Yu Ishige

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.

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.

Single-molecule detection of proteins with antigen-antibody interaction using resistive-pulse sensing of submicron latex particles

We developed a resistive-pulse sensor with a solid-state pore and measured the latex agglutination of submicron particles induced by antigen-antibody interaction for single-molecule detection of proteins. We fabricated the pore based on numerical simulation to clearly distinguish between monomer and dimer latex particles. By measuring single dimers agglutinated in the single-molecule regime, we detected single human alpha-fetoprotein molecules. Adjusting the initial particle concentration improves the limit of detection (LOD) to 95 fmol/l. We established a theoretical model of the LOD by combining the reaction kinetics and the counting statistics to explain the effect of initial particle concentration on the LOD. The theoretical model shows how to improve the LOD quantitatively. The single-molecule detection studied here indicates the feasibility of implementing a highly sensitive immunoassay by a simple measurement method using resistive-pulse sensing.

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).

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

Glucose detection associated with CMOS-based electronics has great potential from the viewpoints of cost, form factor, and simplicity. There are two conventional approaches for emerging CMOS-based glucose detection. One is based on direct charge detection, and another is based on pH detection. Unfortunately, these methods are susceptible to buffer conditions such as the salt concentration and pH buffer capacity, which can seriously degrade their accuracy and reliability.

6.1.4 Redox PotentialSensor Arrayby Extended-Gate FET with Ferrocenyl-Alkanethiol Modified Gold Electrode

We developed a 32×32 array chip of extended-gate FET-based redox potential sensor with 11ferrocenyl-1-undecanethiol (11-FUT) modified gold electrode. The sensor array detected the redox reaction of hexacyanoferrate (II) and hexacyanoferrate (III) as a change in the electric potential of the 11-FUT modified electrode in accordance with the Nernstian response at a slope of 57.9 mV/decade at 25 °C with a dynamic range of more than five orders of magnitude. The stability of the potential was within 0.5 mV/h. Of all 32×32 sensor cells, each potential of 80% and each difference of 92% were within ± 5 mV and ± 1 mV from median, respectively. The 2-dimensional and real time visualization were enabled by imaging of sensor array. Using an enzyme-catalyzed redox reaction, this FET-based sensor array successfully detected a glucose level from 25 to 200 mg/dL.