Sensors and Actuators B 118 (2006) 53–59 DNA biosensor using fluorescence microscopy and impedance spectroscopy Daniel Berdat ∗ , Annick Marin, Fernando Herrera, Martin A.M. Gijs Institute of Microelectronics and Microsystems, Ecole Polytechnique F´ ed´ erale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland Available online 30 May 2006 Abstract Two types of DNA biosensors are presented. Both sensing principles are demonstrated using synthetic oligomer single-stranded DNA (ssDNA) with concentrations in the micromolar range. A first sensor type is based on the detection of fluorescently labeled ssDNA to a complementary probe that is bound to a silicon substrate by a disuccinimidyl terephtalate and aminosilane immobilization procedure. An enhanced fluorescent response is obtained using constructive interference effects in a fused silica layer deposited before immobilization onto the silicon substrate. The selectivity of different DNA probes towards complementary and non-complementary DNA targets is tested. A second type of DNA sensor is based on the impedimetric response of a solution of unlabeled 20-mer ssDNA in de-ionized water. Interdigitated microelectrodes that are 5 m wide and separated by 5 m gaps are microfabricated on glass substrates and the complex impedance of the system in the 100 Hz–100 MHz frequency range is investigated. The proportionality between the measured solution resistance and ssDNA concentration is demonstrated. © 2006 Elsevier B.V. All rights reserved. Keywords: DNA biosensor; Fluorescence; Impedance spectroscopy; Interdigitated microelectrode; DNA immobilization 1. Introduction DNA detection is an important area of research in almost every field of modern life science and is relevant for applica- tions ranging from drug discovery [1], rapid pathogen detection [2], single-nucleotide polymorphism detection [3] to the assess- ment of water and food quality [4]. Hybridization of solution- phase ssDNA to a complementary ssDNA probe that is fixed on a solid-phase support is the basic principle behind modern micro-array technologies [5–7]. Laser-induced fluorescence is the most commonly used method for DNA hybridization detec- tion and provides very high detection sensitivity approaching the single molecule level [8,9], but requires fluorescent label- ing of the target ssDNA. Electrochemical detection methods have also been developed. Here the hybridization is mainly detected by labeling the DNA with metal complexes, enzymes or metal nanoparticles. The detection selectivity often is based on the differences between the electrochemical behavior of dou- ble stranded and single stranded DNA [10]. Also impedance spectroscopy [11,12] which, includes non-Faradic impedance measurements [13,14] is becoming a very powerful tool for the ∗ Corresponding author. Tel.: +41 21 693 65 87; fax: +41 21 693 59 50. E-mail address: daniel.berdat@epfl.ch (D. Berdat). analysis of interfacial property changes of modified electrodes upon biorecognition events [15]. For an amperometric electrical detection, it is required that an electroactive label is appended to the target DNA, while for a voltammetric detection, stable and reliable reference electrodes should be integrated within the system. Direct impedance measurements, where the detection relies on the impedance change of hybridized label-free DNA due to changes in conductivity or dielectric constant, represent a simpler solution [16]. Impedimetric sensors using micro- or nano-scaled electrodes present an improved signal and sensitivity compared to other impedimetric measurement principles. Here, the linear dimen- sion of the probed sample volume, in which a specific bioreaction occurs, can approach the size of the electrode interspacing, resulting in a higher signal level. Label-free dielectric detection of DNA hybridization with nanogap junctions has been demon- strated at micromolar concentrations of target DNA [17,18]. Interdigitated electrodes in Ti and Pt have been proposed by sev- eral groups and their potential for the detection of biomolecules immobilized on surfaces [19,20] or in solution [21,22] has been demonstrated. In this paper, we report on two different approaches where we use microfabrication techniques for the realization of DNA biosensors. A first sensor is schematically shown in Fig. 1(a) and is based on the detection of fluorescently labeled ssDNA 0925-4005/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2006.04.064