to microfluidic valves

This work presents a simple, low‐cost method to fabricate semi‐circular channels using solder paste, which can amalgamate the cooper surface to form a half‐cylinder mold using the surface tension of Sn–Pd alloy (the main component in solder paste). This technique enables semi‐circular channels to be manufactured with different dimensions. These semi‐circular channels will then be integrated with a polymethylmethacrylate frame and machine screws to create miniaturized, portable microfluidic valves for sequential liquid delivery and particle synthesis. This approach avoids complicated fabrication processes and expensive facilities and thus has the potential to be a useful tool for lab‐on‐a‐chip applications.     

Sensitive and Selective Fluorescent and Colorimetric Sensor for Ag+ Based on the Supramolecular Self‐Assembly in Semi‐Water

Specific recognition of ultratrace levels of ions in semi‐water using super‐quicker methods is still a challenge for environmental monitoring. Herein we report a fluorescent and colormetric sensor ( ZH ) based on supramolecular self‐assembly, whose structure was destroyed by the addition of ultratrace of silver ions. The process promoted either naked eye visible color changes or fluorescence intensity quenched in conjunction with a wide pH range. Systematic studies revealed very high selectivity (0.07 µmol/L) for silver ions, and other common cations, e.g ., Hg²⁺, Cu²⁺, Cd²⁺, Pb²⁺ had nearly no influence on the sensing behavior. This sensor also served as a multiple use of component in sensing materials by addition of I⁻ into the mixture of ZH and Ag⁺ (about 5 times). What's more, ZH containing filter paper emerged distinct color and fluorescence changes upon exposure to silver (Ag⁺), which could be used as a portable method to undertake field testing for Ag ⁺ .     

The role of resonances in building cross sections: The Mittag‐Leffler expansion in a two‐channel scattering

The Mittag‐Leffler formalism for identifying resonance contributions to a two‐body scattering cross section is extended here from the one channel case to the two channel case. The reduced partial wave cross sections based on subtracting S‐matrix residues are compared with expressions which can be derived from the Breit‐Wigner approximation. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2009     

A Portable Fill‐and‐Flow Channel Biosensor with an Electrode to Predict the Effect of Interferences

A portable fill‐and‐flow channel biosensor with a “predictor electrode” is described. The predictor electrode, on the opposite channel wall and upstream of the detector electrode, allows direct evaluation of the current due to interfering species at the detector electrode. A model is developed which shows that the reaction at the upstream predictor electrode has no significant effect on the concentration distribution of the interfering species at the detector electrode. This enables a straightforward correction of the current at the detector electrode and a determination of the analyte concentration. The effect of interference from ascorbic acid in the detection of glucose in samples was efficiently removed by using such a concept. The recovery of glucose from samples containing ascorbic acid was 103% (s=12%, n=3). The approach was also applied to a biosensor for the determination of glucose in red wine samples containing polyphenolic interferents.     

PST‐24: A Zeolite with Varying Intracrystalline Channel Dimensionality

Herein we report the synthesis, structure solution, and catalytic properties of PST‐24, a novel channel‐based medium‐pore zeolite. This zeolite was synthesized via the excess fluoride approach. Electron diffraction shows that its structure is built by composite cas‐zigzag (cas‐zz) building chains, which are connected by double 5‐ring (d5r) columns. While the cas‐zz building chains are ordered in the PST‐24 framework, the d5r columns adopt one of two possible arrangements; the two adjacent d5r columns are either at the same height or at different heights, denoted arrangements S and D, which can be regarded as open and closed valves that connect the channels, respectively. A framework with arrangement D only has a 2D 10‐ring channel system, whereas that with arrangement S only contains 3D channels. In actual PST‐24 crystals, the open and closed valves are almost randomly dispersed to yield a zeolite framework where the channel dimensionality varies locally from 2D to 3D.     

Bonding and in‐channel microfluidic functionalization using the huisgen cyclization

The bonding of layers of silicone elastomers during the manufacturing of microfluidic devices is often accomplished using plasma oxidation. The process can be costly, may require a clean room and materials that ensure the flatness of the bonding layers and, as a consequence of hydrophobic recovery, can lead to high variability in the degree of adhesion. As importantly, the process precludes incorporation of chemical functionalities that work as anchors to immobilize biomolecules within the microfluidic channel. We hypothesized that it would be possible to fabricate microfluidic channels using the Huisgen 1,3‐dipolar cycloaddition of azides to alkynes to crosslink silicones and form PDMS elastomers and, in a subsequent step, bond one PDMS layer to another simply by heating, with the added advantage of producing microfluidic channels with embedded chemical functionalities. After thermal bonding, the microfluidic devices underwent cohesive failure (rupture of the elastomer layer) at ∼145 kPa during pressure tests, but did not exhibit adhesive failure (delamination), showing that the azide–alkyne reaction provides a strong bond between elastomer layers. Furthermore, the internal microfluidic channel surfaces retained alkyne and azide functionality during the process, as shown by the grafting of one or more fluorescent dyes, through Huisgen cyclization. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2018 , 56, 589–597     

Multi‐objective optimization of heating channels for rapid heating cycle injection mold using Pareto‐based genetic algorithm

Rapid heating cycle molding (RHCM) technology is a novel polymer injection molding method developed in recent years. In this paper, the principle of RHCM technology was introduced and a RHCM mold for producing a large‐size LCD TV panel was presented. Aiming at achieving a uniform temperature distribution on the cavity surface as well as making sure of the heating efficiency, the factors that influence temperature distribution and heating efficiency were analyzed. The center coordinates of the heating channels were considered as the main design variables. Multi‐objective functions for optimizing the temperature distribution uniformity and heating efficiency were proposed. The layout of the heating channels for a 46‐inch LCD TV panel RHCM mold was optimized by using the finite element method and Pareto‐based genetic algorithm (GA). The temperature distribution uniformity on the stationary mold insert cavity surface was largely improved by using optimal design results and the heating efficiency was also guaranteed. Copyright © 2009 John Wiley & Sons, Ltd.     

Permselectivity of Gramicidin A Channels Based on Single‐channel Recordings

The expression mechanism of permselectivity through a gramicidin A (gA) channel between two aqueous phases (W1 and W2) was investigated. When the concentration of CsCl or CsBr in W1 was equivalent to that in W2, the single‐channel current was proportional to the absolute value of the applied membrane potential. Although the single‐channel current linearly increased with increasing electrolyte concentration in W1 and W2 until about 0.1 M (mol dm^?3), it began to level off around 0.1 M, indicating that ion permeation across the channel pore is the rate‐determining step and that the saturation of the transporting ion within the channel pore provokes the leveling off. In the case of asymmetric composition of the electrolyte in W1 and W2, the monovalent cation and the counter anion were transported in the opposite direction through the gA channel pore or the bilayer lipid membrane around the gA channel. Finally, the experimental data was fitted using the Goldman‐Hodgkin‐Katz equation based on the relationship between the membrane potential and the single‐channel current to define the ratio of the diffusion coefficients of Cs⁺, Cl^?, and Br^? as 5.7 : 1.0 : 0.26.     

Dispersion of charged solute in charged micro‐ and nanochannel with reversible sorption

We study dispersion of a charged solute in a charged micro‐ and nanochannel with reversible sorption and derive an analytical solution for mass fraction in the fluid, transport velocity and dispersion coefficient. Electrical double layer formed on the charged surface gives rise to a charge‐dependent solute transport by modifying the transverse distribution of the solute. We discuss the effect of sorption and electrical double layer on solute transport and show that the coupling between sorption and electrical double layer gives rise to charge‐dependent transport even for a thin double layer. However, in this case, it can be reduced to a simple non‐charge‐dependent case by introducing the intrinsic sorption equilibrium constant.