Radical generation and bactericidal activity of nanobubbles produced by ultrasonic irradiation of carbonated water.

Our previous study showed that nanobubbles (NBs) encapsulating CO2 gas have bactericidal activity due to reactive oxygen species (ROS) (Yamaguchi et al., 2020). Here, we report that bulk NBs encapsulating CO2 can be efficiently generated by ultrasonically irradiating carbonated water using a piezoelectric transducer with a frequency of 1.7 MHz. The generated NBs were less than 100 nm in size and had a lifetime of 500 h. Furthermore, generation of ROS in the NB suspension was investigated using electron spin resonance spectroscopy and fluorescence spectrometry. The main ROS was found to be the hydroxyl radical, which is consistent with our previous observations. The bactericidal activity lasted for at least one week. Furthermore, a mist generated by atomizing the NB suspension with ultrasonic waves was confirmed to have the same bactericidal activity as the suspension itself. We believe that the strong, persistent bactericidal activity and radical generation phenomenon are unique to NBs produced by ultrasonic irradiation of carbonated water. We propose that entrapped CO2 molecules strongly interact with water at the NB interface to weaken the interface, and high-pressure CO2 gas erupts from this weakened interface to generate ROS with bactericidal activity.

Two-dimensional water-molecule-cluster layers at nanobubble interfaces.

HYPOTHESIS: Bulk nanobubbles (NBs) have high surface charge densities and long lifetimes. Despite several attempts to understand the lifetime of NBs, their interfacial layer structure remains unknown. It is hypothesized that a specific interfacial layer exists with a hydrogen bond network that stabilizes NBs. EXPERIMENTS: In situ infrared reflectance-absorption spectroscopy and density functional theory were used to determine the interfacial layer structure of NBs. Furthermore, nuclear magnetic resonance spectroscopy was used to examine the interfacial layer hardness of bubbles filled with N2, O2, and CO2, which was expected to depend on the encapsulated gas species. FINDINGS: The interfacial layer was composed of three-, four-, and five-membered ring clusters of water molecules. An interface model was proposed in which a two-dimensional layer of clusters with large electric dipole moments is oriented toward the endohedral gas, and the hydrophobic surface is adjacent to the free water. The interfacial layer hardness was dependent on the interaction with the gas (N2 > O2 > CO2), which supports the proposed interface model. These findings can be generalized to the structure of water at gas-water interfaces.

New Aspects of Bilayer Lipid Membranes for the Analysis of Ion Channel Functions.

The bilayer lipid membrane (BLM) is the main structural component of cell membranes, in which various membrane proteins are embedded. Artificially formed BLMs have been used as a platform in studies of the functions of membrane proteins, including various ion channels. In this review, we summarize recent advances that have been made on artificial BLM systems for the analysis of ion channel functions. We focus on two BLM-based systems, cell-membrane mimicry and four-terminal BLM systems. As a cell-membrane-mimicking system, an efficient screening platform for the evaluation of drug side effects that act on a cell-free synthesized channel has been developed, and its prospects for use in personalized medicine will be discussed. In the four-terminal BLMs, we introduce "lateral voltage" to BLM systems as a novel input to regulate channel activities, in addition to the traditional transmembrane voltages. Such state-of-the-art technologies and new system setups are predicted to pave the way for a variety of applications, in both fundamental physiology and in drug discovery.

Lateral voltage as a new input for artificial lipid bilayer systems.

In this work, we propose lateral voltage as a new input for use in artificial lipid bilayer systems in addition to the commonly used transmembrane voltage. To apply a lateral voltage to bilayer lipid membranes, we fabricated electrode-equipped silicon and Teflon chips. The Si chips could be used for photodetector devices based on fullerene-doped lipid bilayers, and the Teflon chips were used in a study of the ion channel functions in the lipid bilayer. The findings indicate that the lateral voltage effectively regulates the transmembrane current, in both ion-channel-incorporated and fullerene-incorporated lipid bilayer systems, suggesting that the lateral voltage is a practicable and useful additional input for use in lipid bilayer systems.

Bactericidal Activity of Bulk Nanobubbles through Active Oxygen Species Generation.

We investigated the bactericidal activity of bulk nanobubbles (NBs) using E. coli, a model bacterium. Bulk NBs were produced by forcing gas through a porous alumina membrane with an ordered arrangement of nanoscale straight holes in contact with water. NBs with different gas contents, including CO2, O2, and N2, were generated and evaluated for their bactericidal effects. The survival rate of E. coli was significantly reduced in a suspension of CO2-containing NB (CO2-NB water). The N2-NB water demonstrated a small amount of bactericidal behavior, but its impact was not as significant as that of CO2-NB water. When E. coli was retained in O2-NB water, the survival rate was even higher than that in pure water (PW). We investigated the generation of reactive oxygen species (ROS) in NB suspensions by electron spin resonance spectroscopy. The main ROS generated in the NB water were hydroxyl radicals and OH·, and the production of ROS was the strongest in CO2-NB water, which was consistent with the results of the bactericidal effect measurements. We assumed that NB mediated by ROS would exhibit bactericidal behavior and proposed a kinetic model to explain the retention time variation of the survival rate. The results calculated based on the proposed model matched closely with the experimental results.

Parallel Recordings of Transmembrane hERG Channel Currents Based on Solvent-Free Lipid Bilayer Microarray.

The reconstitution of ion-channel proteins in artificially formed bilayer lipid membranes (BLMs) forms a well-defined system for the functional analysis of ion channels and screening of the effects of drugs that act on these proteins. To improve the efficiency of the BLM reconstitution system, we report on a microarray of stable solvent-free BLMs formed in microfabricated silicon (Si) chips, where micro-apertures with well-defined nano- and micro-tapered edges were fabricated. Sixteen micro-wells were manufactured in a chamber made of Teflon®, and the Si chips were individually embedded in the respective wells as a recording site. Typically, 11 to 16 BLMs were simultaneously formed with an average BLM number of 13.1, which corresponded to a formation probability of 82%. Parallel recordings of ion-channel activities from multiple BLMs were successfully demonstrated using the human ether-a-go-go-related gene (hERG) potassium channel, of which the relation to arrhythmic side effects following drug treatment is well recognized.

Bactericidal Activity of TiO2 Nanotube Thin Films on Si by Photocatalytic Generation of Active Oxygen Species.

The photocatalytic bactericidal activity of titanium dioxide (TiO2) thin films has been extensively studied. In this study, we investigated the bactericidal activities of TiO2 nanotube (NT) thin films using Escherichia coli and Staphylococcus aureus cells as the model bacteria. Metallic titanium (Ti) thin films were anodized on a silicon (Si) wafer substrate to form TiO2 NT thin films. To evaluate the bactericidal activity of the TiO2 NT thin films, bacteria on the TiO2 NT thin films were irradiated with near-ultraviolet light (UV-A) at a wavelength of 365 nm. The bactericidal activity was estimated by the survival rate derived from the number of live cells, which form colonies on the cell culture medium. We demonstrated that the survival rate of the two types of bacteria investigated in this study was significantly reduced by UV light irradiation and that there was a difference in the temporal change in the survival rate between the two types of bacteria. Furthermore, we investigated the generation of reactive oxygen species (ROSs) by UV light irradiation of TiO2 NT thin films using electron spin resonance spectroscopy and fluorescence analysis. We found that the main ROS generated on the surface of the TiO2 NT film was the hydroxyl radical, OH•. In addition, the generation of ROSs increased with an increase in the UV irradiation time. We proposed a kinetic model that reproduces the dependence of bacterial viability on the UV light irradiation time by considering the temporal change in the amount of ROSs generated by UV light irradiation. A comparison of the calculated and experimental results revealed that the bactericidal effect consisted of the direct photolysis of bacteria and the photocatalysis via the generation of hydroxyl radicals, with the latter exhibiting a stronger bactericidal effect than the former.

Self-Assembly of Hybrid Lipid Membranes Doped with Hydrophobic Organic Molecules at the Water/Air Interface.

Because of their unique properties, including an ultrathin thickness (3-4 nm), ultrahigh resistivity, fluidity and self-assembly ability, lipid bilayers can be readily functionalized and have been used in various applications such as bio-sensors and bio-devices. In this study, we introduced a planar organic molecule: copper (II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc) to dope lipid membranes. The CuPc/lipid hybrid membrane forms at the water/air interface by self-assembly. In this membrane, the hydrophobic CuPc molecules are located between the hydrophobic tails of lipid molecules, forming a lipid/CuPc/lipid sandwich structure. Interestingly, an air-stable hybrid lipid bilayer can be readily formed by transferring the hybrid membrane onto a Si substrate. We report a straightforward method for incorporating nanomaterials into a lipid bilayer system, which represents a new methodology for the fabrication of biosensors and biodevices.

Advances in Artificial Cell Membrane Systems as a Platform for Reconstituting Ion Channels.

An artificial cell membrane that is composed of bilayer lipid membranes (BLMs) with transmembrane proteins incorporated within them represents a well-defined system for the analysis of membrane proteins, especially ion channel proteins that are major targets for drug design. Because the BLM system has a high compatibility with recently developed cell-free expression systems, it has attracted attention as a next-generation drug screening system. However, three issues associated with BLM systems, i. e., their instability, the need for non-volatile organic solvents and a low efficiency of ion channel incorporation, have limited their use as a drug screening platform. In this personal account, we discuss our recent approaches to address these issues based on microfabrication. We also discuss the potential for using the BLM system combined with cell-free expression systems as a drug screening system for future personalized medicine.

Modulation of Photoinduced Transmembrane Currents in a Fullerene-Doped Freestanding Lipid Bilayer by a Lateral Bias.

We report on a novel lipid bilayer system, in which a lateral bias can be applied in addition to a conventional transmembrane voltage. Freestanding bilayer lipid membranes (BLMs) doped with [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) were formed in a microaperture, around which metal electrodes were deposited. Using this system, it was possible to modulate and amplify photoinduced transmembrane currents by applying a lateral bias along the BLM. The results indicate that the microfabricated Si chip with embedded electrodes is a promising platform for the formation of transistor-like devices based on PCBM-doped BLMs and have potential for use in a wide variety of nanohybrid devices.

Formation and Characterization of Air-Stable Lipid Bilayer Membranes Incorporated with Phthalocyanine Molecules.

Bilayer lipid membranes (BLMs) are used as basic frameworks for biosensors and biohybrid devices due to their unique properties, which include ultrathin thickness, ultrahigh resistivity, and self-assembling ability. However, BLMs can only form and maintain their structure in aqueous environments, which pose significant limitations to their use. In this work, we report on the formation of highly uniform hybrid BLMs at a water/air interface through self-assembly by simply doping the BLMs with a functional organic molecule, copper(II) 2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine (CuPc). By transferring the membrane onto substrates, we were able to produce stable hybrid BLMs under anhydrous conditions. Atomic force microscopy and X-ray diffraction measurements confirmed that the hybrid membranes were composed of single, highly uniform BLMs or stacks of BLMs. Fluorescence resonance energy transfer measurements indicated that the CuPc molecules were located between the hydrophobic tails of lipid molecules, forming a sandwich structure in the hybrid membranes. The hybrid BLMs fabricated by this method substantially expand the range of applications of BLMs to solid-state devices.

Amphiphobic Septa Enhance the Mechanical Stability of Free-Standing Bilayer Lipid Membranes.

Artificial bilayer lipid membranes (BLMs) provide well-defined systems for investigating the fundamental properties of membrane proteins, including ion channels, and for screening the effect of drugs that act on them. However, the application of this technique is limited due to the low stability and low reconstitution efficiency of the process. We previously reported on improving the stability of BLM based on the fabrication of microapertures having a tapered edge in SiO2/Si3N4 septa and efficient ion channel incorporation based on vesicle fusion accelerated by a centrifugal force. Although the BLM stability and incorporation probability were dramatically improved when these approaches were used, some BLMs were ruptured when subjected to a centrifugal force. To further improve the BLM stability, we investigated the effect of modifying the surface of the SiO2/Si3N4 septa on the stability of BLM suspended in the septa. The modified surfaces were characterized in terms of hydrophobicity, lipophobicity, and surface roughness. Diffusion coefficients of the lipid monolayers formed on the modified surfaces were also determined. Highly fluidic lipid monolayers were formed on the amphiphobic substrates that had been modified with long-chain perfluorocarbons. Free-standing BLMs formed in amphiphobic septa showed a much higher mechanical stability, including tolerance to water movement and applied centrifugal forces with and without proteoliposomes, than those formed in the septa that had been modified with a short alkyl chain. These results demonstrate that highly stable BLMs are formed when the surface of the septa has amphiphobic properties. Because highly fluidic lipid monolayers that are formed on the septa seamlessly connect with BLMs in a free-standing region, the high fluidity of the lipids contributes to decreasing potential damage to BLMs when mechanical stresses are applied. This approach to improve the BLM stability increases the experimental efficiency of the BLM systems and will contribute to the development of high-throughput platforms for functional assays of ion channel proteins.

Mechanically stable solvent-free lipid bilayers in nano- and micro-tapered apertures for reconstitution of cell-free synthesized hERG channels.

The self-assembled bilayer lipid membrane (BLM) is the basic component of the cell membrane. The reconstitution of ion channel proteins in artificially formed BLMs represents a well-defined system for the functional analysis of ion channels and screening the effects of drugs that act on them. However, because BLMs are unstable, this limits the experimental throughput of BLM reconstitution systems. Here we report on the formation of mechanically stable solvent-free BLMs in microfabricated apertures with defined nano- and micro-tapered edge structures. The role of such nano- and micro-tapered structures on the stability of the BLMs was also investigated. Finally, this BLM system was combined with a cell-free synthesized human ether-a-go-go-related gene channel, a cardiac potassium channel whose relation to arrhythmic side effects following drug treatment is well recognized. Such stable BLMs as these, when combined with a cell-free system, represent a potential platform for screening the effects of drugs that act on various ion-channel genotypes.

Fabrication and Characterization of High-Quality Perovskite Films with Large Crystal Grains.

Solution-processable organometal perovskite materials have been widely used in various kinds of devices. In these devices, the perovskite materials normally act as active layers. Grain boundaries and structural disorder in the perovskite layer would interfere the charge transport and increase recombination probability. Here we proposed a novel fabrication method to dramatically increase the crystal size by more than 20 times as compared with previously reported values. Exceptional structural order in the large crystals is illustrated by nanoscale surface morphology and a simple recrystallization method. Because of reduced grain boundaries and increased crystal order in perovskite layers, the lateral charge transport is significantly improved, as demonstrated by conductive atomic-force microscopy and performance of photodetectors. This deposition technology paves the way for future high-performance devices based on perovskite thin films.

Reconstitution of Human Ion Channels into Solvent-free Lipid Bilayers Enhanced by Centrifugal Forces.

Artificially formed bilayer lipid membranes (BLMs) provide well-defined systems for functional analyses of various membrane proteins, including ion channels. However, difficulties associated with the integration of membrane proteins into BLMs limit the experimental efficiency and usefulness of such BLM reconstitution systems. Here, we report on the use of centrifugation to more efficiently reconstitute human ion channels in solvent-free BLMs. The method improves the probability of membrane fusion. Membrane vesicles containing the human ether-a-go-go-related gene (hERG) channel, the human cardiac sodium channel (Nav1.5), and the human GABAA receptor (GABAAR) channel were formed, and the functional reconstitution of the channels into BLMs via vesicle fusion was investigated. Ion channel currents were recorded in 67% of the BLMs that were centrifuged with membrane vesicles under appropriate centrifugal conditions (14-55 × g). The characteristic channel properties were retained for hERG, Nav1.5, and GABAAR channels after centrifugal incorporation into the BLMs. A comparison of the centrifugal force with reported values for the fusion force revealed that a centrifugal enhancement in vesicle fusion was attained, not by accelerating the fusion process but by accelerating the delivery of membrane vesicles to the surface of the BLMs, which led to an increase in the number of membrane vesicles that were available for fusion. Our method for enhancing the probability of vesicle fusion promises to dramatically increase the experimental efficiency of BLM reconstitution systems, leading to the realization of a BLM-based, high-throughput platform for functional assays of various membrane proteins.