Kazuki Matsubara

Single-Molecule Electrical Random Resequencing of DNA and RNA

Two paradigm shifts in DNA sequencing technologies—from bulk to single molecules and from optical to electrical detection—are expected to realize label-free, low-cost DNA sequencing that does not require PCR amplification. It will lead to development of high-throughput third-generation sequencing technologies for personalized medicine. Although nanopore devices have been proposed as third-generation DNA-sequencing devices, a significant milestone in these technologies has been attained by demonstrating a novel technique for resequencing DNA using electrical signals. Here we report single-molecule electrical resequencing of DNA and RNA using a hybrid method of identifying single-base molecules via tunneling currents and random sequencing. Our method reads sequences of nine types of DNA oligomers. The complete sequence of 5′-UGAGGUA-3′ from the let-7 microRNA family was also identified by creating a composite of overlapping fragment sequences, which was randomly determined using tunneling current conducted by single-base molecules as they passed between a pair of nanoelectrodes.

Electrical detection of single methylcytosines in a DNA oligomer.

We report label-free electrical detections of chemically modified nucleobases in a DNA using a nucleotide-sized electrode gap. We found that methyl substitution contributes to increase the tunneling conductance of deoxycytidines, which was attributed to a shift of the highest occupied molecular orbital level closer to the electrode Fermi level by methylation. We also demonstrate statistical identifications of methylcytosines in an oligonucleotide by tunneling current. This result suggests a possible use of the transverse electron-transport method for a methylation level analysis.

Development of microfabricated TiO2 channel waveguides

An optical channel waveguide is a key solution to overcome signal propagation delay. For the benefits of miniaturization, development of microfabrication process for waveguides is demanded. TiO2 is one of the suitable candidates for the microfabricated waveguide because of the high refractive index and the transparency. In the present study, conventional microfabrication processes manufactured TiO2 channel waveguides with 1–20 μm width on oxidized Si substrates and the propagation loss was measured. The prepared channels successfully guided light of 632.8 nm along linear and Y-branched patterns. The propagation loss for the linear waveguide was 9.7 dB/cm.

Atomically controlled fabrications of subnanometer scale electrode gaps

We report electrode gap formations at high temperatures using a self-breaking technique. We obtained narrow distributions of the size of Au electrode gaps dgap centered at about 0.5 nm at temperatures below 380 K. At higher temperatures, on the other hand, we find larger dgap distributing around 0.8 nm. The present results demonstrate the possible use of high temperature Au nanocontact self-breaking processes for controlled fabrications of electrode gaps useful for DNA sequence read out with quantum mechanics.

Embedded TiO2 waveguides for sensing nanofluorophores in a microfluidic channel

Crossed structure of a microfluidic channel and an optical channel waveguide is simple and promising to realize detection of weak fluorescence on an integrated device. Usage of TiO2 waveguides is suitable for the device because of the high numerical aperture. In this study, we developed fabrication processes for the TiO2 channel waveguides traversed by microfluidic channels of 0.5–6 μm widths and investigated the effect of the microfluidic channel to the transmittances. The results elucidated that the photointensity at the microfluidic channel was enough to excite fluorophores. Furthermore, we demonstrated detection of fluorescence from 350 quantum dots.

Nano-scale reactive-ion dry-etching with electron-beam-baked resist

We developed a nano-scale electron-beam (EB) lithography procedure using a high-resistance electron-beam resist for fabrication of nano-biodevices. After a conventional EB image-development procedure, we newly added a resist-baking procedure using an EB exposure with a density of over 50 mC/cm2, and then performed a reactive-ion dry-etching. We found that the EB-baked resists were highly resistant against dry-etchings, resulting in preserving a clear-pattern of the EB-lithographed image up to a sub 50 nm scales. By using this EB baking method, we successfully fabricated nano-fluidics structures, and observed the smooth-translocation of single λ DNA molecules. This nano-scale dry-etching using EB-baked resist would be a general procedure for EB lithography fabrications of DNA nano-fluidics and sensing structures.