Biomedical subjects

Isao Karube

Publications and source records attributed to Isao Karube.

At least 19 recordsLinked to original sources

PubMed2006

Development of a self-sterilizing lancet coated with a titanium dioxide photocatalytic nano-layer for self-monitoring of blood glucose.

A photocatalyst was applied to a lancet for pricking the finger to obtain an antibacterial property. A photocatalytic and uniform nano-layer of titanium dioxide (TiO2) on the surface of the lancet (0.36 mm x 24.5 mm) was formed by sputtering and annealed for crystallization of the TiO2 layer. By elementary analysis of the TiO2 layer, titanium and oxygen were detected. Next, for the estimation of the antibacterial properties resulting from the photocatalytic effect, the lancet was packed into a capillary tube filled with a suspension of Escherichia coli K-12 (non-spore-forming bacterium), and was continuously rolled in a continuous UV-irradiation system under black-light irradiation. Distinct antibacterial effects after irradiation at 0.5 mW cm(-2) for 45 min were observed in the crystallized TiO2 layer on the lancet. Finally, lancing resistances obtained by pricking an artificial skin sheet were examined using control lancets, and lancets with an unannealed TiO2 layer or an annealed TiO2 layer. The results showed almost the same lancing resistances for the control (0.53+/-0 N, n=3) and the lancet with an annealed TiO2 layer (0.51+/-0.018 N), while the lancet with an unannealed TiO2 layer showed a high lancing resistance compared with the other lancets (0.62+/-0.05 N). In conclusion, the lancet coated with a crystallized, velvety nano-layer of TiO2 obtained by annealing had antibacterial properties and a similar lancing resistance compared with the bare lancet, and showed potential for application in monitoring blood glucose in diabetes.

Biosensing Techniques↗

PubMed2006

Application of peptide probe for evaluating affinity properties of proteins using quartz crystal microbalance.

This study proved a possibility of a peptide probe for evaluating affinity properties of proteins. We have designed and synthesized three different peptide probes, H-Ala3-(Gly-Pro5)3-Gly-OH (peptide A), H-Ala3-(Gly-Pro5)-Gly-OH (peptide B) and H-Ala3-Gly-OH (peptide C) for testing their affinities to profilin. Each peptide probe was immobilized on a quartz crystal microbalance (QCM) sensor. The QCM sensor with the peptide A showed a 93 Hz decrease of resonant frequency which indicated profilin bound to the QCM sensor in a single layer. In a successive reaction with actin, the QCM analysis resulted in a 123 Hz decrease of resonant frequency which showed actin bound to the QCM sensor. A fluorescence microscope image of the sensor surface exhibited clear fluorescence after binding a rhodamine labeled actin on the sensor surface. These results supported stepwise reactions of profilin binding to the peptide A and actin binding to profilin. In the three peptide probes, the peptide A showed the highest affinity to profilin, i.e., sequence dependent affinity was confirmed.

Actins↗

PubMed2006

Improving the efficiency of evolutionary de novo peptide design: strategies for probing configuration and parameter settings.

Evolutionary molecular design based on genetic algorithms (GAs) has been demonstrated to be a flexible and efficient optimization approach with potential for locating global optima. Its efficacy and efficiency are largely dependent on the operations and control parameters of the GAs. Accordingly, we have explored new operations and probed good parameter setting through simulations. The findings have been evaluated in a helical peptide design according to "Parameter setting by analogy" strategy; highly helical peptides have been successfully obtained with a population of only 16 peptides and 5 iterative cycles. The results indicate that new operations such as multi-step crossover-mutation are able to improve the explorative efficiency and to reduce the sensitivity to crossover and mutation rates (CR-MR). The efficiency of the peptide design has been furthermore improved by setting the GAs at the good CR-MR setting determined through simulation. These results suggest that probing the operations and parameter settings through simulation in combination with "Parameter setting by analogy" strategy provides an effective framework for improving the efficiency of the approach. Consequently, we conclude that this framework will be useful for contributing to practical peptide design, and gaining a better understanding of evolutionary molecular design.

Algorithms↗

PubMed2005

Electron transfer mediator micro-biosensor fabrication by organic plasma process.

We propose a new strategy for constructing a mediator-type biosensor as a Bio-MicroElectroMechanical Systems (BioMEMS) application. A vinylferrocene plasma-polymerized film (PPF) was deposited directly onto the surface of an electrode under dry conditions. The resulting redox film was extremely thin, adhered well onto a substrate (electrode), and had a highly crosslinked network structure. This technique, capable of polymeric deposition of any kind of monomer, can also serve the purpose of anti-fouling coating, or layer-to-layer interface creation. With a subsequent plasma process, additional polymeric layer of hydrophilic acetonitrile was superimposed onto the existing vinylferrocene-PPF surface to offer crucial features such that the wettability could be adjusted for a better electron transfer, and amino functional groups could be attached to immobilize a large amount of enzyme. Based upon this scheme, the device fabrication could be designed in a manner that the whole procedure was made up of dry wafer-handling processes, which is compatible with mass production. A prototype device was fabricated to have an array of needle-shaped amperometric micro-biosensors. The resultant thin polymer layer carried a large number of the mediator molecules, accomplishing a lower overpotential (+410 mV) and a rapid response time (<5s). Stressing the advantages of the plasma polymerization process together with some additional features accomplished in our device fabrication, we would discuss new possibilities in the field of BioMEMS.

Biosensing Techniques↗

PubMed2005

Development of photocatalytic biosensor for the evaluation of biochemical oxygen demand.

The photocatalytic biosensor of flow system using semiconductor TiO2 was developed to evaluate biochemical oxygen demand (BOD) levels in river water. Photocatalysis of sample was carried out in a photoreactor with TiO2 and a 6W black-light blue fluorescent tube as light source. Sample from a photoreactor outlet was measured by an oxygen electrode with a biofilm. The sensor response of photocatalytic biosensor was between 5 and 10 min depending on concentration of biochemical in the samples. At BOD of 1 mgl-1, the sensor response increased 1.33-fold in comparison with that without photocatalysis. The degradation of tannic acid and humic acid with photocatalysis were 51.8 and 38.4%, respectively. Gum arabic and linear alkylbenzene sulfonate (LAS) were degraded a little, but gave the responses of more than double to the sensor. Free radicals yielded by photocatalysis in a photoreactor did not affect the sensor response because their lifetime is extremely short. Fairly good correlation (r=0.983) between the sensor method and the conventional method was obtained for test samples. This biosensor using photocatalytic pretreatment improved the sensitivity.

Biofilms↗

PubMed2005

A novel method of screening thrombin-inhibiting DNA aptamers using an evolution-mimicking algorithm.

Thrombin-inhibiting DNA aptamers have already been obtained through the systematic evolution of ligands by exponential enrichment (SELEX). However, SELEX is a method that screens DNA aptamers that bind to their target molecules, and it sometimes fails to screen good inhibitors. Therefore, it is necessary to develop a method of screening DNA aptamers based on their inhibitory effects on the target molecules. We developed a novel method of detecting aptamers using an evolution-mimicking algorithm, and we applied it to the search of new aptamers which inhibit thrombin. First, we randomly designed and synthesized ten 15mer oligonucleotides presumed to form G-quartet structures, and then measured their thrombin-inhibiting activities. The aptamers showing high inhibitory activity were selected, and we shuffled and mutated those sequences in silico to generate 10 new sequences of next-generation aptamers. After repeating the cycle five times, we successfully obtained the same aptamers reported previously, and they showed high inhibitory activity. In addition, we added 8mer oligonucleotides to both the 5' and the 3' end of the selected 15mer aptamers, and then repeated the evolution in silico. After two cycles, we were able to obtain aptamers with higher inhibitory activity than that of the 15mer aptamers.

Algorithms↗

PubMed2005

SNPs (single nucleotide polymorphisms) detection based on the formation of cholic-acid binding DNA aptamer.

Simple and rapid methods for the detection of single nucleotide polymorphisms (SNPs) are required to analyze the genetic information. We have previously isolated DNA aptamers that bind to cholic acid by an in vitro selection method. A common feature of the sequences of the aptamers was that they included a secondary structure, a three-way junction, in which three stems were connected at the junction. It was also revealed that three-way junctions with fully matched three stems bind cholic acid whereas single-base substitutions at the junction lose binding affinity completely. Here we report a new method for the detection of single nucleotide mutations that is based on the formation of fully matched three-way junctions which bind to cholic acid.

Aptamers, Nucleotide↗

PubMed2004

Single nucleotide polymorphism typing on DNA array with hydrophobic surface fabricated by plasma-polymerization technique.

A DNA array has been fabricated on glass substrates, which enables high-throughput analysis of single-base mismatches. In this work, microfabrication-compatible plasma-polymerization (PP) method was used for immobilizing probe DNAs to study the hybridization behavior by changing surface properties. The immobilization matrix consisting of 35 A of PP layer, applied additionally on the streptavidin absorbed hexamethyldisiloxane (HMDS)-PP layer, was constructed on the substrates to anchor biotinylated DNA probes onto the surface. The hydrophobic immobilization matrix was considered to enhance hybridization accuracy and efficiency, compared with its hydrophilic acetonitrile-PP layers. The oligonucleotide arrays fabricated on HMDS-PP surface were shown to be effective in detection of single nucleotide polymorphisms (SNPs) of ApoE gene.

Apolipoproteins E↗

PubMed2004

Organic plasma process for simple and substrate-independent surface modification of polymeric BioMEMS devices.

A polymeric bio micro electromechanical systems (BioMEMS) device was fabricated using organic plasma polymerization, by which the surface of a polymeric substrate could easily be modified through vapor-phase deposition of organic thin films. This technique, capable of polymeric deposition of any kind of monomer, can serve the purpose of anti-fouling coating, wettability control, or layer-to-layer interface creation, on the surface of any given chemically-inert polymeric substrate without involving cumbersome surface organic reactions. A prototype device was fabricated to have an array of electrochemical glucose biosensors with the three electrode configuration, each of which has a microfluidic channel (500 microm x 800 microm) for capillary-action-driven sample delivery and the concerned enzymatic reaction. Stressing the advantages of the plasma polymerization process using a polymeric substrate together with some additional features accomplished in our device fabrication, new possibilities in the field of polymeric BioMEMS are discussed.

Biosensing Techniques↗

PubMed2004

Native and sodium dodecyl sulfate-capillary gel electrophoresis of proteins on a single microchip.

Simultaneous electrophoresis of both native and Sodium dodecyl sulfate (SDS) proteins was observed on a single microchip within 20 min. The capillary array prevented lateral diffusion of SDS components and avoided cross contamination of native protein samples. The planar sputtered electrode format provided a more uniform distribution of separation voltage into each of the 36 parallel microchannel capillaries than platinum wire electrodes commonly used in conventional electrophoresis. The customized geometry of the stacking capillary machined into the cover plate of the microchip facilitated reproducible sample injection without the requirement for stacking gel. Polyimide served as a mask and facilitated insulation of the anode and cathode to prevent electrode lift off and deterioration during continuous electrophoresis, even at a constant current of 8 mA. Improved protein separation was observed during capillary electrophoresis at lower currents. Ferguson plot analysis confirmed the electrophoretic mobility of native globular proteins in accordance with their charge and size. Corresponding Ferguson plot analysis of SDS-associated proteins on the same chip confirmed separation of marker proteins according to their molecular weight.

Electrodes↗

PubMed2003

Exploration of structural features of monomeric helical peptides designed with a genetic algorithm.

A genetic algorithm (GA)-based strategy to dissect the determinants of peptide folding into alpha-helix was developed. The structural information of helical peptides was obtained with respect to patterns of sequence variability. In many previously reported studies the intrinsic alpha-helical propensities of amino acids although sequence-dependent are apparently independent of the amino acid position. In this research, monomeric helical peptides selected from possible sequences produced by a GA-chemical synthesis were analyzed to identify possible influential structural features. These hexadeca-peptides were obtained after four successive generations. A total of 128 synthetic peptides were evaluated via circular dichroism (CD) measurements in aqueous solution, while the mean ellipticity at 222 nm confirmed the monomeric state of the peptides. The results presented here show that our GA-based strategy may be useful in the design of proteins with increased alpha-helix content.

Algorithms↗

PubMed2003

A surface plasmon resonance probe with a novel integrated reference sensor surface.

A surface plasmon resonance (SPR) sensor probe with integrated reference surface is described. In order to fabricate the integrated reference surface, two dielectric layers with different thickness were deposited on the single gold SPR sensor surface via plasma polymerization of hexamethyldisiloxane. The working sensor surface was a 34 nm dielectric layer with immobilized bovine serum albumin (BSA) antigen and an adjacent thin 1 nm dielectric layer without BSA provided reference surface. A specific immunoreaction of anti-BSA antibody was detected after immersion of the SPR probe into sample solution. Simultaneous observation of reference and working surface response enabled determination of the immunoreaction without the need for the baseline measurement. Moreover, compensation of nonspecific adsorption could be confirmed using anti-human serum albumin antibody.

Antibodies↗

PubMed2003

Current research activity in biosensors.

Biosensors consist of a molecular recognition element and a transducer. Since the first biosensor was developed by Updike and Hicks many biosensors and their associated techniques have been studied and developed. In this review current research activity on the fundamentals and applications of biosensors is summarized. In discussion of the former, molecular recognition elements, techniques and tools for biosensor construction, and basic biosensor devices are introduced. Coverage of the latter includes description of biosensors for environmental, food, and clinical fields. Chemical sensors developed in our laboratory for environmental monitoring are also described. This review mainly summarizes work performed by our group and by our colleagues, and refers to the main review articles summarizing each field of biosensor research in recent years.

Biosensing Techniques↗

PubMed2003

Electrochemical protein chip with arrayed immunosensors with antibodies immobilized in a plasma-polymerized film.

An electrochemical protein chip was microfabricated. A thin-film three-electrode system, including an array of 36 platinum working electrodes, a set of thin-film Ag/AgCl electrodes, and platinum auxiliary electrodes, was integrated on a glass substrate. Capture antibodies were immobilized in a 4.5-nm-thick double layer of a hexamethyldisiloxane plasma-polymerized film. Because of their highly cross-linked network structure, the capture antibodies could be firmly immobilized. No nonspecific adsorption was observed during a series of procedures to detect target proteins, and electrochemical cross talk between neighboring sites was negligible. The sandwich immunoassay was conducted on a single chip using model proteins, alpha-1-fetoprotein and beta2-microglobulin. A distinct current increase following the oxidation of hydrogen peroxide produced by the enzymatic reaction of glucose oxidase was observed, which indicates that the capture proteins could actually bind the target proteins. Two kinds of protein were detected independently on multiple sites with respective capture antibodies.

Antibodies↗

PubMed2003

Preparation of a whole genome phage library using fragmented Escherichia coli genome and its characterization of protein binding properties by surface plasmon resonance.

A novel phage library has been prepared using the Escherichia coli genome digested with three restriction enzymes. The resulting DNA fragments were ligated to the expression vector pCANTAB5 to obtain the library of recombinant M13 phages displaying relatively long exogenous peptides. The library was screened to isolate recombinant phages with high affinity to alkaline phosphatase (AP) from calf intestine. After four rounds of panning three phages (AP1, AP2 and AP3) were shown to have specific binding properties toward AP by enzyme-linked immunosorbent assay. The phages were further characterized by surface plasmon resonance (SPR). Among the three phages AP3 bound the AP-immobilized sensor chip most and caused the highest resonant angle shift. The sensor response decreased with the decrease of the concentration of AP3 added. Furthermore, displacement of AP3 from the AP-immobilized sensor chip was observed upon injection of AP solution to the SPR system, whereas injection of bovine serum albumin solution led to the great increase of the sensor response. This result indicates the specific binding of AP3 to AP.

Alkaline Phosphatase↗

PubMed2003

Fructose-2,6-bisphosphate contents were increased in response to salt, water and osmotic stress in leaves of Bruguiera gymnorrhiza by differential changes in the activity of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase.

The steady-state level of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphate 2-phosphatase (F6P2K/F26BPase) transcript has been found to be raised in the mangrove Bruguiera gymnorrhiza treated with 500 mM NaCl for 6 h. In the present study, we assayed both F6P2K/F26BPase activity and fructose-2,6-bisphosphate (F26BP) contents in leaves of salt- and water-stressed B. gymnorrhiza. In the plants treated with 500 mM NaCl, no increase in transcript level was observed after 1 day of treatment, while both the ratio between F6P2K and F26BPase activity (K/P ratio) and leaf F26BP level were about two-fold higher than in control plants. Several water stress-associated treatments, including 500 mM NaCl treatment for 6 h, 1 M mannitol treatment for 6 h and dehydration treatment, resulted in increases in leaf F26BP level as compared with water-grown plants. The raised levels of F26BP in osmotically stressed plants treated with NaCl and mannitol were accompanied with increased transcript levels and subsequent increases in both F6P2K and F26BPase activities, while the increase in F26BP levels in dehydrated plants was attributed to an increase in K/P ratio without an increase in transcript levels. These results suggest that, although both treatments resulted in increases in F26BP levels, B. gymnorrhiza differentially responds to osmotic stress and water stress.

Abscisic Acid↗

PubMed2003

Application of plasma-polymerized films for isoelectric focusing of proteins in a capillary electrophoresis chip.

The first use of plasma polymerization technique to modify the surface of a glass chip for capillary isoelectric focusing (cIEF) of different proteins is reported. The electrophoresis separation channel was machined in Tempax glass chips with length 70 mm, 300 microm width and 100 microm depth. Acetonitrile and hexamethyldisiloxane monomers were used for plasma polymerization. In each case 100 nm plasma polymer films were coated onto the chip surface to reduce protein wall adsorption and minimize the electroosmotic flow. Applied voltages of 1000 V, 2000 V and 3000 V were used to separate mixtures of cytochrome c (pI 9.6), hemoglobin (pI 7.0) and phycocyanin (pI 4.65). Reproducible isoelectric focusing of each pI marker protein was observed in different coated capillaries at increasing concentration 2.22-5 microg microL(-1). Modification of the glass capillary with hydrophobic HMDS plasma polymerized films enabled rapid cIEF within 3 min. The separation efficiency of cytochrome c and phycocyanin in both acrylamide and HMDS coated capillaries corresponded to a plate number of 19600 which compares favourably with capillary electrophoresis of neurotransmitters with amperometric detection.

Animals↗

PubMed2003

Novel strategy for DNA aptamers inhibiting enzymatic activity using algorithm mimicking evolution.

We screened the DNA aptamer inhibiting thrombin with novel method using algorithm mimicking evolution. For screening, we first randomly designed and synthesized ten 15-mer oligonucleotides supposed to form G-quartet structure and then measured the inhibitory activity for thrombin. The aptamers showing the high activities were selected and we shuffled and mutated those sequences in silico to generate 10 new sequences of aptamers for next generation. After repeating 5 cycles, we successfully obtained the same aptamers reported previously showing high inhibitory activity. In addition, we added 8-mer oligonucleotides to the both 5' and 3' ends of the selected 15-mer aptamer and then repeated evolution in silico. After 2 cycles, we were able to obtain the aptamer showing better inhibitory activity than the 15-mer aptamer.

Algorithms↗