Beiträge der TFH-Wildau

IMPEDIMETRIC DNA DETECTION ON GOLD FILM ELECTRODES

Oliver Pänke, Jan Kafka, Fred Lisdat*

University of Applied Sciences Wildau,
Biosystems Technology, Bahnhofstr. 1, D-15745 Wildau

flisdat@igw.tfh-wildau.de

DNA analytics based on biochip technology has become an increasingly implemented method in clinical practice, veterinary medicine, agriculture, and basic research. In this regard, impedance spectroscopy is a rapidly developing electrochemical technique which determines the resistive and capacitive properties of sensor surfaces and turned out to be a convenient method to monitor the hybridisation of DNA on electrodes. Here, we present an impedimetric sensor for the specific and quantitative detection of single-stranded DNA (ssDNA).
Gold film electrodes were modified with 5’-thiol-terminated ssDNA acting as probe oligonucleotide. Hybridisation was accomplished by exposing the modified surface to target ssDNA. The electrodes were analysed with impedance spectroscopy providing both the electron transfer resistance of added potassium hexacyanoferrate, Rct, and the capacitance of the modified surface. We observed that Rct increases after hybridisation whereas the capacitance of the surface remained almost constant. Incubation with complementary ssDNA yielded a three-fold increase of Rct on average compared to the non-hybridised sensor surface. Incubation with non-complementary ssDNA did not affect the impedance spectra, indicating a negligible extent of non-specific DNA binding to the electrode. The sensor was reusable and regenerated by incubation in urea solution, allowing up to seven consecutive DNA determinations. The period for a single DNA determination including hybridisation, impedimetric analysis and regeneration was 15 min only. Exposing the sensor surface to different concentrations of complementary ssDNA yielded a half maximum sensor response at 350 ± 30 nM. The respective detection limit was 100 nM.
Furthermore, the sensor was capable of sensing single base pair mismatches within the hybridised DNA duplex. The increase of Rct due to DNA hybridisation was reduced for duplexes carrying a single base pair mismatch even for saturating ssDNA concentrations, which implies that the reduced sensor response was not the result of a reduced amount of ssDNA bound to the electrode. Additionally, the changes for Rct were indicative for the type and the position of the mismatch. The sensitivity as well as the capability of detecting ssDNA targets with sequences larger in length compared to the standard 18mer oligonucleotide allows the analysis of DNA from amplification protocols, like PCR.