Yoshinobu Kohara

Characteristics of liquid electrode plasma for atomic emission spectrometry

Liquid electrode plasma atomic emission spectrometry (LEP-AES) is a recently developed elemental analysis method that uses microplasma. LEP forms in a vapor bubble generated inside a narrow-center microchannel by using high-voltage DC pulse power. We studied the characteristics of LEP and atomic emission of lead (Pb), as an example element, which has not been described in detail. We estimated the plasma parameters and observed the expansion and shrinkage of a vapor bubble with discharge as well as the time course and spatial distribution of the atomic emission of Pb (405.78 nm). The applied voltage was 2.5 kV and the pulse width was less than 3 ms, which produced a current of about 100 mA. We found that the excitation temperature was about 8000 K and the electron density was about 1 × 1015 cm−3. We also found that two quite different emission phases occurred separately during the time course. The first emission phase corresponds to the first expansion and shrinking of the bubble around atmospheric pressure and the second emission phase corresponds to the re-expansion of the bubble and emission at reduced pressure with higher atomic and lower background emissions. Maximum atomic and background emissions were observed at the narrowed center of the microchannel, but there was an additional local maximum atomic emission region at the anode side bubble–liquid interface where the background emission was very low, which would be a better condition for sensitive measurement. The limit of detection determined in our experiment was 4.0 μg L−1 for Pb.

Atomic emission spectrometry in liquid electrode plasma using an hourglass microchannel

Liquid electrode plasma atomic emission spectrometry (LEP-AES) is a new elemental analysis method that uses microplasma. LEP forms in a vapor bubble generated inside a narrow-center microchannel by using high-voltage DC pulse power. In this study, we used a novel hourglass microchannel having a 3-dimensionally and axisymmetrically narrowed shape, which caused a bright emission roughly 200 times that of the flat microchannel used in our previous study. We observed the spatial distribution of atomic emission and determined the limit of detection (LoD) by utilizing the confirmed spatial distribution. We found that the spatial distribution of atomic emission for 41 elements in our experiments could be classified into three patterns in accordance with a maximum emission point: anode side, narrow-center, and cathode side. Atomic emission was measured at the maximum emission point and the calibration curve for each element was made to determine the LoD. The LoD of 25 tested elements in our experiment ranged from 1 μg L−1 for Li to 306 μg L−1 for V.

Single-molecule detection of proteins with antigen-antibody interaction using resistive-pulse sensing of submicron latex particles

We developed a resistive-pulse sensor with a solid-state pore and measured the latex agglutination of submicron particles induced by antigen-antibody interaction for single-molecule detection of proteins. We fabricated the pore based on numerical simulation to clearly distinguish between monomer and dimer latex particles. By measuring single dimers agglutinated in the single-molecule regime, we detected single human alpha-fetoprotein molecules. Adjusting the initial particle concentration improves the limit of detection (LOD) to 95 fmol/l. We established a theoretical model of the LOD by combining the reaction kinetics and the counting statistics to explain the effect of initial particle concentration on the LOD. The theoretical model shows how to improve the LOD quantitatively. The single-molecule detection studied here indicates the feasibility of implementing a highly sensitive immunoassay by a simple measurement method using resistive-pulse sensing.

Rapid multiplex single nucleotide polymorphism genotyping based on single base extension reactions and color-coded beads

A single nucleotide polymorphism (SNP) typing method using color-coded beads is promising because it is easy to use and inexpensive. However, the present protocols are not suitable for clinical and diagnostic applications because they need centrifugation for bead-washing. Here, we developed a simplified protocol without a bead-washing procedure that enables SNP typing of PCR amplified fragments in only 30 min.

Abstract 1574: KRAS genotyping by digital PCR combining melting curve analysis

Background ctDNA is a remarkable liquid biopsy for cancer diagnosis. Highly sensitive quantification method is required to measure a tiny amount of ctDNA. Digital PCR has been developed as a method that can quantify nucleic acids more sensitively than real-time PCR does. However, the digital PCR has large fluctuation in the fluorescence intensity of the droplets or chambers resulting in lower accuracy. Main cause is probably due to the insufficient PCR in the small partitions. In this study, we have proposed a new measurement method combined a digital PCR with melting curve analysis using molecular beacons to solve above mentioned problems, and applied it to KRAS genotyping. Methods Molecular beacons, which have hydrophobic moiety in the stem, were designed for detecting KRAS mutation. The digital PCR with combination of asymmetric PCR was performed using the molecular beacons in the droplets. After the PCR, the fluorescence of the droplets was observed with a microscope while changing the temperature. A melting curve was prepared from the change in fluorescence intensity of the droplet, and the melting temperature (Tm) was calculated from the differential melting curve. Results The melting curve analysis for the KRAS mutation was performed in the droplets where the asymmetric PCR was performed using molecular beacons with hydrophobic stem, which improved signal-to-noise ratio of melting curves. The use of molecular beacons with hydrophobic stem can keep a background fluorescence at a constant value even at high temperature. The change in fluorescence intensity of PCR solution using molecular beacons with hydrophobic stem during the measurement was one-tenth of that using molecular beacon without hydrophobic stem. The asymmetric PCR enabled us to increase the amount of the PCR products hybridized with molecular beacons, resulting in the increase in the fluorescence intensity. The KRAS genotyping of wild-type (WT) and G12D mutant was conducted by the melting curve analysis with a combination of the asymmetric PCR with molecular beacons. The results showed that the peaks of the distributions of the Tm values of DNA in the droplets were 77.9°C for WT and 74.0°C for G12D mutant, which indicates that the WT and the mutant could be successfully discriminated by the proposed method. Conclusion We have proposed a new measurement method combining digital PCR with asymmetric PCR using molecular beacons and melting curve analysis. The genotyping of KRAS mutation was successfully performed by the proposed method. We are planning to prove the concept of this method for the clinical specimens in the future. Citation Format: Junko Tanaka, Yuzuru Shimazaki, Tatsuo Nakagawa, Akiko Shiratori, Masao Kamahori, Takahide Yokoi, Kunio Harada, Yoshinobu Kohara. KRAS genotyping by digital PCR combining melting curve analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1574.

DNA hybridization feature on “Bead-array” — DNA probes on beads arrayed in a capillary

We present a DNA analysis platform named “Bead-array”, where 100-μm-diameter beads are lined with determined order in a capillary, and its hybridization detection features. Model hybridization experiments revealed that as little as 1 amol of fluorescent-labeled oligo DNA was detected and hybridization reaction was completed in one minute irrespective of the amount of target DNA. When the number of target molecules was smaller than that of the probe molecules on the bead, 10 fmol, almost all targets were captured on the bead. “Bead-array” enables reliable and reproducible measurement of the target quantity. This rapid and sensitive platform is very promising for various genetic testing tasks.