Toru Murakami

A Micro Planar Ampehometric Glucose Sensor Using an Isfet as a Reference Electrode

Abstract A very small glucose sensor has been realized, which consists of a gold working electrode with a glucose oxidase immobilized membrane on it, and a gold counter electrode, all made on a sapphire substrate. By using the pH sensitive ISFET as a reference electrode, the potential for a solution, whose pH is constant, can be measured and irreversible metal electrodes, such as gold or platinum, can be used as working electrode and counter electrode. The sensor is very suitable for miniaturizing and mass production, because the Integrated Circuit (IC) fabrication process can be applied. The glucose oxidase immobilized membrane was also deposited by a lift off method, one of the IC processes. A glucose concentration, from 1 to 100 mg/dl, was measured with good linear current output.

Development of an amperometric alcohol sensor based on immobilized alcohol dehydrogenase and entrapped NAD

An amperometric planar alcohol sensor has been developed, which has a triple-layer enzyme-immobilized membrane. The lower layer consists of immobilized alcohol dehydrogenase (ADH), the middle layer contains entrapped mobile nicotinamide adenine dinucleotide (NAD+) molecules, and the upper layer is a denser bovine serum albumin (BSA) layer. Typical sensor response to 500 mM ethanol was 250 nA. The sensor responds sufficiently after 10 measurements and 1 month of storage at −20°C. Using this alcohol sensor, ethanol has been measured, without further addition of NAD+ or any other reactive reagent, and repeatable usage has been realized.

An integrated multibiosensor for simultaneous amperometric and potentiometric measurement

Abstract A multibiosensor, in which a glucose sensor based on amperometry and a urea sensor based on potentiometry are integrated, has been realized by complementary use of ISFETs and micro-patterned planar gold electrodes. This multibiosensor is composed of two ISFETs and three micro-planar gold electrodes on a silicon on sapphire (SOS) device. On the back surface gold layer, one of the electrodes works both as a counter electrode for the amperometric measurement, and as a pseudo reference electrode for the potentiometric measurement. Glucose oxidase (GOD)-immobilized membrane, urease-immobilized membrane, and bovine serum albumin (BSA) crosslinking membranes were deposited in appropriate positions by a lift-off method, one of the integrated circuit (IC) fabrication processes. Consequently the multibiosensor is very suitable for miniaturization and mass production. A glucose concentration in the range 1 to 100 mg/dl and a urea concentration in the range 5 to 100 mg/dl were measured with a good relationship. In the multibiosensor, amperometry and potentiometry exist on a complementary basis and each process provides what the other needs. The multibiosensor can thus be called a ‘complementary multibiosensor (COMBIS)’.

Cytotoxicity testing through cell adhesion to a pattern of collagen matrix

Abstract A pattern of fluorescein isothiocyanate-labeled collagen (FITC-collagen) can be fabricated on a substrate; it is clearly observable by fluorescence microscopy. Human venous endothelial cells separately adhere only to the collagen islands. The stained cells and the collagen islands can be discriminately viewed by fluorescence microscopy with two filter sets. Zinc sulfate in the cell medium produces effects on the cell adhesion; it decreases the number of the cells anchored to the collagen islands, suggesting a new methodology of testing chemical cytotoxicity. The method could also be useful to understand cells’ growth, morphogenesis, differentiation, and death.

Cytotoxicity testing of model compounds through cell adhesion to a pattern of collagen matrix

A pattern of fluorescein isothiocyanate labeled collagen (FITC-collagen) can be fabricated on a substrate by photolithography, which only to human venous endothelial cells specifically adhere. The cytotoxicity of six organic compounds is tested through this adhesion process, by counting the FITC-collagen film islands that have endothelial cells in a binary system, as well as by counting all of the cells anchored to the film islands. The film pattern, 50 μm in diameter with 100 μm spaces, also plays a crucial role to enable chemical cytotoxicity testing with 1 h incubation.

Toxicity testing using lipid monolayers at the air–water interface

Abstract Epifluorescence microscopy can be used to visualize the coexistence of two immiscible fluid phases in lipid monolayers composed of binary mixtures of L- α -dimyristoylphosphatidylcholine (DMPC) and cholesterol at the air–water interface. Fluorescent probes are used to distinguish between the liquid domains rich in DMPC and those domains rich in cholesterol. Dissipation of the cholesterol-rich domains is observed when cholesterol oxidase is dissolved into a subphase. It is shown that, even in the presence of cholesterol oxidase, the monolayer remains an entirely interacting, well-ordered system. Furthermore, the presence of toxic substances in solution can be visualized by means of quantifiable changes in this domain-dissipating phenomenon. In this paper, it is shown that the lipid monolayer system can be used as a sensitive system for detecting the presence of toxic substances in solution. By observing the effects that certain toxic substances have on the enzymatic activity of cholesterol oxidase, and hence the rate of domain dissipation, the presence and relative levels of specific toxins can be quantified.

An immunosensor using a quartz Lamb wave device

A quartz Lamb wave device is applied to an immunosensor, where a surface mass change caused by an immunoreaction is detected as a phase difference change. The benefit of the Lamb wave device is the possibility of operation while immersed in a liquid. Mass change amplification by latex labeled antigens is also used for high sensitive detection. Mass sensitivity of the quartz Lamb wave device, which is made using an 80 μm thick quartz wafer, is about 0.56 degree/(1μm/cm2) at 4.5 MHz. The adsorption amount depends on the concentration of antigen in the sample solution. Concentrations from 0 μg/ml to 4 μg/ml are measured, which shows the Lamb wave device with mass change amplification is useful as an immunosensor.