Keiko Esashika

Optical observation of DNA motion during and immediately after nanopore translocation

Herein, we report an optical nanopore detection system capable of investigating DNA motion near nanopores not only during translocation but also post-translocation with submillisecond resolution. Using our optical nanopore detection system, we observed the voltage dependence of the dwell time of both 10-kbp double-stranded DNA (dsDNA) and lambda DNA in the excitation volume, which can be attributed to the drift-dominated motion. We found that the lambda DNA had slower drift motion than 10-kbp dsDNA, indicating that DNA with longer gyration experiences a lower nonuniform electric force.

Optimized modification of gold nanoparticles with a self-assembled monolayer for suppression of nonspecific binding in DNA assays

Homogeneous DNA assays using gold nanoparticles (AuNPs) require the reduction of nonspecific binding between AuNPs to improve sensitivity in detecting the target molecule. In this study, we employed alkanethiol self-assembled monolayers (SAMs) for modifying the AuNP surface to attain both good dispersability and high hybridization efficiency. The alkanethiol SAMs enhance the repulsive interaction between AuNPs, reducing nonspecific binding and promoting the extension of surface-immobilized ssDNA into the solvent, thus enhancing the hybridization process. Introduction of oligoethylene glycol into the alkanethiol prevented nonspecific binding caused by the entanglement of alkane chains. Finally, the conditions were optimized by controlling the surface charge density through the introduction of a COOH group at the alkanethiol terminus, resulting in the complete blocking of nonspecific binding and the maintenance of high hybridization efficiency.

DNA Hybridization Assay Using Gold Nanoparticles and Electrophoresis Separation Provides 1 pM Sensitivity

The efficiency of gold nanoparticle (AuNP) dimerization mediated by hybridization between two probe DNA molecules and a complementary target DNA molecule was maximized by examining several possible hybridization combinations. The uniformity of the size of the AuNPs, the use of surface modification appropriate for high hybridization efficiency, together with efficient blocking of nonspecific binding, all contributed to achieving a 1 pM detection limit following conventional gel electrophoresis separation of the DNA-modified AuNP multimers. This practical homogeneous DNA hybridization assay methodology will provide a rapid, cost-effective, and field-portable tool for clinical diagnosis.

Optical observation of DNA translocation dynamics through solid-state nanopores

We report an optical nanopore detection system for investigating DNA translocation dynamics through a nanopore at sub-millisecond and sub-100-nm resolutions. The proposed optical nanopore detection scheme enables the observation of both the translocation process and the escape process. We found different correlation between the translocation time and the escape time, depending on whether the translocation occurs in a folded or unfolded configuration.

Highly sensitive measurement of single DNA translocation through an ultraviolet light spot on silicon nanopore

Nanopore-based sensing is an attractive candidate for developing single-molecule DNA sequencing technology. Recently, optical detection with a parallel nanopore array has been demonstrated. Although this method is a promising approach to develop high thorough-put measurement, the approach requires observation at low-background condition. In this paper, we propose a new optical method for nanopore DNA sequencing with high resolution and a high signal-tonoise ratio. We use ultraviolet light for the excitation of a fluorescent probe and a nanopore in a silicon membrane. Because silicon has a large refractive index and an extinction coefficient at ultraviolet wavelengths, light transmission thorough the membrane is negligible. This contributes to low background measurement of fluorescence from fluorophore-labeled DNA strands. In addition, the z-polarization component of the electric field is attributed to generating a large electric field gradient at the nanopore exit due to its boundary condition at the silicon surface. Our numerical electromagnetic simulation revealed that the z-component electric field was dominant compared to the xcomponent electric filed. The intensity of the electric field increased steeply in 2 nm, when ultraviolet light of 375nm wavelength was focused on a 10nm-thick silicon membrane with a 7 nm-diameter nanopore. This steeply increasing electric field can be sufficient resolution for the sequencing of designed DNA polymer. Finally, our experimental results demonstrated optical detection of single DNA translocation events with a high signal-to-noise ratio under applied voltage.