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.
Central Research Laboratory, Hitachi Ltd, Kokubunji-Shi, Tokyo, Japan. masao.kamahori.qj@hitachi.com
Masao
M
Kamahori
Yu
Y
Ishige
Maki
M
Shimoda
D015342:DNA Probes / Q000737:chemistry* / Q000235:genetics*; D004563:Electrochemistry / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D017403:In Situ Hybridization / Q000295:instrumentation* / Q000379:methods; D008839:Microelectrodes; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014173:Transistors, Electronic
D015342:DNA Probes