Masayuki Furuhashi

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.

Transverse electric field dragging of DNA in a nanochannel.

Nanopore analysis is an emerging single-molecule strategy for non-optical and high-throughput DNA sequencing, the principle of which is based on identification of each constituent nucleobase by measuring trans-membrane ionic current blockade or transverse tunnelling current as it moves through the pore. A crucial issue for nanopore sequencing is the fact that DNA translocates a nanopore too fast for addressing sequence with a single base resolution. Here we report that a transverse electric field can be used to slow down the translocation. We find 400-fold decrease in the DNA translocation speed by adding a transverse field of 10 mV/nm in a gold-electrode-embedded silicon dioxide channel. The retarded flow allowed us to map the local folding pattern in individual DNA from trans-pore ionic current profiles. This field dragging approach may provide a new way to control the polynucleotide translocation kinetics.

Detection of post-translational modifications in single peptides using electron tunnelling currents

Post-translational modifications alter the properties of proteins through the cleavage of peptide bonds or the addition of a modifying group to one or more amino acids. These modifications allow proteins to perform their primary biological functions, but single-protein studies of post-translational modifications have been hindered by a lack of suitable analysis methods. Here, we show that single amino acids can be identified using electron tunnelling currents measured as the individual molecules pass through a nanoscale gap between electrodes. We identify 12 different amino acids and the post-translational modification phosphotyrosine, which is involved in the process that switches enzymes on and off. Furthermore, we show that the conductance measurements can be used to partially sequence peptides of an epidermal growth factor receptor substrate, and can discriminate a peptide from its phosphorylated variant.

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.

Diffusion and dissociation mechanisms of vacancy-oxygen complex in silicon

We are examining diffusion mechanisms of the vacancy-oxygen complex (VO) in bulk Si using ab initio calculations based on a 64-atom supercell. We found two atomic mechanisms involved in the VO diffusion; one is caused by migration of an interstitial oxygen atom, another by migration of a vacancy. The energy barrier of the mechanism due to an oxygen migration is 2.02eV, and that caused by a vacancy migration is 1.98eV. These energy barriers are close to the experimental activation energy of 2.0eV required for the dissociation and diffusion of VO. The derived activation energies of the two mechanisms suggest that these mechanisms plausibly occur simultaneously. In addition, we clarify that the dissociation energy of VO, 1.85eV, is lower than the diffusion energy of VO.

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.

Anomalous uphill diffusion and dose loss of ultra-low-energy implanted boron in silicon during early stage of annealing

Uphill diffusion of boron implanted with 0.5 keV in silicon was investigated under nonamorphizing implant conditions. Postimplantation annealing at temperatures above 400°C induces the uphill diffusion of boron, which can be observed in an initial stage of annealing. The uphill diffusion is significantly suppressed by additional Si implantation into the boron implant region, suggesting that the uphill diffusion is mediated by the excess free self-interstitials during the annealing. Also, the uphill diffusion causes severe dose loss of implanted boron to the Si/SiO2 interface.

Additional stress-induced band gap narrowing in a silicon die

Semiconductor devices have intrinsic mechanical stress due to two separate sources, silicon wafer processing and packaging. This intrinsic stress induces band gap narrowing and consequent degradation of device reliability. In this work, a silicon die that included shallow trench isolation structures was used with additional external compression to clarify the interaction effects on band gap narrowing between stresses induced by different factors. It was observed that the pressure coefficient due to the additional external compression is linearly dependent on the intrinsic tensile region/total region ratio of a p-n diode.

High speed DNA denaturation using microheating devices

Denaturation is a first step for further treatment of DNA and is expected to be carried out rapidly on an integrated chip. A microheater is a promising device for the denaturation because of easiness for fabrication and operation. In the present study, we fabricated a microheater and thermometers on a coverslip and investigated response of temperature to application of voltage. In addition, our experiment and simulation proved local heating at an aimed area. Finally, we demonstrated denaturation of DNA in buffer solution, the result of which proved that the DNA around the heater denatured within 60 ms.