Toshiya Sakata

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.

Potentiometric detection of single nucleotide polymorphism by using a genetic field-effect transistor

Potentiometric measurement of allele‐specific oligonucleotide hybridization based on the principle of detection of charge‐density change at the surface of a gate insulator by using of a genetic field‐effect transistor has been demonstrated. Since DNA molecules are negatively charged in aqueous solution, a hybridization event at the gate surface leads to a charge‐density change in the channel of the FET and can be directly transduced into an electrical signal without any labeling of target DNA molecules. One of the unique features of our method is to utilize DNA binders such as intercalators as charged species for double‐stranded DNA after hybridization, since these are ionized and carry positive charges in aqueous solution. Single‐base mismatch of the target DNA could be successfully detected both with the wild‐type and with the mutant genetic FETs by controlling the hybridization temperatures and introducing Hoechst 33258 as DNA binder. The genetic FET platform is suitable as a simple, accurate, and inexpensive system for SNP typing in clinical diagnostics.

Potential behavior of biochemically modified gold electrode for extended-gate field-effect transistor

We propose potentiometric detection of biomolecules using an extended-gate field-effect transistor (EGFET). Using a gold film electrode as the extended gate, the stability of the interface potential was characterized for a shift and a drift, and found to depend on the surface roughness of the gold electrode. The surface of the gold film was coated with self-assembled monolayers (SAMs) of various types of alkanethiol molecules with functional sites such as amino groups, carboxyl groups, hydroxyl groups and oligonucleotides. In all the alkanethiol molecules, the interface potential decreased drastically just after the introduction of each molecule because of the negative charges of thiol groups in aqueous solutions. Moreover, the complementary target deoxyribonucleic acid (DNA) has been introduced to the gold electrode modified with oligonucleotide probes and hybridized with them. The interface potential shifted in the negative direction due to the negative charges of the target DNA. Thus, the charge density change due to DNA hybridization as well as the adsorption of alkanethiol molecules on the gold electrode could be successfully detected using the biochemically modified EGFET.

Direct detection of single-base extension reaction using genetic field effect transistor

We have proposed a novel field effect transistor (FET) in combination with single-base extension for a direct, simple and non-labeled DNA sequencing, which is based on detection of molecular recognition at the gate insulator by the field effect. The intrinsic negative charges generated by DNA polymerase-assisted incorporation of deoxynucleotides can be transduced directly into electrical signal. Mere, we demonstrate that single-base extension at the gate surface can be detected directly as a shift of the threshold voltage of the FET. Moreover, it was possible to determine the base sequence of the target DNA by the iterative cycles of single-base extension with each deoxynucleotides and measurement of the threshold voltage.

Direct detection of single nucleotide polymorphism using genetic field effect transistor

We have been investigating a new approach to realize an clectrochemical detection for DNA chips, although a number of fluorescent detection methods ate widely used for SNP genotyping [l]. The novel concept of a genetic field effect transistor (FET) is proposed in the present study for improving precision, standardization and miniaturization of a DNA chip system. The genetic FET is composed of Si with Si3N4/SiO2 as the gate insulator on which DNA probes are immobilized and subsequently hybridized with target DNA in sample solutions. The potentiometric detection method is based on the direct transduction of surface density change of charged biomolecules into electrical signal by the field effect and is effective for charged species such as DNA molecules. Here, we report the concept of genetic FET and the ability of SNP genotyping by controlling hybridization temperatures, and by the utilization of intercalator or primer extension reaction using the genetic FET.

Detection sensitivity of genetic field effect transistor combined with charged nanoparticle-DNA conjugate

We demonstrated the effectiveness of introduction of charged nanoparticle-DNA conjugate for detection sensitivity of the genetic field effect transistor (FET), of which the principle is based on the potentiometric detection of charge density change on the gate surface. The large amount of negative charges on the gate insulator induced strongly the electrostatic interaction with electrons in silicon crystal by field effect resulting in the bigger shift of threshold voltage. The genetic FET platform combined with the charged nanoparticle-DNA conjugate is suitable for a simple, sensitive, accurate and inexpensive system for DNA analyses in clinical diagnostics

DNA sequencing using genetic field effect transistor

We have proposed a novel field effect transistor (FET) in combination with single-base extension for a direct, simple and non-labeled DNA sequencing, which is based on detection of molecular recognition at the gate insulator by the field effect. The intrinsic negative charges generated by DNA polymerase-assisted incorporation of deoxynucleotides can be transduced directly into electrical signal. Here, we demonstrate that single-base extension at the gate surface can be detected directly as a shift of the threshold voltage of the FET. Moreover, it was possible to determine the base sequence of the target DNA by the iterative cycles of single-base extension with each deoxynucleotides and measurement of the threshold voltage.

Open Sandwich-Based Immuno-Transistor for Label-Free and Noncompetitive Detection of Low Molecular Weight Antigen

In this study, we proposed a new detection method, open sandwich-based immuno-field effect transistor (OS-FET) for label-free and noncompetitive detection of low molecular weight antigen. The principle of OS-FET is based on the detection of intrinsic molecular charges caused by the small antigen-dependent interchain interaction of separated V(L) and V(H) chains from a single antibody variable region using the field effect. Introducing V(H) chain and small antigen bisphenol A into the OS-FET with the immobilized V(L) chain on the gate, we could detect electrically and directly the binding of bisphenol A by V(H) and V(L) chains. Although the detection limit of OS-FET was 1 nM to detect bisphenol A for the standard deviation of control signal, the addition of isothiocyanobenzyl-EDTA with negative charges to the V(L) chain enhanced the detection limit to 1 pM. We could directly transduce the charge density changes based on the capture of target on the gate into the electrical signals using the OS-FET. The platform based on the FETs is suitable for a label-free, noncompetitive, and quantitative detection system for small antigen analysis in environmental, food, and clinical research.

Noninvasive monitoring of transporter-substrate interaction at cell membrane

We report noninvasive monitoring of the transporter-substrate interaction at the cell membrane using an oocyte-based field effect transistor (FET), which is based on detection of extracellular potential change induced as a result of the interaction between transporting peptide and substrate at the cell membrane. The interface potential change at the cell membrane/gate insulator interface can be monitored during the uptake of substrate mediated by transporter without any labeling materials and fracturing oocyte. Moreover, we can discriminate the transporting kinetics of the substrate mediated by the wild-type and the mutant-type transporters by use of the oocyte-based FETs. Our findings on the time course of the interface potential would provide important information to understand the molecular mechanism of the uptake kinetics for the OATP-C transporter.

Chemical-to-Electrical-Signal Transduction Synchronized with Smart Gel Volume Phase Transition.

A stimulus-responsive polymer gel designed on a field-effect transistor gate undergoes a reversible volume phase transition in response to a specific biomolecule. An abrupt permittivity change at the gel/gate interface during the transition gives rise to a chemical to electrical signal conversion; the signal is thus detectable via a transistor without the limit of the Debye length.