Nanopore Filter: A Method for Counting and Extracting Single DNA Molecules Using a Biological Nanopore.

This paper describes a method for the real-time counting and extraction of DNA molecules at the single-molecule level by nanopore technology. As a powerful tool for electrochemical single-molecule detection, nanopore technology eliminates the need for labeling or partitioning sample solutions at the femtoliter level. Here, we attempt to develop a DNA filtering system utilizing an α-hemolysin (αHL) nanopore. This system comprises two droplets, one filling with and one emptying DNA molecules, separated by a planar lipid bilayer containing αHL nanopores. The translocation of DNA through the nanopores is observed by measuring the channel current, and the number of translocated molecules can also be verified by quantitative polymerase chain reaction (qPCR). However, we found that the issue of contamination seems to be an almost insolvable problem in single-molecule counting. To tackle this problem, we tried to optimize the experimental environment, reduce the volume of solution containing the target molecule, and use the PCR clamp method. Although further efforts are still needed to achieve a single-molecule filter with electrical counting, our proposed method shows a linear relationship between the electrical counting and qPCR estimation of the number of DNA molecules.

DNA Nanopore-Tethered Gold Needle Electrodes for Channel Current Recording.

Synthetic DNA nanopores are attracting attention as alternatives to conventional biological nanopores in nanopore sensors because of the high designability of their pore structures and functionability. However, the efficient insertion of DNA nanopores into a planar bilayer lipid membrane (pBLM) remains challenging. Although hydrophobic modifications such as the use of cholesterol are required to insert DNA nanopores into pBLMs, these modifications also induce negative effects, including the undesired aggregation of DNA structures. Herein, we describe an efficient method to insert DNA nanopores into pBLMs and measure the channel currents of DNA nanopores using a DNA nanopore-tethered gold electrode. When the pBLM is formed at the electrode tip by immersing the electrode into a layered bath solution comprising an oil/lipid mixture and an aqueous electrolyte, the electrode-tethered DNA nanopores are physically inserted into the pBLM. In this study, we designed a DNA nanopore structure that can be immobilized on the gold electrode based on a reported six-helix bundle DNA nanopore structure and prepared DNA nanopore-tethered gold electrodes. Thereafter, we demonstrated the channel current measurements of the electrode-tethered DNA nanopores, and a high insertion probability of the DNA nanopores was achieved. We believe that this efficient DNA nanopore insertion method can accelerate the application of DNA nanopores in stochastic nanopore sensors.

Microcavity volume control on a tip of Ag/AgCl electrodes for stable channel current measurements of biological nanopores.

A probe-type channel current measurement system with a planer bilayer lipid membrane (pBLM) at the tip of a probe provided several advantages for pBLM formation and channel current measurements. The procedure for preparing pBLMs was simple (i.e., the probe was submerged into a bath solution layered by an oil/lipid mixture and buffer solution). The probe systems offered local detection of analytes by nanopore sensing. Nevertheless, the current decay caused by changing the ion concentration in the electrolyte held on the tip of the probes influenced the sensing accuracy. Here we applied a cavity microelectrode (CME) technique to fabricate pBLM probes with larger electrolyte volume on the tip. We fabricated silver CMEs with different cavity volumes and measured channel currents of biological nanopores. Furthermore, we evaluated the channel current decay as a function of cavity volume by analyzing the step signals of α-hemolysin nanopores. Consequently, the channel current decay was extended by increasing the cavity volume, indicating that the volume of the electrolyte solution was important for channel current measurements of nanopores. We concluded that the pBLM system using CMEs is useful for channel current measurements of biological nanopores. Additionally, the fundamental evaluation of the relationship between the electrolyte volume and channel current decay will be helpful in the design of pBLM systems made by microfabrication and microfluidic techniques.

De-Insertion Current Analysis of Pore-Forming Peptides and Proteins Using Gold Electrode-Supported Lipid Bilayer.

Analysis of the pore formation mechanisms of transmembrane peptides and proteins potentially provides an insight into cellular biology because of their significant roles in biological systems. Here, we describe the procedures to analyze the pore formation mechanisms of transmembrane peptides and proteins by measuring de-insertion currents of biological nanopores that can be obtained by unzipping the lipid bilayers.

Simple Fabrication of Solid-State Nanopores on a Carbon Film.

Solid-state nanopores are widely used as a platform for stochastic nanopore sensing because they can provide better robustness, controllable pore size, and higher integrability than biological nanopores. However, the fabrication procedures, including thin film preparation and nanopore formation, require advanced micro-and nano-fabrication techniques. Here, we describe the simple fabrication of solid-state nanopores in a commercially available material: a flat thin carbon film-coated micro-grid for a transmission electron microscope (TEM). We attempted two general methods for nanopore fabrication in the carbon film. The first method was a scanning TEM (STEM) electron beam method. Nanopores were fabricated by irradiating a focused electron beam on the carbon membrane on micro-grids, resulting in the production of nanopores with pore diameters ranging from 2 to 135 nm. The second attempt was a dielectric breakdown method. In this method, nanopores were fabricated by applying a transmembrane voltage of 10 or 30 V through the carbon film on micro-grids. As a result, nanopores with pore diameters ranging from 3.7 to 1345 nm were obtained. Since these nanopores were successfully fabricated in the commercially available carbon thin film using readily available devices, we believe that these solid-state nanopores offer great utility in the field of nanopore research.

Analysis of Membrane Protein Deinsertion-Associated Currents with Nanoneedle-Supported Bilayers to Discover Pore Formation Mechanisms.

Analysis of the pore formation mechanisms of biological nanopores can provide insight into pore-forming peptide-induced diseases and into the characterization of nanopores employed in sensing methods. Evaluation of pore formation mechanisms is typically performed using microscopy including atomic force microscopy, transmission electron microscopy, as well as electrically via channel current measurements using a patch-clamp amplifier. However, due to the relatively low temporal resolution of the above-mentioned microscopy techniques and the low analysis accuracy of the channel current measurements, new analytical methods are required. Here, we describe a new analytical strategy to measure and analyze both ionic currents associated with biological nanopore insertion and deinsertion into and out of lipid bilayers to determine pore formation mechanisms for several representative proteins. The current changes associated with protein deinsertion are monitored as the lipid membrane leaflets are pulled apart-a unique phenomenon enabled by our gold nanoneedle measurement probe. This deinsertion current analysis (DiCA) is performed using a gold nanoneedle-supported lipid bilayer at which a bilayer membrane is formed by bringing together two lipid monolayers on the surface of the nanoneedle and at the interface of an aqueous solution and a lipid/oil mixture. The lipid bilayer can be pulled apart by removing the nanoneedle from this interface. In this study, we demonstrate the determination of pore formation mechanisms for four different pore-forming proteins and peptides-α-hemolysin, streptolysin O, alamethicin, and amyloid ß 1-42 using DiCA. As a result, we successfully discern the pore formation mechanism, either addition or expansion, for each protein/peptide by analyzing the ratio and magnitude of insertion and deinsertion current events.

Recent Advances in Liposome-Based Molecular Robots.

A molecular robot is a microorganism-imitating micro robot that is designed from the molecular level and constructed by bottom-up approaches. As with conventional robots, molecular robots consist of three essential robotics elements: control of intelligent systems, sensors, and actuators, all integrated into a single micro compartment. Due to recent developments in microfluidic technologies, DNA nanotechnologies, synthetic biology, and molecular engineering, these individual parts have been developed, with the final picture beginning to come together. In this review, we describe recent developments of these sensors, actuators, and intelligence systems that can be applied to liposome-based molecular robots. First, we explain liposome generation for the compartments of molecular robots. Next, we discuss the emergence of robotics functions by using and functionalizing liposomal membranes. Then, we discuss actuators and intelligence via the encapsulation of chemicals into liposomes. Finally, the future vision and the challenges of molecular robots are described.

Competing Roles of Two Kinds of Ligand during Nonclassical Crystallization of Pillared-Layer Metal-Organic Frameworks Elucidated Using Microfluidic Systems.

To diversify metal-organic frameworks (MOFs), multi-component MOFs constructed from more than two kinds of bridging ligand have been actively investigated due to the high degree of design freedom afforded by the combination of multiple ligands. Predicting the synthesis conditions for such MOFs requires an understanding of the crystallization mechanism, which has so far remained elusive. In this context, microflow systems are efficient tools for capturing non-equilibrium states as they facilitate precise and efficient mixing with reaction times that correspond to the distance from the mixing point, thus enabling reliable control of non-equilibrium crystallization processes. Herein, we prepared coordination polymers with pillared-layer structures and observed the intermediates in the syntheses with an in-situ measurement system that combines microflow reaction with UV/Vis and X-ray absorption fine-structure spectroscopies, thereby enabling their rapid nucleation to be monitored. Based on the results, a three-step nonclassical nucleation mechanism involving two kinds of intermediate is proposed.

Microfluidic Formation of Honeycomb-Patterned Droplets Bounded by Interface Bilayers via Bimodal Molecular Adsorption.

Assembled water-in-oil droplets bounded by lipid bilayers are used in synthetic biology as minimal models of cell tissue. Microfluidic devices successfully generate monodispersed droplets and assemble them via droplet interface bilayesr (DIB) formation. However, a honeycomb pattern of DIB-bounded droplets, similar to epithelial tissues, remains unrealized because the rapid DIB formation between the droplets hinders their ability to form the honeycomb pattern. In this paper, we demonstrate the microfluidic formation of a honeycomb pattern of DIB-bounded droplets using two surfactants with different adsorption rates on the droplet surface. A non-DIB forming surfactant (sorbitan monooleate, Span 80) was mixed with a lipid (1,2-dioleoyl-sn-glycero-3-phosphocholine, PC), whose adsorption rate on the droplet surface and saturated interfacial tension were lower than those of Span 80. By changing the surfactant composition, we established the conditions under which the droplets initially form a honeycomb pattern and subsequently adhere to each other via DIB formation to minimize the interfacial energy. In addition, the reconstituted membrane protein nanopores at the DIBs were able to transport molecules. This new method, using the difference in the adsorption rates of two surfactants, allows the formation of a honeycomb pattern of DIB-bounded droplets in a single step, and thus facilitates research using DIB-bounded droplet assemblies.

Recessed Ag/AgCl Microelectrode-Supported Lipid Bilayer for Nanopore Sensing.

Biological nanopores reconstituted into supported lipid bilayer membranes are widely used as a platform for stochastic nanopore sensing with the ability to detect single molecules including, for example, single-stranded DNA (ssDNA) and miRNA. A main thrust in this area of research has been to improve overall bilayer stability and ease of measurements. These improvements are achieved through a variety of clever strategies including droplet-based techniques; however, they typically require specific microfabrication techniques to prepare devices or special manipulation techniques for microdroplets. Here, we describe a new method to prepare lipid bilayers using a recessed-in-glass Ag/AgCl microelectrode as a support structure. The lipid bilayer is formed at the tip of the microelectrode by immersing the microelectrode into a layered bath solution consisting of an oil/lipid mixture and an aqueous electrolyte solution. In this paper, we demonstrate this stable, supported lipid bilayer structure for channel current measurements of pore-forming toxins and single-molecule detection of ssDNA. This Ag/AgCl-supported lipid bilayer can potentially be widely adopted as a lipid membrane platform for nanopore sensing because of its simple and easy procedure needed to prepare lipid bilayers.

Biological Nanopore Probe: Probing of Viscous Solutions in a Confined Nanospace.

This paper describes a nanospace probing system constructed with a pore-forming toxin and a hairpin DNA (hpDNA) molecule. The single hpDNA molecule can be inserted and can move in the confined nanospace of the alpha-hemolysin (αHL) pore. The molecular motion of the hpDNA can be determined based on the fluctuation of the blocking current via channel current measurements. Using this system, we investigated the effect of viscosity of the aqueous solution in the macrospace (bulk) and in the confined nanospace with a small molecule (glycerol) and a polymer (PEG600). The molecular motion of the hpDNA in the nanospace differed in glycerol and PEG600 solutions, while the viscosity remained the same in the bulk solution. The fundamental factors for the viscosity in glycerol and PEG600 solutions are hydrogen bonding and the entanglement of polymer chains, respectively. This difference in factors becomes significant in confined nanospaces, and our system allows us to observe its effect. Additionally, we constructed a spatially resolved nanopore probe integrated into a gold nanoneedle. The αHL-hpDNA nanoprobe system was constructed with the nanoneedle and can be used to monitor the nanospace with nanometer spatial resolution.

Osmotic-engine-driven liposomes in microfluidic channels.

Self-propelled underwater microrobots that locomote without external sources of energy have potential application as drug carriers and probes in narrow spaces. In this study, we focused on an osmotic engine model, which is a migration mechanism, and applied it as a negative chemotaxis mechanism to induce liposome displacement. First, we confirmed the osmotic flow across the lipid bilayer and calculated the osmotic flow velocity to be 8.5 fL min-1 µm-2 when a salt concentration difference was applied to the lipid bilayer. Next, we designed and fabricated a microchannel that can trap a giant liposome and apply a salt concentration difference to the front and rear of the liposome. Then, we demonstrated the movement of the liposome by flowing it to the microchannel. The liposome successfully moved in the direction of the lower ion concentration at a speed of 0.6 µm min-1 owing to the osmotic pressure difference. Finally, we visualized the inner flow in the liposome by encapsulating microbeads in the liposome and observed the movement of the microbeads to verify that an osmotic flow was generated on the liposome. As a result, we observed the circulation of the microbeads in the liposome when the concentration difference was applied to the front and rear of the liposome, suggesting that the movement of the liposome was driven by the osmotic flow generated by the osmotic pressure difference. These results indicate that the osmotic-pressure-based migration mechanism has the potential to be utilized as the actuator of molecular robots.

Spatially Resolved Chemical Detection with a Nanoneedle-Probe-Supported Biological Nanopore.

In this article, we describe the quantitative characterization of a gold nanoneedle ion channel probe and demonstrate the utility of this probe for spatially resolved detection of a small molecule using ion channel activity. Our report builds on recent reports of Ide and co-workers, who reported the use of an etched gold wire modified with a poly(ethylene) glycol monolayer as a support for a lipid bilayer and subsequent single ion channel recordings. Although this nanoneedle electrode approach was reported previously, in our report, we investigate the effects of several operational parameters on the performance of the ion channel measurement and electrochemical phenomenon that occur in the nanoconfined space between the supported bilayer and the gold electrode. More specifically, we address the effects of length of the supporting monolayer and the composition of the electrolyte baths on channel current measurements and provide a quantitative description of what carries current at the working electrode (double-layer charging). In addition, we demonstrate the ability to control the direction of protein insertion (tip side vs bath side) with freely diffusing protein, which has not been previously reported, with the former method (tip side) enabling single-molecule detection of ß-cyclodextrin (ßCD) using a reconstituted α-hemolysin channel. Finally, anticipating future use of a nanoneedle-based biological nanopore probe in a scanned-probe microscopy, we demonstrate the ability to quantify and spatially resolve the concentration of ßCD molecules in a microfluidic channel. We believe, in the long term, the described nanoneedle-based biological nanopore probe can be employed in, for example, scanning ion conductance microscopy using ion channels.

Microfluidic Formation of Double-Stacked Planar Bilayer Lipid Membranes by Controlling the Water-Oil Interface.

This study reports double-stacked planar bilayer lipid membranes (pBLMs) formed using a droplet contact method (DCM) for microfluidic formation with five-layered microchannels that have four micro guide pillars. pBLMs are valuable for analyzing membrane proteins and modeling cell membranes. Furthermore, multiple-pBLM systems have broadened the field of application such as electronic components, light-sensors, and batteries because of electrical characteristics of pBLMs and membrane proteins. Although multiple-stacked pBLMs have potential, the formation of multiple-pBLMs on a micrometer scale still faces challenges. In this study, we applied a DCM strategy to pBLM formation using microfluidic techniques and attempted to form double-stacked pBLMs in micro-meter scale. First, microchannels with micro pillars were designed via hydrodynamic simulations to form a five-layered flow with aqueous and lipid/oil solutions. Then, pBLMs were successfully formed by controlling the pumping pressure of the solutions and allowing contact between the two lipid monolayers. Finally, pore-forming proteins were reconstituted in the pBLMs, and ion current signals of nanopores were obtained as confirmed by electrical measurements, indicating that double-stacked pBLMs were successfully formed. The strategy for the double-stacked pBLM formation can be applied to highly integrated nanopore-based systems.

Biofuel cell backpacked insect and its application to wireless sensing.

This study investigated an enzymatic biofuel cell (BFC) which can be backpacked by cockroaches. The BFC generates electric power from trehalose in insect hemolymph by the trehalase and glucose dehydrogenase (GDH) reaction systems which dehydrogenate ß-glucose obtained by hydrolyzing trehalose. First, an insect-mountable BFC (imBFC) was designed and fabricated with a 3D printer. The electrochemical reaction of anode-modified poly-L-lysine, vitamin K3, diaphorase, nicotinamide adenine dinucleotide, GDH and poly(sodium 4-styrenesulfonate) in the imBFC was evaluated and an oxidation current of 1.18 mAcm(-2) (at +0.6 V vs. Ag|AgCl) was observed. Then, the performance of the imBFC was evaluated and a maximum power output of 333 µW (285 µW cm(-)(2)) (at 0.5 V) was obtained. Furthermore, driving of both an LED device and a wireless temperature and humidity sensor device were powered by the imBFC. These results indicate that the imBFC has sufficient potential as a battery for novel ubiquitous robots such as insect cyborgs.

Insect biofuel cells using trehalose included in insect hemolymph leading to an insect-mountable biofuel cell.

In this paper, an insect biofuel cell (BFC) using trehalose included in insect hemolymph was developed. The insect BFC is based on trehalase and glucose oxidase (GOD) reaction systems which oxidize ß-glucose obtained by hydrolyzing trehalose. First, we confirmed by LC-MS that a sufficient amount of trehalose was present in the cockroach hemolymph (CHL). The maximum power density obtained using the insect BFC was 6.07 µW/cm(2). The power output was kept more than 10 % for 2.5 h by protecting the electrodes with a dialysis membrane. Furthermore, the maximum power density was increased to 10.5 µW/cm(2) by using an air diffusion cathode. Finally, we succeeded in driving a melody integrated circuit (IC) and a piezo speaker by connecting five insect BFCs in series. The results indicate that the insect BFC is a promising insect-mountable battery to power environmental monitoring micro-tools.