Ayumi Hirano-Iwata

Free-Standing Lipid Bilayers in Silicon Chips-Membrane Stabilization Based on Microfabricated Apertures with a Nanometer-Scale Smoothness

In the present study, we propose a method for preparing stable free-standing bilayer lipid membranes (BLMs). The BLMs were prepared in a microfabricated aperture with a smoothly tapered edge, which was prepared in a nanometer-thick Si(3)N(4) septum by the wet etching method. Owing to this structure, the stress on lipid bilayers at the contact with the septum was minimized, leading to remarkable membrane stability. The BLMs were not broken by applying a constant voltage of +/-1 V. The membrane lifetime was 15-45 h with and without an incorporated gramicidin channel. Gramicidin single-channel currents were recorded from the same BLM preparation when the aqueous solutions surrounding the BLM were repeatedly exchanged, demonstrating the tolerance of the present BLM to repetitive solution exchanges. Such stable membranes enable analysis of channel functions under various solution conditions from the same BLM, which will open up a variety of applications including a high throughput drug screening for ion channels.

In situ real-time monitoring of apoptosis on leukemia cells by surface infrared spectroscopy

We have investigated in situ real-time monitoring of apoptosis on human promyelocytic leukemia (HL-60) cells using infrared absorption spectroscopy with the multiple internal reflection (MIR-IRAS) geometry. Actinomycin D (Act D)-induced apoptosis on HL-60 cells was monitored for 24 h. Apoptotic cells showed two strong peaks around the protein amide I and amide II bands probably due to the leakage of cytoplasmic proteins, while growing viable cells showed a peak corresponding to the secretion of metabolites and two downward peaks corresponding to uptake of nutrients from culture media. In addition, IR absorption peak intensity of the amide I and amide II bands was proportional to the extracellular concentration of lactate dehydrogenase, a marker protein for cell damage. These results demonstrate that our MIR-IRAS method is useful for discrimination of apoptotic cells from viable ones and cell apoptotic processes can be monitored in situ by analyzing the amide I and amide II peak intensity.

In situ real-time monitoring of biomolecular interactions by using surface infrared spectroscopy

This paper reviews our recent approaches for in situ label-free detection of biomolecules and their interactions by using infrared absorption spectroscopy (IRAS) in the multiple internal reflection (MIR) geometry. Biomolecular interactions, such as DNA hybridization, DNA hydration, protein-protein interaction, cell growth and cell death, were characterized and monitored in situ both in D2O and H2O media. Combination of MIR-IRAS and various sensing platforms, such as Si, GaAs, porous Si, and porous amunina, was also discussed in terms of sensitivity and applicability to chip analysis. It is demonstrated that MIR-IRAS is a promising tool not only for the label-free detection of biomolecules but also for the accurate discrimination between specific and nonspecific interactions, which is critically important when we are monitoring complex and dynamic biological samples.

Annealing-induced chemical and structural changes in tri-iodide and mixed-halide organometal perovskite layers

The annealing process is crucial for obtaining high-quality perovskite layers used in highly efficient planar perovskite solar cells. In this study, we have investigated the annealing-induced chemical and structural changes of tri-iodide (TI) and mixed-halide (MH) organometal perovskite layers using infrared absorption spectroscopy, scanning electron microscopy and X-ray diffraction measurements. For TI layers, the solvent molecules, dimethylformamide (DMF), remained in the form of the PbI2/DMF compound after drying at room temperature. During annealing, the DMF evaporated to form PbI2 crystals. When the MH perovskite film was annealed, both CH3NH3PbCl3 and CH3NH3PbI3 crystals were initially formed from an amorphous phase. With further annealing, the CH3NH3PbI3 crystals gradually grew through the incorporation of source materials supplied from the CH3NH3PbCl3 crystals and the amorphous phase and the slow evaporation of methylammonium (MA) and chloride ions. The resultant MH perovskite layer after annealing was mainly composed of large CH3NH3PbI3 grains with a trace of chloride ions. We suggest that the difference in composition and structure leads to different charge transport properties of the TI and MH perovskite layers.

Micro- and Nano-Technologies for Lipid Bilayer-Based Ion-Channel Functional Assays

Ion channel proteins provide gated pores that allow ions to passively flow across cell membranes. Owing to their crucial roles in regulating transmembrane ion flow, ion channel proteins have attracted the attention of pharmaceutical investigators as drug targets for use in the studies of both therapeutics and side effects. In this review, we discuss the current technologies that are used in the formation of ion channel-integrated bilayer lipid membranes (BLMs) in microfabricated devices as a potential platform for next-generation drug screening systems. Advances in BLM fabrication methodology have allowed the preparation of BLMs in sophisticated formats, such as microfluidic, automated, and/or array systems, which can be combined with channel current recordings. A much more critical step is the integration of the target channels into BLMs. Current technologies for the functional reconstitution of ion channel proteins are presented and discussed. Finally, the remaining issues of the BLM-based methods for recording ion channel activities and their potential applications as drug screening systems are discussed.

Reconstitution of Human Ether-a-go-go-Related Gene Channels in Microfabricated Silicon Chips

This paper reports on the reconstitution of human ether-a-go-go-related gene (hERG) channels in artificial bilayer lipid membranes (BLMs) formed in micropores fabricated in silicon chips. The hERG channels were isolated from Chinese hamster ovary cell lines expressing the channels and incorporated into the BLMs formed by a process in which the two lipid monolayers were folded into the micropores. The characteristic features of hERG channels reported by the patch-clamp method, including single-channel conductance, voltage dependence, sensitivity to typical drugs and dependence on the potassium concentration, were investigated in the BLM reconstitution system. The BLM with hERG channels incorporated exhibited a lifetime of ~65 h and a tolerance to repetitive solution exchanges. Such stable BLMs containing biological channels have the potential for use in a variety of applications, including high-throughput drug screening for various ion-channel proteins.

Real-time monitoring of cell death by surface infrared spectroscopy

We have developed a method for real-time monitoring of the cell responses to cytotoxicants using Fourier transform infrared spectroscopy with the multiple internal reflection (MIR-FTIR) geometry. To prevent cell damages induced by measurement environments, we have constructed specialized chambers, in which temperature was maintained at (37±0.5)°C and humidified air containing 5% CO2 was supplied. We monitored cell death induced by cytotoxic surfactant Tween20 using MIR-FTIR spectroscopy. It was found that cell death can be monitored by the absorption intensity of amide II band. This result suggests that our method has a potential to be applied for real-time cytotoxicity assay.

Effects of interfacial chemical states on the performance of perovskite solar cells

We showed that the widely used solvent molecule, N,N-dimethyl-formamide (DMF), readily adsorbs on the surface of TiO2 electrodes of perovskite solar cells (PSCs), and that the adsorbed DMF molecules remain intact on the TiO2 surface even after long-term annealing of the perovskite layer, resulting in an increase in the contact resistance of the PSCs. We found that the absorption of DMF is significantly suppressed by modifying the TiO2 electrode surface with a fullerene derivative, [6,6]-phenyl-C61-butyric acid (PCBA). We also suggested that the high electron affinity of PCBA enhances the charge transportation at the perovskite/TiO2 interface and reduces the contact resistance.

Stable lipid bilayers based on micro- and nano-fabrication

In this review, we will discuss our recent approaches for improving the mechanical stability of free-standing bilayer lipid membranes (BLMs) by combining with BLM formation and micro- and nano-fabrication techniques. BLMs were prepared across a microaperture fabricated in silicon (Si) chips or nanoporous alumina films, and their mechanical stability and electric properties were investigated. BLMs spanned over the porous alumina showed background noise currents small enough for recording activities of low-conductance channels, though further stability enhancement of porous alumina films was necessary. BLMs suspended in a thin Si3N4 septum showed a dramatic improvement of BLM stability. The BLMs were resistant to a voltage of ± 1 V and the membrane lifetime was 15–43 h with and without incorporated channels. The membrane containing gramicidin channel exhibited tolerance to repetitive solution exchanges, though the electric properties of the BLMs are necessary to be improved. The realisation of BLMs having both mechanical stability and proper electric properties will open up a variety of applications including highly sensitive biosensors and high-throughput drug screenings for ion channels.

Lipid bilayer array for simultaneous recording of ion channel activities

This paper describes an array of stable and reduced-solvent bilayer lipid membranes (BLMs) formed in microfabricated silicon chips. BLMs were first vertically formed simultaneously and then turned 90° in order to realize a horizontal BLM array. Since the present BLMs are mechanically stable and robust, the BLMs survive this relatively tough process. Typically, a ∼60% yield in simultaneous BLM formation over 9 sites was obtained. Parallel recordings of gramicidin channel activities from different BLMs were demonstrated. The present system has great potential as a platform of BLM-based high throughput drug screening for ion channel proteins.