Biosensors and Bioelectronics 21 (2005) 645–654 Label-free impedance detection of oligonucleotide hybridisation on interdigitated ultramicroelectrodes using electrochemical redox probes V. Dharuman a , T. Grunwald b , E. Nebling a,∗ , J. Albers a , L. Blohm b , R. Hintsche a,b a Department of Biotechnical Microsystems, BTMS, Fraunhofer Institute of Silicon Technology, BTMS, Fraunhoferstrasse 1, D-25524 Itzehoe, Germany b eBiochip Systems GmbH, Fraunhoferstrasse 1, D-25524 Itzehoe, Germany Received 18 October 2004; received in revised form 22 December 2004; accepted 23 December 2004 Available online 30 January 2005 Abstract The direct detection of oligodeoxynucleotide (ODN) hybridisation using electrochemical impedance spectroscopy was made on interdigi- tated array (IDA) gold (Au) ultramicroelectrodes manufactured by silicon technology. The immobilisation of single stranded ODNs (ssODNs) was accomplished by self-assembling of thiol-modified ODNs onto an Au-electrode surface. Faradaic impedance was measured in the pres- ence of K 3 [Fe(CN) 6 ]. Double strand formation was identified by a decrease of approximately 50% in impedance in the low frequency region in the presence of K 3 [Fe(CN) 6 ], compared to the spectrum of single stranded ODN. The frequency dependent diffusion of Fe(CN) 6 3- ions through defects in the ODN monolayer determines the impedance of Au–ssODN surface. The influence of DNA intercalator methylene blue on the impedance of both, single and double strands, was examined along with K 3 [Fe(CN) 6 ] and confirmed by cyclic voltammetry. The layer densities and the hybridisation have been further corroborated by chronoamperometric redox recycling of para-aminophenol (p-AP) in ELISA like experiments. It can be concluded, that a performed impedance spectroscopy did not change the layer density. The impedance spectroscopy at ultramicroelectrodes combined with faradaic redox reactions enhances the impedimetric detection of DNA hybridisation on IDA platforms. © 2005 Elsevier B.V. All rights reserved. Keywords: ODN hybridisation; Label-free detection; IDA electrodes; Si-technology; Impedance; Electrical biochips 1. Introduction The detection of biomolecular interactions using electro- chemical impedance spectroscopy (EIS) has attracted recent interest in biosensor applications (Katz and Willner, 2003; Guan et al., 2004). Because the interdigitated microarray electrodes (IDAs) are more sensitive than conventional gold electrodes and compatible with microarray construction of DNA sensors through silicon technology, IDAs have been applied as impedimetric sensors. This includes the detec- tion of the cellular behavior (Ehret et al., 1997), enzyme activity (Saum et al., 1998; Katz and Willner, 2003) and antigen–antibody interactions (Lillie et al., 2001). However, the detection of DNA hybridisation without enzyme labelling ∗ Corresponding author. Tel.: +49 4821 174312; fax: +49 4821 174350. E-mail address: nebling@isit.fhg.de (E. Nebling). on IDAs is still limited to the use of EIS (Paeschke et al., 1996a,b; Jacobs, 1998; Frace et al., 2002). In the EIS, the label-free affinity binding of target molecules to capture probes on the electrode surface is indicated by a shift in the impedance or a change in capacitance or admittance of the bulk electrode (Montelius et al., 1995; Van Gerwen et al., 1998; Souteyrand et al., 2000). But in practice, the separa- tion and detection of an electrical response arising from the biomolecular recognition is still limited by the low sensitivity of the EIS technique. The sensitive detection requires a more intimate association between the molecular recognition layer and the transduction technique. Two different approaches have been applied to label-free detection of the hybridisation event on IDA platforms. In the first method, polypyrrole, a conducting polymer which acts as a potential reporter of biomolecular interactions, is in- tegrated on IDA surfaces and used for label-free detection 0956-5663/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2004.12.020