Impedance-based detection of DNA sequences using a silicon transducer with PNA as the probe layer

Electrochemical impedance measurements were used for the detection of single-strand DNA sequences using a peptide nucleic acid (PNA) probe layer immobilized onto Si/SiO2 chips. In our approach, the PNA is covalently linked to the surface of Si/SiO2 chips that have been functionalized with a silane, 3-glycidoxypropyltrimethoxysilane (GPTS). The functionalization procedure has been optimized to ensure maximum available sites for probe attachment. The PNA probe is hybridized with complementary solution-phase target DNA.;Impedance measurements allow for the detection of the changes in charge distribution at the oxide/solution interface following modifications to the oxide surface. Due to these modifications, there are significant shifts in the semiconductor's flat-band potential after immobilization and hybridization. The results obtained using this direct and rapid approach are supported by fluorescence measurements according to classical methods for the detection of nucleic acid sequences.;One of the main challenges to achieving highly reproducible and more sensitive silicon-based sensor devices is the optimization of the probe layer immobilization procedures. Hybridization may be kinetically or sterically hindered at high surface probe densities. Varying the amount of time that the solid support is exposed to the PNA solution and controlling the probe solution concentration can control the probe density of the probe layer. Fifteen minutes for immobilization appears to be sufficient enough to obtain a single strand layer with a good balance between density and steric hindrance.;The second focus of this thesis is the determination of the melting temperature (Tm) of a complex sequence PNA 10-mer by measuring the impedance of the electrochemical system at different temperatures. The Tm for perfect matched duplexes is 52.2 +/- 0.3°C. This compared well to the theoretical one of 50°C. The introduction of a single base mismatch in the complementary DNA oligomer results in a 13°C lowering of the observed Tm. The Tm for a single base mismatch is 39.7 +/- 0.6°C.