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 Kd(s) on the surface was 0.38 mu M, 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 mu M 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-control ling gold surface. (C) 2007 Elsevier B.V. All rights reserved.