Masao Kamahori

DNA analysis chip based on field-effect transistors

We have been developing a genetic field-effect transistor (FET) based on the potentiometric detection of hybridization and intercalation on the Si3N4 gate insulator. In this study, we demonstrated the detection of charge density change as a result of hybridization and intercalation using genetic FETs. Since the electrical output signal is obtained with the genetic FET without any labeling reagent, as compared with the conventional fluorescence-based DNA chips, the genetic FET platform is suitable for a simple and inexpensive system for genetic analysis in clinical diagnostics.

Extended-gate FET-based enzyme sensor with ferrocenyl-alkanethiol modified gold sensing electrode

We developed a field-effect transistor (FET)-based enzyme sensor that detects an enzyme-catalyzed redox-reaction event as an interfacial potential change on an 11-ferrocenyl-1-undecanethiol (11-FUT) modified gold electrode. While the sensitivity of ion-sensitive FET (ISFET)-based enzyme sensors that detect an enzyme-catalyzed reaction as a local pH change are strongly affected by the buffer conditions such as pH and buffer capacity, the sensitivity of the proposed FET-based enzyme sensor is not affected by them in principle. The FET-based enzyme sensor consists of a detection part, which is an extended-gate FET sensor with an 11-FUT immobilized gold electrode, and an enzyme reaction part. The FET sensor detected the redox reaction of hexacyanoferrate ions, which are standard redox reagents of an enzymatic assay in blood tests, as a change in the interfacial potential of the 11-FUT modified gold electrode in accordance with the Nernstian response at a slope of 59 mV/decade at 25 degrees C. Also, the FET sensor had a dynamic range of more than five orders and showed no sensitivity to pH. A FET-based enzyme sensor for measuring cholesterol level was constructed by adding an enzyme reaction part, which contained cholesterol dehydrogenase and hexacyanoferrate (II)/(III) ions, on the 11-FUT modified gold electrode. Since the sensitivity of the FET sensor based on potentiometric detection was independent of the sample volume, the sample volume was easily reduced to 2.5 microL while maintaining the sensitivity. The FET-based enzyme sensor successfully detected a serum cholesterol level from 33 to 233 mg/dL at the Nernstian slope of 57 mV/decade.

Immobilization of DNA Probes onto Gold Surface and its Application to Fully Electric Detection of DNA Hybridization using Field-Effect Transistor Sensor

A field-effect transistor (FET) sensor with a gold sensing electrode (extended-gate FET sensor), on which DNA probes can be immobilized via an Au–S bond, was designed. A method of controlling the surface density of DNA probes immobilized on the gold electrode was developed using a competitive reaction between DNA probes and alkanethiols. The immobilized DNA probes were characterized using voltammetry and a single-base extension reaction combined with bioluminescence detection. The relationship between DNA probe density and hybridization efficiency was clarified, and it was found that the optimum density for FET sensors was about 2.6×1012 molecules/cm2. The fully electric detection of hybridized target DNA (about 7 fmol) was achieved by the extended-gate FET sensor with the above DNA probe density. In addition, the surface potential in proportion to the density of both single-stranded DNA and double-stranded DNA immobilized on the gold electrode was successfully obtained using the extended-gate FET sensor.

Miniaturized pyrosequencer for DNA analysis with capillaries to deliver deoxynucleotides.

As the human genome project proceeds, various types of DNA analysis tools are required for life sciences and medical sciences including DNA diagnostics. For example, a small DNA sequencer for sequencing a short DNA is required for bed‐side DNA testing as well as DNA analysis in a small laboratory. Here, a new handy DNA sequencing system (pyrosequencer) based on the detection of inorganic pyrophosphate (PPi) released by polymerase incorporation is demonstrated. The system uses the bioluminescence detection system. The key point for the miniaturized DNA sequencer is to make a deoxynucleotide triphosphate (dNTP) delivery system small and inexpensive. It has been realized by using narrow capillaries to connect a reaction chamber and four dNTP reservoirs. Each dNTP is introduced into the reaction chamber by applying a pressure to the reservoir. Compared with other microdispensers, it is much cheaper and easier. By optimizing the conditions, an excellent sequencing ability is achieved while it is a simple and inexpensive system. In most cases, more than 40 bases can be successfully sequenced. A homopolymeric region, which can not be easily sequenced by a conventional gel‐based DNA sequencer, is readily sequenced with this system. The new system is successfully applied to sequence a GC rich region or a region close to a priming region where misreading frequently occurs. A rapid analysis for a short DNA was easily achieved with this small instrument.

Analysis of plasma catecholamines by high-performance liquid chromatography with fluorescence detection: simple sample preparation for pre-column fluorescence derivatization.

Analysis of plasma catecholamines (norepinephrine, epinephrine and dopamine) by high-performance liquid chromatography using 1,2-diphenylethylenediamine as a fluorescent reagent is described. We have developed an automatic catecholamine analyser, based on pre-column fluorescence derivatization and column switching. The analysis time for one assay was 15 min. The correlation coefficients of the linear regression equations were greater than 0.9996 in the range 10-10,000 pg/ml. The detection limit, at a signal-to-noise ratio of 3, was 2 pg/ml for dopamine. A new method of sample preparation for the pre-column fluorescence derivatization of plasma catecholamines was used. In order to protect the catecholamines from decomposition, an ion-pair complex between boric acid and the diol group in the catecholamine was formed at a weakly alkaline pH. The stabilities of plasma catecholamines were evaluated at several temperatures. After complex formation, the catecholamines were very stable at 17 degrees C for 8 h, and the coefficients of variation for norepinephrine, epinephrine and dopamine were 1.2, 4.2 and 9.3%, respectively.

An extended-gate CMOS sensor array with enzyme-immobilized microbeads for redox-potential glucose detection

An extended-gate CMOS sensor array with enzyme-immobilized microbeads for redox-potential glucose detection is demonstrated for the first time. Redox-potential detection has the possibility to achieve high accuracy because it is not affected by the buffer conditions. Despite this high-accuracy property, redox-potential detection requires a sufficient amount of enzyme, which leads to increased cost. In order to reduce the enzyme consumption while maintaining the detection capability, we have introduced enzyme-immobilized microbeads. By using the microbeads, the enzyme can be efficiently positioned and reused several times. Thus, the required amount of enzyme can be reduced dramatically. To verify the proposed concept, we have developed and measured a prototype with a 0.6-μm CMOS test chip including the microfluidics. Measurements successfully demonstrate glucose detection with a sensitivity of -61.6 mV/decade while reusing identical enzyme-immobilized microbeads.

Direct detection of enzyme-catalyzed products by FET sensor with ferrocene-modified electrode

An FET-based biosensor with a ferrocene-modified gold electrode detects the enzyme-produced electrons by using mediators that transfer the electrons from the enzyme to the sensor. Since an extended-gate FET sensor with a light-shielding mask can be operated without a light-shielding box, a small portable instrument will soon be realised. However, when the FET sensor detected enzyme-catalyzed products with the mediators under light conditions, measurements fluctuated due to photo-reduction of the mediators, resulting in decreased sensitivity. To improve sensitivity by reducing the fluctuation, we developed a procedure for directly detecting enzyme-catalyzed products without using the mediators. The key technique used in this procedure was a measurement technique using our developed potential-keeping method, in which the modified electrode of the FET sensor was oxidised by ferricyanide solution to make its surface the same high potential every time, and this high potential was kept until measurement because of the high input impedance of the FET structure. After this method was applied, the interfacial potential of the gold electrode decreased depending on the amount of enzyme-catalyzed products due to the ferrocene molecules immobilised on the gold electrode directly reacting with the products. The results obtained in light conditions indicated that model compounds of the products were detected from 10 μM to 10 mM with the Nernstian response of 59.2 mV/decade. Also, this method was applied to pesticide detection by using the enzyme inhibition by pesticide, and 5 ppb of diazinon was successfully detected by using only a sensor chip.

A wireless biosensing chip for DNA detection

A wireless sensing chip for biological assay is fully operational in a sample solution. The IC monolithically integrates a biological sensor, an RF communication circuit, and a coupling coil on a 2.5/spl times/2.5 mm/sup 2/ chip in 0.35 /spl mu/m CMOS technology. Detection of single nucleotide polymorphism (SNP) in DNA is successfully carried out by the chip.

A new single nucleotide polymorphisms typing method and device by bioluminometric assay coupled with a photodiode array

Easy and inexpensive single nucleotide polymorphisms (SNPs) typing systems are required for the practice of genetic testing as well as genetic medicine. Most of the SNPs typing systems use laser-induced fluorescence detection coupled with fluorophore tagging on DNA, which are expensive. A new simple and inexpensive SNPs typing system is presented. It uses a bioluminometric assay coupled with modified primer extension reactions and an inexpensive photodiode array for the luminometric detection. The reagents consumed in the assay are also inexpensive. Although the system is very small, simple and inexpensive, it gives enough sensitivity for detecting target DNAs as small as 10 fmol, which is good enough for SNPs typing.

High-sensitivity micro ultraviolet absorption detector for high-performance liquid chromatography

Abstract A UV absorption detector with a 0.6-μl flow cell for high-performance liquid chromatography (HPLC) was developed. In order to improve the signal-to-n