Search

This paper describes 3D printed microfluidic devices for the rapid implementation of lipid bilayer recordings. We examined 3D printing materials from the perspective of measurement performances and presented feasible applications.

Liposomes, molecular self-assemblies resembling biological membranes, are a promising scaffold to investigate the physicochemical logic behind the complexity of living cells. Despite elaborate synthetic studies constructing cell-like chemical systems using liposomes, less attention has been paid to the proactive role of the membrane emerging as dynamics of the molecular self-assembly. This study investigated the liposomes containing anionic phospholipids by exposing them to steady flow conditions using a newly constructed automatic microfluidic observation platform. We demonstrated that the liposomes accumulated even macromolecules under the microfluidic condition without pore formation. By investigating the effect of composition of liposomes and visualizing negatively charged phospholipids upon the flow, we presumed that the external flow caused a compositional asymmetry of anionic phospholipids between the inner/outer leaflets, and the asymmetry enabled a rapid accumulation of those molecules against the concentration gradient. The current study opens new research interests regarding the nature of biological membranes under steady flow conditions.

Structures that are periodic on a microscale in three dimensions are abundant in nature, for example, in the cellular arrays that make up living tissue. Such structures can also be engineered, appearing in smart materials1–4, photonic crystals5, chemical reactors6, and medical7 and biomimetic8 technologies. Here we report that fluid–fluid interfacial energy drives three-dimensional (3D) structure emergence in a micropillar scaffold. This finding offers a rapid and scalable way of transforming a simple pillar scaffold into an intricate 3D structure that is periodic on a microscale, comprising a solid microscaffold, a dispersed fluid and a continuous fluid. Structures generated with this technique exhibit a set of unique features, including a stationary internal liquid–liquid interface. Using this approach, we create structures with an internal liquid surface in a regime of interest for liquid–liquid catalysis. We also synthesize soft composites in solid, liquid and gas combinations that have previously not been shown, including actuator materials with temperature-tunable microscale pores. We further demonstrate the potential of this method for constructing 3D materials that mimic tissue with an unprecedented level of control, and for microencapsulating human cells at densities that address an unresolved challenge in cell therapy. The revelation that fluid–fluid interfacial energy can drive structure formation in micropillar scaffolds offers a scalable way of synthesizing soft composites, which may have applications in building materials that mimic biological tissue.

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.

Droplet Contact Method (DCM) has widely been used to form a planar lipid bilayer between a pair of aqueous droplets for measurement of the functions of membrane proteins. However, the fabrication of DCM devices was laborious, and the integration of 3D structures into the devices remains difficult. To solve these issues, we have applied stereolithography to the fabrication of DCM devices. As a model structure, a DCM device integrated with microfluidic channels was monolithically fabricated, and a nanopore-forming protein $\alpha$ -hemolysin was incorporated into the planar lipid bilayer while perfusing one of the droplets. We further compared several stereolithography materials to maximize the measurement performance.

Cell-based sensors using olfactory receptors have attracted attention because of their high sensitivity and selectivity. A challenge is efficient partition of gaseous samples to the aqueous solution where the cell-based sensors reside. In this paper, we developed a device with a gas-flow channel, which allowed inflow of gaseous odorants over a well filled with aqueous solution. We considered that shear stress induced by the gas-flow at the gas-liquid interface initiate convectional mixing inside liquid and in turn, promotes partitioning of gaseous odorants into the solution. Solubility of a gaseous odorant was experimentally found to increase with the mixing strength. We further demonstrated efficient detection of a gaseous odorant using the sensor cells with the developed gas-flow device.

Biohybrid odorant sensors that directly integrate a biological olfactory system have been increasingly studied and are suggested to be the next generation of ultrasensitive sensors by taking advantage of the sensitivity and selectivity of living organisms. In this review, we provide a detailed description of the recent developments of biohybrid odorant sensors, especially considering the requisites for their perspective of on-site applications. We introduce the methodologies to effectively capture the biological signals from olfactory systems by readout devices, and describe the essential properties regarding the gaseous detection, stability, quality control, and portability. Moreover, we address the recent progress on multiple odorant recognition using multiple sensors as well as the current screening approaches for pairs of orphan receptors and ligands necessary for the extension of the currently available range of biohybrid sensors. Finally, we discuss our perspectives for the future for the development of practical odorant sensors.

A highly sensitive odorant sensor was developed by using insect olfactory receptors reconstituted into a lipid bilayer membrane., This paper reports a volatile organic compound (VOC) sensor based on olfactory receptors that were reconstituted into a lipid bilayer and used in a specifically designed gas flow system for rapid parts per billion (ppb)–level detection. This VOC sensor achieves both rapid detection and high detection probability because of its gas flow system and array design. Specifically, the gas flow system includes microchannels and hydrophobic microslits, which facilitate both the introduction of gas into the droplet and droplet mixing. We installed this system into a parallel lipid bilayer device and subsequently demonstrated parts per billion–level (0.5 ppb) detection of 1-octen-3-ol in human breath. Therefore, this system extends the various applications of biological odorant sensing, including breath diagnosis systems and environmental monitoring.

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.

Sloshing is the movement of liquid in a container induced by an external force. The phenomenon has been extensively studied at the macroscopic scale because of the importance of liquid control in tanks. Sloshing at the microliter scale should be explored for microdroplets in Lab-on-a-chip applications. We therefore verified the sloshing characteristics of droplets in microliter-sized wells. A water-in-oil droplet in the well was vibrated horizontally at between 20 and 100 Hz. Circular and elliptic wells between 2 and 5 mm sizes were applied for the well geometry. The surface chemistry of the well was additionally changed by using amphiphobic coatings to examine the effect of the interfacial tension between the well surface and the liquid, because we considered it a dominant factor at the microliter scale. Decreasing the well diameter caused the natural frequency to increase and the sloshing to suppress. This agreed with Housner’s equation, which is conventionally used for macroscopic scale. The experiments also revealed that decreasing the interfacial tension caused the natural frequency to increase and resulted in drastic suppression of the sloshing, which was characterized by Bond number, similarly with the cases under microgravity. We experimentally verified that at the microliter scale, the interfacial tension is dominant to control the sloshing phenomenon.

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.

We evaluated the speed profile of self-propelled underwater oil droplets comprising a hydrophobic aldehyde derivative in terms of their diameter and the surrounding surfactant concentration using a microfluidic device. We found that the speed of the oil droplets is dependent on not only the surfactant concentration but also the droplet size in a certain range of the surfactant concentration. This tendency is interpreted in terms of combination of the oil and surfactant affording spontaneous emulsification in addition to the Marangoni effect.

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.

This paper proposes a nanopore-based sensor exploiting the solution exchange of a droplet-based lipid bilayer driven by a superabsorbent polymer. Biological nanopores are candidates for use in portable sensors because of their potential to recognize and detect single molecules. One of the current challenges in the development of portable nanopore sensors is the inability to achieve continuous detection. To achieve continuous detection, we have exploited the suction force of a superabsorbent polymer to drive the continuous microfluidic flow required to wash the analyte out of the droplet. The superabsorbent polymer drives the microfluidic flow without electricity, and the developed solution exchange system remains compact. To demonstrate solution exchange in the droplet containing the lipid bilayer, the concentration of heptakis(6-O-sulfo)-β-cyclodextrin was monitored in a time-dependent manner using α-hemolysin nanopores. A reduction in the concentration, attributable to solution exchange, was successfully observed. We believe that the proposed system will increase the portability and usability of nanopore sensors.

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.

Voltage-clamp fluorometry (VCF) has been combined with artificial lipid bilayer systems to optically measure the dynamics and contribution of membrane proteins on molecular transport at the clamped membrane potential. However, the previous methods for VCF require expensive apparatus, such as total internal reflection fluorescence microscope, or require extensive experience for voltage-clamping, or possibly lead to imprecise membrane potential. This study describes VCF performed using a pico-liter-sized lipid bilayer chamber with an embedded Ag/AgCl microelectrode. Owing to the size of the picoliter order, molecular transport through the lipid bilayer can be detected using a normal fluorescent microscope. Easy electrical access into the lipid bilayer chamber can be realized because the microelectrode is spontaneously embedded inside when forming the lipid bilayer chamber. The microelectrode is made of Ag/AgCl; therefore, it does not tend to polarize, which results in reliable regulation of the membrane potential. Using the developed system, we performed the VCF of molecular transport through the lipid bilayer by incorporating the pore-forming membrane protein, α-hemolysin. This proposed method will increase the number of researches that can perform the VCF of molecular transport through artificial lipid bilayers.

We developed a highly sensitive point-of-care testing (POCT) chip using an antigen-antibody complex reaction for the early diagnosis of infectious disease. The central concept of the chip was to balance the sensitivity enhancement with appropriate safety measures for infectious risk during POCT. The chip consisted of a sheet of glass fiber sealed in a polymethylmethacrylate microfluidic channel. Antibody-immobilized microbeads were accumulated in a transparent location in the channel that served as the test line, trapping the target antigen bound to fluorescently labeled antibodies and allowing diagnosis by a portable fluorescence scanner. In this work, we applied a superabsorbent polymer to drive a stable and continuous fluidic flow for both the sample and a rinsing solution without an external power supply. The generated flow enabled thorough bound/free separation of the antigen-antibody interactions and significantly improved the signal-to-noise ratio and the reproducibility. Moreover, the polymer had the advantage of retaining the potentially infectious sample within the chip, contributing to its safety for practical use. An influenza test using a virus protein exhibited a sensitivity enhancement of more than a thousand-fold compared with commercial immunochromatographic test strips.

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.