Biomedical subjects

Kenichi Yamashita

Publications and source records attributed to Kenichi Yamashita.

8 recordsLinked to original sources

PubMed2005

Specific molecule localization in microchannel laminar flow and its application for non-immobilized-probe analysis.

Microfluidic systems enable superior control of fluidics. We have developed a novel size-separation method utilizing secondary flow within a microchannel. Using confocal fluorescence microscopy and computer simulation, we confirmed that separation occurred as a result of specific molecular localization in the curving part of the microchannel. Maximum separation efficiency was achieved by optimizing microchannel design and flow rate for individual separation targets. In addition, more effective separation was achieved by use of plural microchannel curves. This method was used for sequence-selective DNA sensing. Double-stranded DNA formed by hybridization between target DNA and a complementary probe had different elution profiles from those of the single-stranded non-complementary sequence. Moreover, the response depends on the length of the DNA molecules. This method does not require immobilization of either probe or target DNA, because all reactions occurred in the solution phase. Such features may reduce experimental error and the difference between data from different operators.

Biosensing Techniques↗

PubMed2005

Genotyping of the human lipoprotein lipase gene by ferrocenylnaphthalene diimide-based electrochemical hybridization assay.

A ferrocenylnaphthalene diimide (FND)-based electrochemical hybridization assay (FND-EHA) was applied to the detection of two mutations in human lipoprotein lipase (LPL) gene, G188E (one base transition) and Arita (one base deletion). A probe oligodeoxyribonucleotide of 13 bases representing the wild type (WT) sequence of LPL was immobilized on a gold electrode, followed by hybridization with a sample PCR product of 350 base pairs under conditions in which both WT and mutated (MT) sequences could form a duplex with the probe. The hybridized electrodes were soaked in an electrolyte containing FND under conditions in which only the mismatched duplex could undergo dissociation. FND was concentrated in proportion to the amount of the duplex remaining on the electrode to give rise to a current signal. Blind tests were run to judge the genotype (WT/WT, WT/MT, or MT/MT) of 10 samples each for the G188E and Arita mutations and then, 8 and 10 of them were judged correctly, respectively.

Base Sequence↗

PubMed2005

Efficient immobilization of enzymes on microchannel surface through His-tag and application for microreactor.

We developed a simple immobilisation method for His-tagged enzymes on a microchannel surface. It facilitates immobilisation of protein molecule on microchannel surface through Ni-complex, using crude or purified protein solutions. By this method, we could immobilize proteins on microcapillary constantly. This method might be useful for further development of microreactor with reversibly immobilized enzymes.

Animals↗

PubMed2004

Direct observation of long-strand DNA conformational changing in microchannel flow and microfluidic hybridization assay.

Conformational control of macromolecules is useful for efficient chemical and biochemical reactions. This article reports a direct observation method for macromolecules, such as long-strand DNA, in microchannel flow as well as a simple method for stretching DNA strands by microfluidics. Stretching and orientation of DNA molecules by control of flow within a microchannel was observed by optical microscopy. This DNA stretching is explained by coil-stretch transition of polymer molecules. This technique is useful for creating chemical reactions with macromolecules. It offers high selectivity and efficiency that are impossible to achieve in bulk solution. We also demonstrate that our microfluidic stretching method can accomplish efficient hybridization of long-strand DNA. This method will be useful for direct hybridization assay of long-strand DNA.

DNA↗

PubMed2003

Sequence-selective DNA detection using multiple laminar streams: a novel microfluidic analysis method.

On-site detection methods for DNA have been demanded in the pathophysiology field. Such analysis requires a simple and accurate method, rather than high-throughput. This report describes a novel microfluidic analysis method and its application for simple sequence-selective DNA detection. The method uses a microchannel device with a serpentine structure. Sequence-specific binding of probe DNA can be detected at one side of the microchannel. This method is capable of sequence-specific detection of DNA with high accuracy. Single base mutations can also be analyzed. Combination of laminar stream and laminar secondary flow in the microchannel enable specific detection of probe-bound DNA.

Base Sequence↗

PubMed2003

Direct detection of single nucleotide polymorphism (SNP) with genomic DNA by the ferrocenylnaphthalene diimide-based electrochemical hybridization assay (FND-EHA).

A ferrocenylnaphthalene diimide-based electrochemical hybridization assay (FND-EHA) was applied to the direct detection of a C-to-G transition in a codon (TCA) for Ser-447 of the human lipoprotein lipase (LPL) gene, which resulted in the termination of the LPL protein there. Either one of two 13-meric oligonucleotide probes, S447 WT and S447X MT, representing sequences complementary to those of the wild type (WT) and mutated (MT) forms, was immobilized on a gold electrode, followed by hybridization with chromosomal DNA extracted from human leukocytes under the condition in which both WT- and MT-type sequences can form a duplex. These two electrodes were soaked in an electrolyte containing FND under a condition [0.1 M HOAc/KOAc (pH 5.6) containing 0.1 KCl and 0.05 mM FND at 40 degrees C], in which only the MT duplex could undergo dissociation. FND was concentrated in proportion to the amount of the duplex formed on the electrode to give rise to a current signal. The electrochemical signal ratios obtained for WT/WT, WT/MT and MT/MT were close to the theoretical 2:1:0 with the S447 WT-modified electrode, and was again close to 0:1:2 with the S447X MT-modified one.

Base Sequence↗

PubMed2002

Electrochemical detection of nucleic base mismatches with ferrocenyl naphthalene diimide.

Electrochemical detection of nucleic base mismatches was attempted successfully with ferrocenyl naphthalene diimide (FND) in a model system with 20-meric double-stranded oligonucleotides with or without a mismatch(es). Thus, dA(20) or a 20-meric sequence of the lac Z gene was immobilized on a gold electrode and complementary oligonucleotides with different numbers of mismatches were allowed to hybridize in the presence of FND to give rise to an electrochemical signal. The signal intensity varied depending on the number of unpaired bases on the DNA duplex. From experiments with a quartz crystal microbalance, eight molecules of FND were found to bind to the 20-meric double-stranded oligos and this number decreased as the number of mismatches increased. These findings were further supported by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy. This novel method will be useful for the analysis of single-nucleotide polymorphisms present on human genes.

Base Pair Mismatch↗

PubMedDate not supplied

Ferrocenylnaphthalene diimide-based electrochemical hybridization assay for a heterozygous deficiency of the lipoprotein lipase gene.

An electrochemical hybridization assay has been devised that enables the rapid analysis of a heterozygous deficiency of the human lipoprotein lipase (LPL) gene. PCR products of 350 base pairs (bp) containing the wild-type sequence, a mutated G(818) --> A transition or a G(916) deletion of the LPL gene were subjected to hybridization with a probe DNA of 13 or 15 bases that represented either the wild-type or the mutated sequence immobilized on a gold electrode. The differential pulse voltammetry of the electrode before and after hybridization was determined in the presence of ferrocenylnaphthalene diimide (FND) at 460 mV. The measured change in peak current, Deltai, was defined by (i - i(o))/i(o) x 100%, where i(o) and i represent the current before and after hybridization, respectively. Matched combinations of sample and probe gave Deltai values of 40-90%, whereas mismatched combinations gave values of 20-35%, enabling the discrimination of matched hybrids from mismatched ones across a slim margin. Because the heterozygote contains both the wild-type and mutated sequences, however, it alone gives large Deltai values with both the wild- and mutant-type probes. This system was validated on 10 unknown samples of each of the two types of LPL mutation, which were correctly identified in every case.

Base Sequence↗