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.

In this study, we have proposed and demonstrated experimentally a novel monitoring device of single mouse embryo activity after in vitro fertilization (IVF) using a semiconductor-based field effect transistor (FET). The FET biosensor realized to detect it noninvasively, quantitatively, and continuously by change of hydrogen ions with positive charges, which were induced by dissolved carbon dioxide due to cellular respiration activity during cleavage. The electrical signal of FET biosensor should become an effective indication to evaluate objectively single embryo activity as its morphology is observed subjectively after IVF. The platform based on the FET biosensor will contribute to promote elective single embryo transfer (eSET) in human assisted reproductive technology (ART).

Gene functional analyses have proceeded remarkably in the fields of molecular biology, pharmacogenomics, and clinical research, on the basis of completion of the decoding of the human genome. The analysis of nucleotide variation has become increasingly important for the assembly of a highresolution map of disease-related loci and for clinical diagnostics. The most common form of genomic variation is single-nucleotide polymorphism (SNP), which is an important marker in personalized medicine that affects disease susceptibility and resistance. Although a number of methods for SNP analysis have been developed, [1][2][3][4][5][6][7] DNA sequencing techniques still need to be improved in terms of cost, simplicity, and throughput to analyze not only SNPs but also genomic variations, such as insertion/deletion and short tandem repeats.We have been investigating a new approach to the direct, simple, and highly sensitive detection of nonlabeled molecular recognition events on a miniaturized and arrayed solidstate device.[8] Recently, several types of field-effect devices have been used for the electrochemical detection of hybridization events on a solid surface.[9] As DNA molecules are negatively charged in an aqueous solution, the number of negative charges at the gate surface of field-effect devices increases as a result of hybridization and extension reactions. The charge-density change is directly transduced into an electrical signal by the field effect. Based on this principle, point-mutation analysis was carried out by using the PCR products amplified with allele-specific primers.[9e] In this case, the overall specificity was determined from that of the allelespecific PCR. A single-base mismatch could also be distinguished by hybridization with complementary and mismatched DNA probes immobilized on the capacitor-type field-effect devices.[9b]We propose a new method for DNA sequencing of known as well as unknown sequence variants, which is based on detection of the intrinsic charges of DNA molecules by using the field effect. Herein, we report the direct transduction of single-base extension at the gate surface into an electrical signal, and the possibility of label-free DNA sequencing based on the intrinsic charges of DNA molecules.Oligonucleotide probes are immobilized on the Si 3 N 4 gate surface, and the complementary target DNA is hybridized with these probes. The hybridization events are followed by the introduction of DNA polymerase and one of each deoxynucleotide (dCTP, dATP, dGTP, or dTTP). DNA polymerase extends the immobilized oligonucleotide probes in a template-dependent manner ( Figure 1). As a result of the extension reaction, the number of negative charges increases at the gate surface of the field-effect transistor (FET) because of the intrinsic negative charges of the incorporated molecules. This change in charge density can be detected as a shift in the threshold voltage (V T ) of the FET. Thus, iterative addition of each deoxynucleotide and measurement of the threshold voltage allow direct, simple,...

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.

We proposed an interface molecule for immobilization of DNA probes on solid substrates of DNA chips. We have designed and synthesized tripodal thiol derivatives for stable immobilization of oligonucleotide probes on a gold surface. On the basis of the tetrahedral structure of tripod, the tripodal thiol derivatives were bonded upright to the gold substrate, which would control the orientation of oligonucleotide probes. When the gold substrate with oligonucleotide probes tethered using the thiol derivatives was exposed to deionized water at higher temperatures, the tripodal interface molecules were attached to the gold surface more stably than the single contact molecules. The DNA chip platform combined with the functional interface molecule is suitable for a reproducible, inexpensive, and high-throughput detection system for genetic analyses in clinical diagnostics.