Peptide−Nucleic Acid-Modified Ion-Sensitive Field-Effect Transistor-Based Biosensor for Direct Detection of DNA Hybridization

Here, we report the development of a peptide-nucleic acid (PNA)-modified ion-sensitive field-effect transistor (IS-FET)-based biosensor that takes advantage of the change in the surface potential upon hybridization of a negatively charged DNA. PNA was immobilized on a silicon nitride gate insulator by an addition reaction between a maleimide group introduced on the gate surface, the succinimide group of N-(6-maleimidocaproyloxy) succinimide, and the thiol group of the terminal cysteine in PNA. The surface was characterized after each step of the reaction by X-ray photoelectron spectroscopy analysis, and the kinetic analysis of the hybridization events was assessed by surface plasmon resonance. In addition, we measured the -potential before and after PNA-DNA hybridization in the presence of counterions to investigate the change in surface charge density at the surface-solution interface within the order of the Debye length. On the basis of the zeta-potential, the surface charge density, DeltaQ, calculated using the Grahame equation was approximately 4.0 x 10(-3) C/m2 and the estimated number of hybridized molecules was at least 1.7 x 10(11)/cm2. The I-V characteristics revealed that the PNA-DNA duplexes induce a positive shift in the threshold voltage, VT, and a decrease in the saturated drain current, ID. These results demonstrate that direct detection of DNA hybridization should be possible using a PNA-modified IS-FET-based biosensor. PNA is particularly advantageous for this system because it enables highly specific and selective binding at low ionic strength.