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.

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.

Enzyme immunoassay using a reusable extended-gate field-effect-transistor sensor with a ferrocenylalkanethiol-modified gold electrode.

A reusable extended-gate field-effect transistor (FET) sensor with an 11-ferrocenyl-1-undecanethiol (11-FUT) modified gold electrode was developed for applying to enzyme immunoassay. It was found that the 11-FUT modified FET sensor detected a thiol compound 50 times or more repeatedly after a treatment with a 5% hydrogen peroxide solution. The gate-voltage shift of the FET sensor showed a fairly good linearity (R(2) = 0.998) within a range from 10(-2) to 10(-6) M on the concentration of 6-hydroxyl-1-hexanethiol, which is a thiol compound, at a Nernstian response of 58.5 mV/decade. The FET-based immunoassay was constructed by combining the 11-FUT modified-FET sensor with the enzyme-linked immunosorbent assay (ELISA), in which the enzyme chemistry of acetylcholinesterase (AChE) was used to generate a thiol compound. The 11-FUT modified FET sensor with an AC voltage at 1 MHz superimposed onto the reference electrode detected the AChE-catalyzed product corresponding to a serum concentration of interleukin 1beta from 10 to 5000 pg/mL. In addition, all measurements were successfully performed by using the same FET-sensor chip after a treatment with a 5% hydrogen peroxide solution.

Detection of DNA hybridization and extension reactions by an extended-gate field-effect transistor: characterizations of immobilized DNA-probes and role of applying a superimposed high-frequency voltage onto a reference electrode.

As we have already shown in a previous publication [Kamahori, M., Ihige, Y., Shimoda, M., 2007. Anal. Sci. 23, 75-79], an extended-gate field-effect transistor (FET) sensor with a gold electrode, on which both DNA probes and 6-hydroxyl-1-hexanethiol (6-HHT) molecules are immobilized, can detect DNA hybridization and extension reactions by applying a superimposed high-frequency voltage to a reference electrode. However, kinetic parameters such as the dissociation constant (K(d)(s)) and the apparent DNA-probe concentration (C(probe)(s)) on a surface were not clarified. In addition, the role of applying the superimposed high-frequency voltage was not considered in detail. In this study, the values of K(d)(s) and C(probe)(s) were estimated using a method involving single-base extension reaction combined with bioluminescence detection. The value of K(d)(s) on the surface was 0.38 microM, which was about six times that in a liquid phase. The value of C(probe)(s), which expressed the upper detection limit for the solid phase reaction, was 0.079 microM at a DNA-probe density of 2.6 x 10(12)molecules/cm(2). We found that applying the superimposed high-frequency voltage accelerated the DNA molecules to reach the gold surface. Also, the distance between the DNA-probes immobilized on the gold surface was controlled to be over 6 nm by applying a method of competitive reaction with DNA probes and 6-HHT molecules. This space was sufficient to enable the immobilized DNA-probes to lie down on the 6-HHT monolayer in the space between them. Thus, the FET sensor could detect DNA hybridization and extension reactions by applying a superimposed high-frequency voltage to the DNA-probes density-controlling gold surface.

A novel enzyme immunoassay based on potentiometric measurement of molecular adsorption events by an extended-gate field-effect transistor sensor.

We developed a novel enzyme immunoassay based on a potentiometric measurement of molecular adsorption events by using an extended-gate field-effect transistor (FET) sensor. The adsorbing rate of a thiol compound on a gold surface was found to depend on the concentration of the compound. To construct an electrochemical enzyme immunoassay system by using the sensor, the enzyme chemistry of acetylcholinesterase (AChE) to generate a thiol compound was used and combined with the enzyme-linked immunosorbent assays (ELISA). After the AChE-catalyzed reaction, the amount of the antigen was obtained by detecting the adsorbing rate of the generated thiol compound on the gold electrode using the FET sensor. The measurement stability was also found to improve when a high frequency voltage of 10 kHz or more was superimposed to the reference electrode. The signal corresponding to a range between 1 and 250 pg/mL of Interleukin 1 beta was obtained by the FET sensor when a voltage of 1 MHz was superimposed onto the reference electrode. The FET sensor based ELISA used in this measurement technique can successfully detect Interleukin 1 beta at concentrations as low as 1 pg/mL.

DNA detection by an extended-gate FET sensor with a high-frequency voltage superimposed onto a reference electrode.

An extended-gate field-effect-transistor (FET) sensor with a gold-sensing electrode, to which a gold-thiol bond could easily be applied, was developed for DNA detection. Because the gold electrode is located in a different area from the FET, it can be operated without a light-shielding box by masking only the FET. However, when the FET sensor is used in an aqueous solution, fluctuation of the interface potential on the gold surface occurs, which results in decreased sensitivity. In DNA detection, 1 h or more was required to stabilize the drain current of the FET sensor after dipping it into the solution. To improve the sensitivity by reducing the fluctuation, we devised a measurement technique using a high-frequency voltage superimposed onto a reference electrode. With a superimposed high frequency voltage of over 1 kHz, the time required to stabilize the drain current of the FET sensor after dipping it in the solution was not only shortened to 5 min, but the fluctuation of the drain current was also reduced. As a result of applying this method, the FET sensor could successfully detect DNA hybridization and the extension reaction.

Bond strength between four luting systems and enamel modified with phosphoric acid.

The purpose of this study was to investigate the effect of phosphoric acid etching on the bond strength between enamel and three luting materials employing self-etching primer (PanaviaF2.0, Linkmax, and Multibond). A luting material without self-etching primer (Super-Bond) was used as a control. Two etching agents (K-etchant and Red Activator) were prepared. The surfaces of bovine enamel were ground, etched with either K-etchant or Red Activator, and then bonded to a stainless steel rod. Tensile bond strengths were determined following 24-hour immersion in water. Without etching, all of the luting materials showed the same statistical bond strength. When K-etchant was applied, the bond strengths of PanaviaF2.0, Linkmax, Multibond, and Super-Bond were significantly greater than that of non-etched control. No significant differences were found between K-etchant and Red Activator. Strongest bonds were obtained for Super-Bond in conjunction with K-etchant (23.6 +/- 6.3 MPa) or Red Activator (21.0 +/- 6.5 MPa), whereby the values were statistically comparable.