Precise Dispensing Technology Using Electroformed Tubes for Micro-Volume Blood Diagnosis.

Precise dispensing of droplets is a crucial step for acquiring reliable blood diagnostic results. When the source sample volume is limited, the need for precise dispensing of submicroliter and nanoliter quantities is especially important. In this paper, we developed a positive-displacement high-precision dispensing technique using a nickel electroformed tube with an inner diameter accuracy of [Formula: see text] or less. When dispensing variation of 100 nL was evaluated by using a photometric method, the most stable coefficients of variation (CV) were observed for a tube thickness of [Formula: see text] with hydrophobic treatment, where the average CV value was 1.3%, i.e., 1.3 nL. Furthermore, the glucose concentration of 100- and 200-nL animal-based control serum was colorimetrically measured using enzymatic reactions without drying and mixing reagents. The CV value was approximately 6.36% at 100 nL and 3.23% at 200 nL, suggesting that several biochemical panels can be precisely measured even from less than one drop of blood. This positive-displacement dispenser ensures zero contamination and almost-zero dead volume, thus it would be useful for multi-panel clinical blood testing.

Intercalation Compounds as Inner Reference Electrodes for Reproducible and Robust Solid-Contact Ion-Selective Electrodes.

With billions of assays performed every year, ion-selective electrodes (ISEs) provide a simple and fast technique for clinical analysis of blood electrolytes. The development of cheap, miniaturized solid-contact (SC-)ISEs for integrated systems, however, remains a difficult balancing act between size, robustness, and reproducibility, because the defined interface potentials between the ion-selective membrane and the inner reference electrode (iRE) are often compromised. We demonstrate that target cation-sensitive intercalation compounds, such as partially charged lithium iron phosphate (LFP), can be applied as iREs of the quasi-first kind for ISEs. The symmetrical response of the interface potentials towards target cations ultimately results in ISEs with high robustness towards the inner filling (ca. 5 mV dec(-1) conc.) as well as robust and miniaturized SC-ISEs. They have a predictable and stable potential derived from the LiFePO4/FePO4 redox couple (97.0±1.5 mV after 42 days).

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.

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.