Yoshio Ishimori

Novel DNA sensor for electrochemical gene detection

Abstract Some commercially available intercalators have been found to be electrochemically active through voltammetric experiments. For example, daunomycin was oxidized at a low potential (446 mV) with a high current density (6.5 μA/cm2 in a 10 μM solution) on a basal plane pyrolytic graphite electrode with an adsorbed DNA probe. Daunomycin was able to electrochemically distinguish between double and single stranded DNAs using linear sweep voltammetry. After the hybridization reaction of a model targeted gene with DNA probes adsorbed on the electrode, the electrochemical signal of daunomycin intercalated between base pairs of the formed hybrids on the electrode was measured. The use of daunomycin resulted in the detection of the targeted gene of 10−8 g/ml in the buffer solution.

Quantitative analysis for solid-phase hybridization reaction and binding reaction of DNA binder to hybrids using a quartz crystal microbalance

Abstract A 20-mer DNA probe complementary to a part of an oncogene v-myc, having a mercaptohexyl group at the 5′-end, was immobilized on a gold electrode of a quartz crystal microbalance (QCM). The quantity of the probe immobilized on the electrode depended on the pH. Under acidic or basic immobilization conditions, the probe was immobilized more than under neutral conditions. The largest quantity of the target DNA containing v-myc was hybridized when the probe was immobilized under neutral conditions. Using the QCM on which the probe was immobilized under neutral conditions, the quantity of the hybridized target increased with the quantity of the immobilized probe. In the region where the QCM was saturated with immobilized probes, around 70% of the given target was hybridized, and the ratio of target/probe was about 1:100. After the hybridization, the QCMs were reacted with Hoechst 33258 (a DNA binder), which bound to the DNA hybrids formed on the QCMs. The quantity of the bound Hoechst 33258 increased with the quantity of the hybridized target, and the anodic current derived from linear sweep voltammetry of the bound Hoechst 33258 also increased with the quantity of the hybridized target. These results suggested that QCMs could be applied to quantitative analysis for the solid-phase hybridization and the binding of a DNA binder to hybrids.

Liposome immune lysis assay (LILA): a simple method to measure anti-protein antibody using protein antigen-bearing liposomes

A new simple immunoassay technique using immune lysis of liposomes was developed to measure antibody against protein antigens. Multilamellar liposomes were composed of dipalmitoylphosphatidylcholine, cholesterol and phosphatidylethanolamine substituted with the hetero-bifunctional cross-linking reagent N-hydroxysuccinimidyl 3-(2-pyridyldithio)propionate (SPDP). The protein antigen (human IgG) was coupled to these liposomes after treatment with SPDP and mild reduction. As a release marker, carboxyfluorescein (CF) was entrapped in the liposomes. The CF release was specific to anti-human IgG antibody and depended on the presence of complement. This technique could detect 10(-15) mol of anti-human IgG antibody or human IgG. The liposomes were stable over 8 months at 4 degrees C under nitrogen gas.

DNA sensor: A novel electrochemical gene detection method using carbon electrode immobilized DNA probes

Abstract The authors have developed a novel, rapid, convenient, and specific gene detection method, named the ‘DNA sensor,’ using a graphite electrode loaded with DNA probes. Synthesized oligonucleotide (5-TGCAGTTCCGGTGGCTGATC-3′) complementary to oncogene v-myc was employed for a model probe. The oligonucleotide was chemically adsorbed on a basal plane pyrolytic graphite (BPPG) electrode. The sensor was able to be applied to a hybridization reaction (40°C) in a linearized pVM623 solution carrying the Pst I fragment of v-myc (1.5 kbp). After the hybridization reaction, the sensor was immersed into an acridine orange solution (1 μM) and washed with a phosphate buffer (pH 7.0). Acridine orange intercalated between base pairs of the formed double stranded DNAs on the electrode. The anodic peak potential of acridine orange that interacted with the DNAs on the electrode was measured. The positive shift of the peak potential increased in proportional to the pVM623 concentration in the hybridization reaction. 10...

Development of an eco-sensor for the continuous monitoring of environmental volatile organic chlorinated compounds

In recent years, we have developed an advanced environmental monitoring system (AEMS) containing an eco-sensor, meaning a sensor for the measurement of environmental pollutants, based on lipid membranes, for continuous monitoring of underground water in industrial areas such as semiconductor factories (Ishimori Y, Tamura H, Kawano K, Aoyama N and Tamiya E 2000 Proc. Photonic East 2000 pp 43-50). The AEMS project is made up of three components as follows: (1) the eco-sensor, (2) prediction of plume propagation using a computer simulation technique, and (3) the environmental protection method. In this paper, we would like to focus on the study of the eco-sensor. We considered modified lipid membranes to serve as good models for cell membranes because they would be ideal hosts for receptor molecules of biological origin or disruptive environmental pollutants. Thus, we selected the lipid membrane as an environmental sensing element. In attempting to improve the applicability and the responsivity of bilayer lipid membranes (BLMs) in the eco-sensor, we have investigated automatic BLM preparation devices. An automatic BLM preparation device was made by use of an inkjet mechanism. The reproducibility of the BLM preparation was remarkably improved. The sensitivity to volatile organic chlorinated compounds such as cis-1, 2-dichloroethylene was of the order of 10 ppb using mono-olein BLMs even in real underground water. We have also been developing a smaller eco-sensor for practical use.

Overview of the AEMS project

High-tech industries are often the cause of groundwater contamination that affects surrounding areas. While steps must be taken to prevent this type of contamination, high-tech industries should be able to procure the required amounts of high-quality groundwater for their manufacturing processes. The objective of the Advanced Environmental Monitoring System (AEMS) project is to develop a new integrated groundwater monitoring system based on innovative technologies in order to facilitate effective management of groundwater contamination in and around high-tech industrial facilities. It will be possible to use the biosensors developed in this project not only to monitor ground and other fresh water from various sources for contamination, but also to assess the toxicity and environmental hazards arising from industrial effluents. The AEMS project provides high-tech industries with the means to fulfill their commitments to modern society. Through this project they can pursue sustainable development, compliance with environmental regulations, responsible corporate citizenship, effective life-cycle management, and improved worker safety.

Development of eco-sensor for the continuous monitoring of environmental volatile organic chlorinated compounds

In recent years, we have developed an advanced environmental monitoring system (AEMS) containing the eco-sensor, which means a sensor for the measurement of environmental pollutants, based on lipid membranes for continuous monitoring of underground water in industry areas such as semiconductor factories. The AEMS project is composed of three work packages followed by 1)Eco-sensor, 2)Prediction of plume propagation using a computer simulation technique, and 3)Environmental protection method. In this presentation, we would like to focus on the study of the eco-sensor. The reason why lipid membranes selected as a sensing element for environmental pollutants is that the pollutants should be interacted with cell membranes because cells are surrounded by cell membranes containing lipid components. Improving the applicability and the responsibility of bilayer lipid membranes (BLMs) in the eco-sensor, we have investigated the automatic BLMs preparation device. An automatic BLMs preparation was remarkably improved. The sensitivity to volatile organic chlorinated compounds such as cis-1,2-dichloroethylene was in the order of 10ppb using the monoolein BLMs even in real underground water. We also have been developing a smaller sized eco-sensor for the practical use.

Experimental investigation on the behavior of a microdroplet jet

This paper deals with the generation, measurement and control of micro liquid droplet jet in water. In connection with the development of a lipid membrane biosensor device for underground water pollution, a method of working liquid transportation by micro droplet jet has been proposed. The generation and the behavior of micro droplet jet have been investigated by the method of flow visualization. Experimental results show that the behavior of micro droplet is controllable by changing the driven pressure and the duration time of jet ejection. It is feasible to transport the working liquid to a given place by arranging the relative position according to the trajectory of droplet jet.

Development of an eco-sensor based on bilayer lipid membrane for the continuous monitoring of environmental pollutants

In recent years, we have developed an advanced environmental monitoring system (AEMS) containing the eco-sensor, which means a sensor for the measurement of environmental pollutants, based on lipid membranes for continuous monitoring of ground water in industry areas such as semiconductor factories. The AEMS project is composed of three work packages as follows, 1) Eco -sensor, 2) Prediction of plume propagation using a computer simulation technique, and 3) Environmental protection method. In this paper, we would like to focus on the study of the eco-sensor. We considered that modified lipid membranes serve as good models for cell membranes because they would be ideal hosts for receptor molecules of biological origin or disruptive environmental pollutants. Thus, we selected lipid membrane as a sensing element for environmental pollutants. We have already confirmed that the eco-sensor could detect a 10 ppb level of volatile organic chlorinated compounds (VOCs) such as trichloroethylene in ground water. Here, we tried to apply the eco-sensor to measure other environmental pollutants containing pesticides and endocrine disrupting chemicals. We made a novel automatic bilayer lipid membrane preparation device and a new system for the continuous measurement of environmental pollutants in ground water.

Advanced environmental monitoring system using ecosensor based on bilayer lipid membrane

In recent years, we have developed an advanced environmental monitoring system (AEMS) containing the eco-sensor, which means a sensor for the measurement of environmental pollutants, based on lipid membranes for continuous monitoring of underground water in industry areas such as semiconductor factories. The AEMS project is composed of three work packages followed by 1) Eco-sensor, 2) Prediction of plume propagation using a computer simulation technique, and 3) Environmental protection method. In this presentation, we would like to focus on the study of the eco-sensor. The reason why lipid membranes were selected as a sensing element for environmental pollutants is that the pollutants should be interacted with cell membranes because cells are surrounded by cell membranes containing lipid components. Improving the applicability and the responsibility of bilayer lipid membranes (BLMs) in the eco-sensor, we have investigated the automatic BLMs preparation device. An automatic BLMs preparation device was made by use of an inkjet mechanism. The reproducibility of the BLMs preparation was remarkably improved. The sensitivity to volatile organic chloride compounds such as cis-1,2-dichloroethylene was in the order of 10 ppb using the monoolein BLMs even in real underground water. We also have been developing a smaller sized eco-sensor for the practical use.