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Artificial cell membranes such as liposomes and lipid vesicles have been developed using microfluidic technologies. These technologies have advantages for generating cell-sized liposomes, including a monodispersed and asymmetric lipid composition. Cell-sized asymmetric liposomes with various combinations of asymmetric lipid leaflets are sequentially produced using an improved pulsed-jetting device with the six wells arranged on a circular rotation table by manual manipulation. In this study, we reveal optimal parameters of rotation speed and jetting timing for the cell-sized liposome formation using an automatic sequential liposome generation system driven by a stepping motor. First, we optimize the rotation speed of the stepping motor for forming the planar lipid bilayer under control of a microcomputer. Next, we generate cell-sized liposomes using this automated system at the optimized rotation speed. This automatic system will contribute to high-throughput investigations of membrane protein activities and functions using the electrophysiological or the asymmetric liposome analyses.

An artificial cell membrane is applied to study the pore formation mechanisms of bacterial pore-forming toxins for therapeutic applications. Electrical monitoring of ionic current across the membrane provides information on the pore formation process of toxins at the single pore level, as well as the pore characteristics such as dimensions and ionic selectivity. However, the efficiency of pore formation detection largely depends on the encounter probability of toxin to the membrane and the fragility of the membrane. This study presents a bilayer lipid membrane array that parallelizes 4 or 16 sets of sensing elements composed of pairs of a membrane and a series electrical resistor. The series resistor prevents current overflow attributed to membrane rupture, and enables current monitoring of the parallelized membranes with a single detector. The array system shortens detection time of a pore-forming protein and improves temporal stability. The current signature represents the states of pore formation and rupture at respective membranes. The developed system will help in understanding the toxic activity of pore-forming toxins.

MicroRNAs have critical roles in a number of serious diseases and, as a result, are of major interest as clinical diagnostic targets. Conventionally, microRNAs are collected from blood and urine samples and are measured by either quantitative reverse-transcription polymerase chain reaction or microarray. Recently, nanopore sensing techniques have been applied for measuring microRNAs at the single-molecule level. However, existing techniques are technically complex, needing several tools and requiring purification and/or labeling of microRNA samples prior to use. Here we report a method for microRNA detection in a simple procedure requiring neither purification nor labeling. This system utilizes magnetic beads anchored with DNA and nanopores on a liposome membrane. In the presence of the target microRNA, it forms a duplex with complementary DNA, which is then cleaved by a duplex-specific nuclease (DSN). The cleaved DNA, which harbors a liposome on its terminus, is subsequently released from the magnetic bead, fuses to the lipid bilayer on chip, and emits an electrical signal derived from the formation of a nanopore. Because of a property of the DSN, the signals derived from microRNAs are expected to be amplified in an isothermal reaction. Our system possesses the specificity to detect target microRNAs from mixtures containing >106 different microRNA sequences and readily uses blood or urine samples. Although the limit of detection is above 10 nM and needs to be improved for practical diagnosis, because purification and labeling are not required, the presented system proposes a possible schematic for the development of easy and on-site diagnosis.

Current methods of generating asymmetric lipid vesicles produce only single types of vesicles, which poses a challenge for investigation of vesicles with different lipid leaflet combinations using a single device. Here, we describe a device for sequentially generating asymmetric lipid giant vesicles (GVs) with various combinations of asymmetric lipid leaflets. Various combinations of planar asymmetric lipid bilayers are formed by sliding and contacting the water in oil (phospholipid) (W/O) droplets in the collecting and jetting wells of our device. Next, we generate asymmetric lipid vesicles using a pulsed-jetting method. We sequentially generate three types of GVs: two asymmetric GVs containing fluorescent-conjugated phospholipids (either (i) rhodamine or (ii) BODIPY) on the outer leaflet, and (iii) a symmetric GV with phosphatidylcholine (PC) on the inner and outer leaflets. The use of asymmetric GVs with various combinations of asymmetric lipid leaflets reveals that increase in membrane phosphatidylethanolamine (PE) concentration influences cinnamycin activity and promotes the phospholipid flip-flop dynamics. This system will be useful for investigating activities of proteins or peptides on GV membranes with various combinations of lipid leaflets.

We report the functional synthesis and quantification of membrane proteins-α-hemolysin from Staphylococcus aureus and the multidrug transporter EmrE from Escherichia coli-at the stabilized droplet interface bilayer using an in vitro transcription-translation system. The system developed here can expand the list of integral membrane proteins applicable for quantitative functional analysis.

In investigations of the emergence of protocells at the origin of life, repeatable and continuous supply of molecules and ions into the closed lipid bilayer membrane (liposome) is one of the fundamental challenges. Demonstrating an abiotic process to accumulate substances into preformed liposomes against the concentration gradient can provide a clue. Here we show that, without proteins, cell-sized liposomes under hydrodynamic environment repeatedly permeate small molecules and ions, including an analogue of adenosine triphosphate, even against the concentration gradient. The mechanism underlying this accumulation of the molecules and ions is shown to involve their unique partitioning at the liposomal membrane under forced external flow in a constrained space. This abiotic mechanism to accumulate substances inside of the liposomal compartment without light could provide an energetically up-hill process for protocells as a critical step toward the contemporary cells. How small molecules could have accumulated within hypothetical protocells on the early Earth is an open question. Here automated microfluidic experiments provide evidence for abiotic accumulation of small molecules within cell-sized liposomes under hydrodynamic flow evoking a surface-mediated mechanism.

This work describes a gas flow system for an odorant sensor based on a droplet interface bilayer (DIB) device. In the previous study, the olfactory receptor (OR) was reconstituted in DIB device and odorant, 1-octen-3-ol (octenol), was successfully detected. As a next challenge, we aimed at the continuous monitoring of odorant. Here, we designed a gas flow pathway at the bottom of the droplet forming the bilayer, to effectively absorb the gas into the aqueous phase. The pathway consisted of a microchannel and a slit-structure at the bottom of a well that kept the droplet on top. This gas flow system was integrated as a 16-ch DIB device. By sequentially infusing the octenol gas and air into the channel, we confirmed the increase and decrease of octenol concentration in the droplet. Thus, the designed system was able to absorb and exchange odorant in the droplet. Remarkably, the controlled gas flow stirred inside the droplet, resulting in promoting the gas absorption.

This paper proposes a technique to reconstitute a lipid bilayer membrane by injecting an air bubble onto the raptured spot. Our group has been studying the artificial bilayer that can host membrane proteins and can be a fundamental to analyze their functions. The only drawback of the lipid bilayer formed at the interface of two water-in-oil droplets is its sustainability, namely, the bilayer often collapses and the droplets merges by the disturbance like self-exerted oscillation or sloshing. To overcome this issue, the newly proposed technique utilizes a bubble at approximately $4\ \mu \mathrm{L}$ in volume. When the bubble is injected and retracted, the new artificial bilayer is reconstituted. The major advantage of this technique is the in-situ reconstitution of the bilayer during the electrical measurement of the membrane proteins. The proposed technique was successfully verified by confirming the emergence of nanopore membrane proteins.

Lipid distribution in intracellular vesicles is different from that in the plasma membrane of eukaryotic cells. The lipid components in the intracellular vesicles are composed of phosphatidylserine and phosphatidylethanolamine in the outer leaflet and phosphatidylcholine and sphingomyelin in the inner leaflet. The lipid asymmetricities both in the intracellular vesicle membrane and the plasma membrane contribute to synaptic transmission functions. In this study, we developed a cell-sized asymmetric lipid vesicle system containing small-sized asymmetric lipid vesicles (of diameter 200–1000 nm) (asymmetric vesicles-in-a-vesicle), emulating lipid components in the plasma membrane and intracellular vesicle membrane of eukaryotic cells, using microfluidic technology. We successfully constructed an artificial exocytosis system using the asymmetric vesicles-in-a-vesicle system. This asymmetric vesicles-in-a-vesicle system will be helpful in understanding the mechanisms of vesicle transport, such as neurotransmission and exocytosis.

This work designs and demonstrates the lipid bilayer-based ultra-sensitive biosensor capable of short-time detection in a quantitative manner, which is achieved by connecting multiple sensor elements in parallel.Artificial cell membranes with functional membrane proteins can form sensitive/selective biosensors. But the sensors suffer lengthy detection time at low concentration of analytes because the sensing mechanism relies on stochastic phenomena.In this work, we connected independent membrane sensors in parallel, where the ionic current through the multiple membranes was monitored by a single detector. With this format, the detection time is shortened based on the number of the array and the sensing can be quantitative. We developed a quad sensor assembling four membranes in parallel, and examined detection time of a single membrane sensor, the quad sensor, and four parallel quad-sensors. 1-GΩ resistor was installed in series with each membrane to avoid overload current caused by membrane rupture. The results showed significant improvement in the detection time and deviation by the parallelization, which promises quantitative monitoring with the stochastic sensor.

This paper describes an odorant sensor based on artificial cell membrane device integrated with a system of solution exchange for repeatable odorant detection. The olfactory receptor (OR) was reconstituted in the membrane on the developed device and the target odorant, 1-octen-3-ol, was detected by the opening current of the ionotropic receptor (OR). Solution exchange, which is to refresh the absorbed odorant from OR, was implemented by continuous flow of a buffer solution, driven by suction force of superabsorbent polymer. Note that no electricity is required for the exchange system. To demonstrate the solution exchange, a biological nanopore was reconstituted in the membrane, and blocking phenomena was observed using hepta-6-sulfato-β-cyclodextrin. The frequency of current steps that correspond to blocking of the nanopore decreased over the solution exchange. We believe that this solution exchange system is applicable to the odorant sensor based on artificial cell membrane and realizes repeatable odorant detection.

This paper describes a method for a bilayer lipid membrane (BLM) formation using a perforated sheet along with an open chamber. Microscopic observation of the formed membrane showed a typical droplet interface bilayer. We proved that the formed membrane was a BLM based on electrical measurements of the membrane protein &alpha, hemolysin, which produces nanopores in BLMs. Unlike the conventional approach for BLM formation based on the droplet contact method, this method provides aqueous surfaces with no organic solvent coating layer. Hence, this method is suitable for producing BLMs that facilitate the direct addition of chemicals into the aqueous phase.

Nano-sized lipid vesicles (50–300 nm in diameter) are widely used in drug delivery systems and cosmetics. Recently, exosomes have been shown to be important as the carriers of biomolecules. The membrane structures of exosomes exhibit the same asymmetric lipid distributions as those of the plasma membranes of eukaryotic cells. The asymmetric lipid distributions of the exosome membranes play a key role in the interactions with and recognition of living cells. The asymmetric lipid distributions of nano-sized lipid vesicles facilitate delivery in advanced drug delivery systems, which emulate the cellular transportation systems of exosomes. In this study, we generate nano-sized asymmetric lipid vesicles using a pulsed-jet method for cell-sized asymmetric lipid vesicle formation. They were generated from an asymmetric planar lipid bilayer by applying pulsed-jet flows of longer duration and higher pressure than those used in the formation of micro-sized lipid vesicles. Using a transmission electron microscope, nano-sized lipid vesicles with thicknesses of approximately 5–6 nm (approximately 70 % of the generated sample), unilamellarity, and asymmetry were observed to be produced by this method. The nano-sized asymmetric lipid vesicles using this method have the potential to be useful as carriers in drug delivery systems.

This paper describes an odorant sensor based on mosquito olfactory receptors (ORs) that is sensitive to the volatile organic compound octenol. The ORs and OR coreceptors were reconstructed in the lipid bilayer membrane in a chamber device equipped with electrodes. Using this odorant sensor, we obtained ion current changes caused by specific OR responses to octenol. We installed the odorant sensor into a mobile robot and succeeded in the demonstration of coupling octenol gas detection and robot actuation. We believe that this biohybrid odorant sensing system will be a key technology for future artificial olfaction.

Ion channels are located in plasma membranes as well as on mitochondrial, lysosomal, and endoplasmic reticulum membranes. They play a critical role in physiology and drug targeting. It is particularly challenging to measure the current mediated by ion channels in the lysosomal and the endoplasmic reticulum membranes using the conventional patch clamp method. In this study, we show that our proposed device is applicable for an electrophysiological measurement of various types of ion channel in plasma and organelle membranes. We designed an on-chip device that can form multiple electrical contacts with a measurement system when placed on a mount system. Using crude cell membranes containing ion channels extracted from cultured cells without detergents, we detected open/close signals of the hERG, TRPV1, and NMDA channels on plasma membranes, those of the TRPML1 channels on lysosomal membranes, and open/close signals of the RyR channels on SR membranes. This method will provide a highly versatile drug screening system for ion channels expressed by various cell membranes, including plasma, SR, mitochondrial, Golgi, and lysosomal membranes.

This paper describes automation of planar lipid bilayer formation by introducing a stepping motor to a microfluidic device. Planar lipid bilayers or lipid vesicles are useful to understand biological reactions and to investigate the interaction between lipids and proteins. Therefore, to acquire large amount of the information, high-throughput production of planar lipid bilayers or giant vesicles (GVs) is necessary. The droplet split-and-contact method, which enhances the efficiencies of both planar lipid bilayer formation and GV generation, needs to be automated for increasing the throughput. Previous droplet split-and-contact devices were manipulated manually; hence, the influence of manipulation on planar lipid bilayer formation was not evaluated quantitatively. First, to develop an automated system for generating asymmetric planar lipid bilayers, a stepping motor, which allows to control the angular speed of the rotor, is integrated into the droplet split- and-contact device (Fig. $1(\mathrm{b)$). Next, we assessed planar lipid bilayer generation at various angular speeds and found the speed limit for bilayer formation. Finally, we generated asymmetric planar lipid bilayers that have different lipid composition on outer and inner leaflets using this automated device and confirmed the asymmetry of the planar lipid bilayers by generating GVs.

This paper shows a fabrication of a shallow chamber device for formation of bilayer lipid membranes (BLMs). The chamber device is composed of microperforated polytetrafluoroethylene thin film, and two magnetic rings that work as both electrodes and the chamber walls. Comparing to conventional open chamber devices for BLM formation that is based on droplet contact method, our approach can reduce the water depth, and it becomes that much easier to access the BLM via liquid ab extra. We confirmed that the formed membrane was BLM using membrane protein that made nanopore in BLMs. In addition, gas chromatographic analyses implied that adsorption of odorant into the buffer solution was more effective at shallower water level than deep position. We expect that this shallow chamber is potentially useful for odorant sensing based on olfactory receptors reconstructed in BLMs.

This paper proposes a formation method for oil-layer-free bilayer lipid membranes (BLM) by using a compressive force of DC electric field. Using a droplet contact device previously developed, constant-voltage steps were applied to the pre-bilayer membrane and carefully controlled the electric stress in response to the membrane features, based on optical and current monitoring. We examined the pathways of the membrane formation and disruption, and discovered a qualitative protocol for oil-layer-free BLM formation. We succeeded in BLM formation using 1:1-mixture of hexadecane and n-decane as an oil example, which was hardly available for the former studies using the device. The method would contribute to produce vivo-like membranes appropriate for membrane protein functionalities.

Artificial lipidic bilayers are widely used as a model for the lipid matrix in biological cell membranes. We use the Pockels electro-optical effect to investigate the properties of an artificial lipidic membrane doped with nonlinear molecules in the outer layer. We report here what is believed to be the first electro-optical Pockels signal and image from such a membrane. The electro-optical dephasing distribution within the membrane is imaged and the signal is shown to be linear as a function of the applied voltage. A theoretical analysis taking into account the statistical orientation distribution of the inserted dye molecules allows us to estimate the doped membrane nonlinearity. Ongoing extensions of this work to living cell membranes are discussed.

We present a microarray system that enables simultaneous monitoring of multiple ionic currents through transmembrane alpha-hemolysin nanopores arrayed at bilayer lipid membranes. We applied the self-assembling ability of lipid molecules interfaced between an aqueous solution and organic solvent to induce bilayer membrane formation at a microfluidic device; the device consists of a hydrophobic polymer film that serves to suspend the lipid-containing solvent at micrometer-sized apertures as well as to separate the aqueous solution into two chambers. In this study, we confirmed that expeditious and reproducible bilayer formation is realized by control of the composition of the solvent, a mixture of n-decane and 1-hexanol, which permits simultaneous incorporation of the alpha-hemolysin nanopores to the membrane array. Monitoring the eight wells on the array at once, we obtained a maximum of four relevant, synchronous signals of translocating ionic current through the nanopores. The system was also able to detect translocation events of nucleic acid molecules through the pore via the profile of a blocked current, promising its potential for high-throughput applications.

We develop a device adapted for repetitive formation of horizontal lipid bilayer membranes. This device allows simultaneous optical and electrophysiological measurements of the formed membranes. We integrated a rotational chamber on a fluidic channel separated by a parylene micropore. The rotational motion is designed to form a bilayer repeatedly. This rotational process emulates the conventional painting method, in which a thick lipid-oil layer at a micropore was made thinner by hand work to obtain a bilayer. The bilayer formation with our device was examined optically and electrically. The simultaneous measurement device will be useful for better understanding of bilayer features and membrane protein incorporation.

In this study, we examined the dielectric properties of an intermediate layer in a bipolar membrane, which is composed of a negatively charged layer and a positively charged layer joined in series. As a result of the time-dependent impedance measurements of charged membranes, the negative increment in electric conductivity and the positive increment in electric capacity were observed only in the case of a bipolar membrane under the application of reverse-biased voltages, which were quite different from the behavior of both monopolar membranes and of a bipolar membrane under forward-biased voltages. Further, the observed shifts showed a nearly constant value against the reverse-biased voltage. It is concluded that these characteristics coincide with the process of ion exclusion in the intermediate layer and are attributed to the water splitting mechanism.

This letter describes the integration of an electrical connection onto a lipid bilayer membrane microchip for the electrical detection/regulation of transmembrane phenomena together with fluorescence monitoring. Arrayed microchambers with a volume of 3 pL were fabricated within a poly(dimethyl siloxane) microfluidic device, and electrical access was achieved by designing channels adjacent to the chambers that were bridged by gold patterns on a glass substrate. We succeeded in the formation of a lipid bilayer membrane at the mouth of the chamber and applied electrical potential to the membrane with the integrated component. We confirmed the reconstitution of a transmembrane nanopore protein into the formed lipid bilayer membrane by signal monitoring with the electrical connection.

Asymmetric lipid giant vesicles have been used to model the biochemical reactions in cell membranes. However, methods for producing asymmetric giant vesicles lead to the inclusion of an organic solvent layer that affects the mechanical and physical characteristics of the membrane. Here we describe the formation of asymmetric giant vesicles that include little organic solvent, and use them to investigate the dynamic responses of lipid molecules in the vesicle membrane. We formed the giant vesicles via the inhomogeneous break-up of a lipid microtube generated by applying a jet flow to an asymmetric planar lipid bilayer. The asymmetric giant vesicles showed a lipid flip-flop behaviour in the membrane, superficially similar to the lipid flip-flop activity observed in apoptotic cells. In vitro synthesis of membrane proteins into the asymmetric giant vesicles revealed that the lipid asymmetry in bilayer membranes improves the reconstitution ratio of membrane proteins. Our asymmetric giant vesicles will be useful in elucidating lipid-lipid and lipid-membrane protein interactions involved in the regulation of cellular functions.

The transport of 5-fluorouracil (an anti-cancer medicine, 5FU) through a protein membrane was investigated. The membrane was prepared from silk fibroin. The permeability coefficients of 5FU were measured at different pH values ranging from 3.0 to 9.0. The protein membrane was composed of both weak acid and weak base groups—a so-called amphoteric membrane. The isoelectric point of this membrane is about pH 4.0. The measured results showed that the permeability coefficient of 5FU abruptly decreased above about pH 7 because 5FU became anionic in an alkaline solution and the membrane become a cation exchanger above pH 4.0. The abrupt decrease in permeation can be theoretically explained in terms of the Donnan equilibrium and the Nernst-Planck equation. Fibroin membranes are suggested to be potentially very useful in drug delivery systems.

The effect of polymer component and structure in the membrane interface on rectification and water splitting is discussed in order to examine the water splitting mechanism. Various types of bipolar membranes which had different amino groups in the anion exchange layer, different polyelectrolytes in the membrane interface, and different interface thickness of polystyrene were prepared. The current–voltage characteristics and membrane resistance were measured. If the space between the cation and anion exchange layers was produced, the water splitting effect was reduced. Water splitting effect was accelerated even if a quaternary amino group was inserted in the interface region instead of a secondary or ternary amino group, which implied that the water dissociation occurred not only due to the catalytic effect of the secondary or ternary amino group but also to the electric field. It was evidenced that the membrane which had a thick interface region showed a high conductivity before the critical point of thickness of the interface region, which meant an increase in the reactive region of the water. It was proved that if the anion exchange layer was composed of only binary or ternary amino groups, the water splitting effect was also reduced due to the low anion selectivity.

This report describes preliminary results of a reconstitution and function of membrane proteins (flippases) into asymmetric lipid giant liposomes (GLs) that were prepared by deforming a planar asymmetric bilayer using a pulsed jet flow. Frist, we confirmed the expression of flippases on the baculovirus membrane by a western blot analysis. Next, we successfully reconstituted flippases into the asymmetric GL of phosphatidylserine by a baculovirus-liposome fusion method. Finally, we observed the translocation of phosphatidylserine from the extracellular leaflet to the cytoplasmic leaflet which was catalyzed by flippases. The flippase-reconstituted asymmetric liposomes that emulate the function of cell membranes will be useful for the study of elementary lipid-lipid or lipid-protein interactions.

Although ion channels are attractive targets for drug discovery, the systematic screening of ion channel-targeted drugs remains challenging. To facilitate automated single ion-channel recordings for the analysis of drug interactions with the intra- and extracellular domain, we have developed a parallel recording methodology using artificial cell membranes. The use of stable lipid bilayer formation in droplet chamber arrays facilitated automated, parallel, single-channel recording from reconstituted native and mutated ion channels. Using this system, several types of ion channels, including mutated forms, were characterised by determining the protein orientation. In addition, we provide evidence that both intra- and extracellular amyloid-beta fragments directly inhibit the channel open probability of the hBK channel. This automated methodology provides a high-throughput drug screening system for the targeting of ion channels and a data-intensive analysis technique for studying ion channel gating mechanisms.

International audience; We present a molecular resolved image of a supported lipid bilayer using atomic force microscopy in liquid conditions. Due to the well-known molecular diffusion processes with velocities of a few nm/s occurring in free-standing membranes, this type of mapping is difficult to achieve. We have obtained a sub-nanometer scale resolution of a dioleoyl-sn-glycero-phosphocholine membrane because of the high sensitivity of our phase modulated microscope and the slowed down diffusion due to the interaction with the substrate. A pair function analysis has revealed local disordered and gel phases with an order range limited to the first neighbour.

Monitoring complex biological assays such as in vitro protein synthesis over long periods in micrometer-sized cavities of poly(dimethyl siloxane) (PDMS) microfluidic devices requires a strategy that solves the adsorption and absorption problems on PDMS surfaces. In this study, we developed a technique that instantaneously arrays aqueous microdroplets coated with a phospholipid membrane within a single microfluidic device. The simple lipid bilayer coating effectively inhibits the adsorption of proteins and DNA, whereas the encapsulation of the droplet reduces the area in contact with the PDMS surface, resulting in decreased absorption in part. Although the size becomes smaller during the first few hours, a lipid-coated microdroplet array demonstrated a temporal stability of more than 20 h and a size uniformity of CV 3% in the device. Furthermore, we succeeded in expressing a green fluorescent protein by confining an in vitro translation system in the microdroplets, which was confirmed by scanning the fluorescence spectrum of the droplets, demonstrating that the lipid coat secured the synthetic reaction from the adsorption problem.

This work presents a strategy that realizes efficient incorporation of membrane proteins in liposomes into a planar bilayer lipid membrane by using dielectrophoretic (DEP) force. The device consists of a chamber and a channel separated by a µm-sized aperture that suspends a planar bilayer membrane. Applying an AC electric field across the membrane, the DEP force concentrates at the bilayer area therefore attracts the liposomes. We also observed fusion of liposomes autonomously occurs into the lipid membrane under the DEP effect. This DEP-based fusion method will be highly applicable to membrane protein study.

This paper describes a microfluidic device for a high through-put ion channel recordings, in which planar bilayer lipid membranes (BLMs) are spanning across a nanometer-sized poly(p-xylylene) (parylene) pore (Figure 1). To make the nanopore, a micrometer-sized pore of parylene was prepared by photolithography, and then parylene was additionally deposited on to the micropore with the isotropic vaporization. As a result, we successfully obtained an 800 nm diameter pore in the parylene film. In addition, BLM was able to be formed stably at the parylene nanpores that was built into the microfluidic device with upper and lower fluidic channels. We also demonstrated to reconstitute of alpha-hemolysin (αHL) into the BLMs. This device will provide a high reliability platform for the ion-channel recordings.

In this paper, we examined two types of devices for the electrical recording of transmembrane currents: the material used for the electrodes in the first device was agarose gel, and that in the second device was gold. In the first device, we embedded the agarose gel into microfluidic channels, which becomes a “salt-bridge” connecting a micrometer-sized chamber to the external circuit electrically. Next, in the second device, the micrometer-sized chamber was connected to the external microfluidic channel through a gold pattern. As a result, we expect that the gold sheet connection device will more significantly contribute to membrane ion-channel study.

We report the successful formation of supported multicomponent lipid bilayer membranes (sLBMs) on polymer cushions consisting of a set of alternating maleic acid copolymers. The formation of sLBMs was triggered by a transient reduction of the electrostatic repulsion between the polymer cushions and the lipid vesicles by lowering the solution's pH to 4. Upon formation, the stability of the sLBMs was not affected by subsequent variations of the environmental pH. The degree of hydrophilicity and swelling of the anionic polymer cushions was found to determine both the kinetics of the membrane formation and the mobility of the lipid bilayer with lipid diffusion coefficients in the range from 0.26 to 2.6 microm2s(-1). The introduced polymer cushion system is concluded to provide a versatile base for the integration of active transmembrane proteins in sLBMs.

This paper describes microfluidic logic gates which use DNA and biological nanopores. Single-stranded DNA (ssDNA) can pass through αHL, a biological nanopore, incorporated in bilayer lipid membranes (BLMs), whereas double-stranded DNA (dsDNA) cannot. In this study, these passing and non-passing phenomena were applied as the binary system and logic gates. Two types of ssDNA were used as inputs, while the output was obtained by electrical signals across the nanopores, which recognizes whether ssDNA passed through the nanopores or not. NAND gate was successfully demonstrated by exploiting the mechanism. The proposed approach herein is significantly different from the conventional computation using DNA in the respect that electrical signals are directly obtained as the output, which drastically facilities the microfluidic system to connect to electrical systems for fast and accurate computing. In addition, it is not required to use fluorescence, enzyme or PCR in order to obtain outputs. We believe that this method leads to a rapid computing system using biomolecules.

The membrane potential characteristics of a bipolar membrane are discussed in an examination of the contribution of an intermediate phase to time-dependent behavior. Bipolar membranes, which consist of a poly(sulfone) base polymer with a quaternary amino group and a poly(styrene-co-divinylbenzene) cation-exchange membrane, were prepared in this study. The membrane potentials in various external concentration differences, the facing directions of the membrane, and the external electrolyte solutions were measured as a function of time. In this study, the time course of the membrane potential was simulated by using the equation based on the Teorell-Meyer-Sievers theory and by assuming the concentration in the intermediate phase, which is much higher or lower than that in the external solution. The membrane facing direction and the intermediate phase condition will alter the direction of the membrane potential change. The intermediate phase in a bipolar membrane seems to act as an alteration barrier for the membrane potential according to the membrane facing direction. Copyright 2001 Academic Press.

This paper proposes a solution exchange of a droplet-based lipid bilayer system, in which the inner solution of a droplet is replaced for the purpose of efficient ion channel analyses. In our previous report, we successfully recorded the channel conductance of alpha-hemolysin in a bilayer lipid membrane using a droplet contact method that can create a spontaneous lipid bilayer at the interface of contacting droplets; this method is widely used as highly efficient method for preparing planar lipid membranes. When only pipetting droplets of the solution, this method is highly efficient for preparing lipid membranes. However, the drawback of droplet-based systems is their inability to exchange the solution within the droplets. To study the effect of inhibitors and promoters of ion channels in drug discovery, it would be beneficial to conduct a solution exchange of droplets to introduce membrane proteins and to apply or wash-out the chemicals. In this study, we propose a droplet contact method that allows for the solution exchange of droplets via microfluidic channels. We experimentally and numerically investigated the bilayer stability with respect to exchanging flow rates, and then demonstrated a binding assay of an alpha-hemolysin using one of its blockers. The solution exchange in this system was conducted in less than 20 s without rupturing the membrane. We believe that the proposed system will enhance the efficiency of ion channel analyses.

ATP-binding cassette (ABC) transporters, a protein superfamily that ranges from prokaryotes to eukaryotes, transport a wide variety of substrates across membranes, including metabolic products, lipids, and drugs. They utilize the energy of ATP hydrolysis for transport, and are involved in various human diseases, such as cancer and cystic fibrosis. Much attention has been paid in recent years to ABC-transporters as potential drug targets, and high-throughput drug-screening systems for ABC-transporters are in great demand. We have succeeded in detecting ATP-dependent transport by the immobilized P-glycoprotein, an ABC transporter, in polydimethylsiloxane (PDMS) microfluidic channels. We here present a first report of the on-chip accurate analyses of the inhibition of P-glycoproteins with several drugs, digoxin, verapamil, cyclosporine A and quinidine. The half-maximal inhibitory concentration (IC50) values for the Rh123 transport, determined by the microchip were 1.82 ± 0.36 μM (digoxin), 1.91 ± 0.28 μM (verapamil), 0.45 ± 0.01 μM (cyclosporine A) and 0.66 ± 0.04 μM (quinidine). Because our procedure detects the fluorescence of immobilized ABC transporter-containing vesicles and their size and intensity can be directly measured from the images, we could estimate the substrate transport at the single-transporter level.

Summary Emulation of biological ion channels by synthetic molecules is not only a challenge for chemists in designing highly complex (supra)molecules but also key to developing a new tool for exploring subcellular electrochemical activity. Despite efforts to create a single pore in a lipid bilayer by synthetic channels, a general synthetic strategy for realizing more complex two-pore channels has yet to be proposed. Here, we demonstrate two distinct ion conductance states by embedding a single metal-organic porous molecule with the geometry of an Archimedean cuboctahedron into an artificially reconstructed lipid bilayer membrane in which triangular and square apertures in the cuboctahedron work independently as ion-transporting pathways. By changing the aliphatic chain length introduced on the periphery of the cuboctahedron, we found that the rotational dynamics of the cuboctahedron regulate the open pore time of each conductance state through distinct apertures and the switching between them.

As the main component of cellular membranes, lipid bilayers were widely investigated. A prerequisite approach may consist in studying an artificial bi-layer, which is able to mimic some aspects of a natural membrane through its constituents. Methods were proposed to produce these artificial membranes using different biochip technologies.