Maki Shimoda

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.

Molecule counting with alkanethiol and DNA immobilized on gold microplates for extended gate FET

Several molecule counting methods based on electrochemical characterization of alkanethiol and thiolated single-stranded oligonucleotide (HS-ssDNA) immobilized on gold microplates, which were used as extended gates of field effect transistors (FETs), have been investigated in this paper. The surface density of alkanethiol and DNA monolayers on gold microplates were quantitatively evaluated from the reductive desorption charge by using cyclic voltammetry (CV) and fast CV (FCV) methods in strong alkali solution. Typically, the surface density of 6-hydroxy-1-hexanethiol (6-HHT) was evaluated to be 4.639 molecules/nm(2), and the 28 base-pair dsDNA about 1.226-4.849 molecules/100 nm(2) on Au microplates after post-treatment with 6-HHT. The behaviors on surface potential and capacitance of different aminoalkanethiols on Au microplates were measured in 0.1 mol/L Na2SO4 and 10 mmol/L Tris-HCl (pH=7.4) solutions, indicating that the surface potential increases and the double-layer capacitance decreases with the length of carbon chain increased for the thiol monolayers, which obey a physics relationship for a capacitor. Comparably, a simple sensing method based on the electronic signals of biochemical reaction events on DNA immobilization and hybridization at the Au surface of the extended gate FET (EGFET) was developed, with which the surface density of the hybridized dsDNA on the gold surface of the EGFET was evaluated to be 1.36 molecules per 100 nm(2), showing that the EGFET is a promising sensing biochip for DNA molecule counting.

Electrical Characteristics of Extended Gate FET Sensing Chip Constructed for Detection of DNA

An extended gate field effect transistor (EGFET) sensing chip has been constructed by using one gold plate electrode for molecule recognition and FET part for signal transduction. By using a 70.7mV DC voltage onto a Ag/AgCl reference electrode, the electrical characteristics of immobilization of the oligonucleotide probe of P1 and hybridization with the target single strand DNA of P2 on the EGFET sensing chip were examined in detail. The electrical signals on the change of a threshold voltage (VT) shift at a constant ID (3000μA) in VG-ID characteristic were obtained, and the VT shift value due to hybridization was calculated to be 12 mV, which may be attributed to the decreased negative charges after hybridization occurred at the gate surface. The surface density of hybridized dsDNA on gold surface of the FET was evaluated to be about 1 × 1012 molecules/cm2, indicating that the EGFET was a promising sensing element for biochip.

Immobilization of DNA molecules on a gold plate for an extended gate FET sensing chip

We have proposed an electrochemical detection method for DNA molecules based on an extended gate field effect transistor (EGFET) sensing chip which consists of one gold plate for molecule recognition and FET part for signal transduction. DNA Probes were immobilized on the gold plate by forming mixed monolayers of thiolated single-stranded oligonucleotide (HS-ssDNA) and alkanethiols, like 6-hydroxy-1-hexanethiol (6-HHT). Electrode reaction corresponding to the reductive desorption of the adsorbed monolayers in strong alkali solution was presented for quantification of surface density of DNA by using fast cyclic voltammetry (FCV). The passivation effects of surface modification with different functional groups on the potential behavior of the gold electrode were also examined. It was feasible to use EGFET chip for detection of DNA hybridization reaction, that the hybridization efficient was estimated to be about 40%.