Open-source and low-cost miniature microscope for on-site fluorescence detection.

The development of a compact and affordable fluorescence microscope can be a formidable challenge for growing needs in on-site testing and detection of fluorescent labeled biological systems, especially for those who specialize in biology rather than in engineering. In response to such a situation, we present an open-source miniature fluorescence microscope using Raspberry Pi. Our fluorescence microscope, with dimensions of 19.2 × 13.6 × 8.2 cm3 (including the display, computer, light-blocking case, and other operational requirements), not only offers cost-effectiveness (costing less than $500) but is also highly customizable to meet specific application needs. The 12.3-megapixel Raspberry Pi HQ Camera captures high-resolution imagery, while the equipped wide-angle lens provides a field of view measuring 21 × 15 mm2. The integrated wireless LAN in the Raspberry Pi, along with software-controllable high-powered fluorescence LEDs, holds potential for a wide range of applications. This open-source fluorescence microscope offers biohybrid sensor developers a versatile tool to streamline unfamiliar mechanical design tasks and open new opportunities for on-site fluorescence detections.

Lipid Bilayer Reformation Using the Wiping Blade for Improved Ion Channel Analysis.

The measurement of ion permeation activity across planar lipid bilayers is a useful technique for the functional analysis and drug evaluation of ion channels at the single-molecule level. To enhance the data throughput, parallelization of lipid bilayers is desirable. However, existing parallelized approaches face challenges in simultaneously and efficiently measuring ion channel activities under various conditions on one chip. In this study, we propose an approach to overcome these limitations by developing a device capable of repeated measurements of ion channels incorporated into individually arrayed lipid bilayers. Our device forms an array of a lipid bilayer at a micropore on a separator by merging two lipid monolayers assembled on the surface of aqueous droplets. We introduce a vertically moving, blade-shaped module─referred to as a "wiping blade"─which enables controlled disruption and reformation of the bilayer at the micropore. By optimizing the surface properties and clearance of the wiping blade, we successfully achieved repeated bilayer formation. The arrayed lipid bilayer device with the integrated wiping blade module demonstrates a 5-fold improvement in data throughput during ion channel activity measurements. Finally, we validate the practical utility of our device by evaluating the effects of an ion channel inhibitor. The developed device opens new avenues for high-throughput analysis and screening of ion channels, leading to significant advancements in drug discovery and functional studies of membrane proteins. It offers a powerful tool for researchers in the field and holds promise for accelerating drug development by targeting ion channels.

Real-time quantitative characterization of ion channel activities for automated control of a lipid bilayer system.

This paper describes a novel signal processing method to characterize the activity of ion channels on a lipid bilayer system in a real-time and quantitative manner. Lipid bilayer systems, which enable single-channel level recordings of ion channel activities against physiological stimuli in vitro, are gaining attention in various research fields. However, the characterization of ion channel activities has heavily relied on time-consuming analyses after recording, and the inability to return the quantitative results in real time has long been a bottleneck to incorporating the system into practical products. Herein, we report a lipid bilayer system that integrates real-time characterization of ion channel activities and real-time response based on the characterization result. Unlike conventional batch processing, an ion channel signal is divided into short segments and processed during the recording. After optimizing the system to maintain the same characterization accuracy as conventional operation, we demonstrated the usability of the system with two applications. One is quantitative control of a robot based on ion channel signals. The velocity of the robot was controlled every second, which was around tens of times faster than the conventional operation, in proportion to the stimulus intensity estimated from changes in ion channel activities. The other is the automation of data collection and characterization of ion channels. By constantly monitoring and maintaining the functionality of a lipid bilayer, our system enabled continuous recording of ion channels over 2 h without human intervention, and the time of manual labor has been reduced from conventional 3 h to 1 min at a minimum. We believe the accelerated characterization and response in the lipid bilayer systems presented in this work will facilitate the transformation of lipid bilayer technology toward a practical level, finally leading to its industrialization.

3D printed microfluidic devices for lipid bilayer recordings.

This paper verifies the single-step and monolithic fabrication of 3D structural lipid bilayer devices using stereolithography. Lipid bilayer devices are utilized to host membrane proteins in vitro for biological assays or sensing applications. There is a growing demand to fabricate functional lipid bilayer devices with a short lead-time, and the monolithic fabrication of components by 3D printing is highly anticipated. However, the prerequisites of 3D printing materials which lead to reproducible lipid bilayer formation are still unknown. Here, we examined the feasibility of membrane protein measurement using lipid bilayer devices fabricated by stereolithography. The 3D printing materials were characterized and the surface smoothness and hydrophobicity were found to be the relevant factors for successful lipid bilayer formation. The devices were comparable to the ones fabricated by conventional procedures in terms of measurement performances like the amplitude of noise and the waiting time for lipid bilayer formation. We further demonstrated the extendibility of the technology for the functionalization of devices, such as incorporating microfluidic channels for solution exchangeability and arraying multiple chambers for robust measurement.

Role of Negatively Charged Lipids Achieving Rapid Accumulation of Water-Soluble Molecules and Macromolecules into Cell-Sized Liposomes against a Concentration Gradient.

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.

Biohybrid sensor for odor detection.

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.

Highly sensitive VOC detectors using insect olfactory receptors reconstituted into lipid bilayers.

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.

CYK4 relaxes the bias in the off-axis motion by MKLP1 kinesin-6.

Centralspindlin, a complex of the MKLP1 kinesin-6 and CYK4 GAP subunits, plays key roles in metazoan cytokinesis. CYK4-binding to the long neck region of MKLP1 restricts the configuration of the two MKLP1 motor domains in the centralspindlin. However, it is unclear how the CYK4-binding modulates the interaction of MKLP1 with a microtubule. Here, we performed three-dimensional nanometry of a microbead coated with multiple MKLP1 molecules on a freely suspended microtubule. We found that beads driven by dimeric MKLP1 exhibited persistently left-handed helical trajectories around the microtubule axis, indicating torque generation. By contrast, centralspindlin, like monomeric MKLP1, showed similarly left-handed but less persistent helical movement with occasional rightward movements. Analysis of the fluctuating helical movement indicated that the MKLP1 stochastically makes off-axis motions biased towards the protofilament on the left. CYK4-binding to the neck domains in MKLP1 enables more flexible off-axis motion of centralspindlin, which would help to avoid obstacles along crowded spindle microtubules.

Efficient Lipid Bilayer Formation by Dipping Lipid-Loaded Microperforated Sheet in Aqueous Solution.

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 α-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.

A Lipid-Bilayer-On-A-Cup Device for Pumpless Sample Exchange.

Lipid-bilayer devices have been studied for on-site sensors in the fields of diagnosis, food and environmental monitoring, and safety/security inspection. In this paper, we propose a lipid-bilayer-on-a-cup device for serial sample measurements using a pumpless solution exchange procedure. The device consists of a millimeter-scale cylindrical cup with vertical slits which is designed to steadily hold an aqueous solution and exchange the sample by simply fusing and splitting the solution with an external solution. The slit design was experimentally determined by the capabilities of both the retention and exchange of the solution. Using the optimized slit, a planar lipid bilayer was reconstituted with a nanopore protein at a microaperture allocated to the bottom of the cup, and the device was connected to a portable amplifier. The solution exchangeability was demonstrated by observing the dilution process of a blocker molecule of the nanopore dissolved in the cup. The pumpless solution exchange by the proposed cup-like device presents potential as a lipid-bilayer system for portable sensing applications.

Rapid and Resilient Detection of Toxin Pore Formation Using a Lipid Bilayer Array.

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.

Perfusion Chamber for Observing a Liposome-Based Cell Model Prepared by a Water-in-Oil Emulsion Transfer Method.

For the construction of a chemical model of contemporary living cells, the so-called water-in-oil emulsion transfer (WOET) method has drawn much attention as one of the promising preparation protocols for cell-sized liposomes encapsulating macromolecules and even micrometer-sized colloidal particles in high yields. Combining the throughput and accuracy of the observation is the key to developing a synthetic approach based on the liposomes prepared by the WOET method. Recent advances in microfluidic technology can provide a solution. By means of surface modification of a poly(dimethylsiloxane)-type microfluidic device integrating size-sorting and trapping modules, here, we enabled a simultaneous direct observation of the liposomes with a narrow size distribution, which were prepared by the WOET method. As a demonstration, we evaluated the variance of encapsulation of polystyrene colloidal particles and water permeability of the cell-sized liposomes prepared by the WOET method in the device. Since the liposomes prepared by the WOET method are useful for constructing cell models with an easy protocol, the current system will lead to a critical development of not only supramolecular chemistry and soft matter physics but also synthetic biology.

Hydrodynamic accumulation of small molecules and ions into cell-sized liposomes against a concentration gradient.

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.

A pumpless solution exchange system for nanopore sensors.

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)-ß-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.

Construction of a Biohybrid Odorant Sensor Using Biological Olfactory Receptors Embedded into Bilayer Lipid Membrane on a Chip.

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.

Formation of vesicles-in-a-vesicle with asymmetric lipid components using a pulsed-jet flow method.

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.

Electrophysiological measurement of ion channels on plasma/organelle membranes using an on-chip lipid bilayer system.

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.

Automatic Planar Asymmetric Lipid Bilayer Membrane Formation toward Biological High-Throughput Assay.

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.

Quantitative analysis of cell-free synthesized membrane proteins at the stabilized droplet interface bilayer.

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.

Purification-Free MicroRNA Detection by Using Magnetically Immobilized Nanopores on Liposome Membrane.

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.