Ryo-taro Yamaguchi

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.

Fabrication of Titanium Oxide Nanotubes by Rapid and Homogeneous Anodization in Perchloric Acid/Ethanol Mixture

We have synthesized anodic titanium oxide nanotubes with a high aspect ratio by anodizing a metallic titanium sheet in a perchloric acid based electrolyte, and fabricated a dye-sensitized solar cell (DSSC) by using the nanotubes as its negative electrode. Rapid and homogeneous anodization has been accomplished using a mixture of perchloric acid and ethanol as the electrolyte. Furthermore, it was demonstrated that the diameter of anodic titanium oxide nanotubes could be varied by adjusting the anodization conditions, such as the electrolyte and the anodic potential. The measured characteristic of the DSSC showed that anodic titanium oxide nanotubes have a potential for improving the performance of a DSSC.

In situ observation of DNA hybridization and denaturation by surface infrared spectroscopy

We have investigated in situ the hybridization and denaturation of DNA in aqueous solution using infrared absorption spectroscopy (IRAS) in the multiple internal reflection (MIR) geometry. We demonstrate that conformational changes of DNA strands due to hybridization (binding of two complementary single-stranded DNAs) and denaturation (separation of double helix at elevated temperatures) are reflected in the infrared absorption spectra in the frequency region where vibrational modes of the bases of DNA appear. Comparison with results of ab initio cluster calculation shows that hybridization produces the specific CO carbonyl stretching vibration modes in the hydrogen-bonded base pairs. The ratio of absorbance of the CO stretching peak at 1690cm−1 to the absorbance at 1660cm−1 provides a definitive metric for determining DNA hybridization. We also reveal that the CO stretching vibration modes of the bases of a single strand is strongly influenced by the surrounding water molecules that may interact with ...

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.

DNA hybridization detection by porous silicon-based DNA microarray in conjugation with infrared microspectroscopy

A method is described for the label-free detection of DNA hybridization on porous silicon (por-Si), based upon the pairing of oligonucleotide chemistry and standard silicon nanotechnology. Por-Si with a pore diameter of approximately 30 nm was used to immobilize probe DNA. Infrared microspectroscopy was employed to monitor the hybridization of probe-DNA immobilized on pore surfaces with its complementary DNA (target-DNA). The immobilization of probe DNA on por-Si facilitates hybridization detection for a small sensing area (approximately 50×50 μm2) with a high detection efficiency. In this study, we fabricated a porous silicon-based DNA microarray (por-Si-microarray) using photolithographic and Si anodizing techniques. We demonstrate that DNA hybridization can be detected on a por-Si-microarray through the analysis of infrared absorption spectral profiles in the region where the vibration modes of the bases appear. This present approach demonstrates that por-Si-microarray in conjugation with infrared micr...

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.

Room-temperature observation of a Coulomb blockade phenomenon in aluminum nanodots fabricated by an electrochemical process

An aluminum nanodot was self-organized between two electrodes using the anodization process of an aluminum microelectrode of 3μm in width. The authors observed a clear Coulomb staircase with a very large Coulomb energy of about 2eV at room temperature. This very large Coulomb energy is attributed to the device structure which depends strongly on the aluminum nanodot formation mechanism. The authors’ results indicate that a single electron transistor operating at room temperature can be fabricated at an appropriate position using both bottom-up and top-down processes.

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.

Self-formation of bilayer lipid membranes on agarose-coated silicon surfaces studied by simultaneous electrophysiological and surface infrared spectroscopic measurements

Self-formation process of bilayer lipid membranes (BLMs) cushioned on agarose-coated Si surfaces was in situ monitored by simultaneous electrophysiological and infrared absorption spectroscopic (IRAS) measurements using IRAS with the multiple internal reflection geometry. IRAS signals corresponding to self-thinning of lipid solution to form BLMs were demonstrated. It was found that the appearance of IRAS bands due to C=O modes of phosphstidylcholine is related to formation of BLMs with a gigaohm seal. The functionality of the present BLM system was also demonstrated by incorporating gramicidin into the BLMs and recording its channel activities.

Real-time monitoring of mitochondrial adenosine 5′-triphosphate synthesis and hydrolysis by surface infrared spectroscopy

We have developed a method for in situ real-time monitoring of adenosine 5′-triphosphate (ATP) synthesis in mitochondria using infrared absorption spectroscopy with the multiple internal reflection geometry. Spectral changes corresponding to ATP synthesis and hydrolysis were monitored under oxygenation and constant stirring condition. It was demonstrated that the reversible process of ATP synthesis in mitochondria can be monitored by analyzing stretching modes of α- and β-PO2− in adenine nucleotides. Our method has potential to evaluate mitochondrial toxicity in terms of mitochondrial activities of ATP synthesis and hydrolysis.