Biosensors and Bioelectronics 25 (2010) 2172–2176 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios Short communication Rapid, multistep on-chip DNA hybridisation in continuous flow on magnetic particles Martin Vojtíˇ sek, Alexander Iles, Nicole Pamme ∗ The University of Hull, Department of Chemistry, Cottingham Road, Hull HU6 7RX, UK article info Article history: Received 13 November 2009 Received in revised form 14 January 2010 Accepted 27 January 2010 Available online 4 February 2010 Keywords: DNA hybridisation DNA isolation Continuous flow Magnetic particles Microfluidic chip abstract DNA hybridisation is an important tool for bioanalytical research and clinical diagnostics; conventional methods, however, require long incubation times and numerous washing steps, rendering the procedure time consuming and labour intensive. In this paper, we report on a rapid method for DNA hybridisation and isolation within a microfluidic device, where all reaction and washing steps are performed in con- tinuous flow in an automated fashion within less than two minutes. Magnetic particles were used as a solid support and manipulated through laminar flow streams containing reagents and buffers by means of an external magnet. Thus, hybridisation, washing, intercalation, fluorescence detection and isolation were performed in continuous flow on the surface of the particles. Initially, the sensitivity of the system was investigated for a one-step DNA hybridisation of Alexa Fluor 555 labelled target DNA to a capture probe immobilised on the particle surface. Hybridisation and washing steps were performed in half a minute and target DNA was readily detected down to 20 nmol L -1 . Then a two-step assay, label-free DNA hybridisation followed by intercalation with PicoGreen was performed. All reaction and washing steps were carried out in continuous flow with a total assay time of about 1 min. This is a significant reduction in procedural time compared to conventional methods and opens the door for developing fully automated continuous flow integrated DNA analysis platforms. © 2010 Elsevier B.V. All rights reserved. 1. Introduction DNA hybridisation is a widely utilised analytical technique to detect the presence of specific nucleotide sequences within a sam- ple. The method is an indispensable tool in biochemical research, clinical diagnostics and forensic science. DNA hybridisation assays are often performed on microarrays which enable multiplexed analysis of thousands or tens-of-thousands of sequences and have been applied to gene expression, pathogen detection and genotyp- ing (Kim et al., 2008; Peplies et al., 2003; Stoughton, 2005). While such arrays are a powerful resource in many research and clini- cal laboratories, their widespread use is limited by their relatively high cost. Drawbacks such as high chip-to-chip variation (Hsiao and Chen, 2009) and the requirement for sample amplification via PCR (Cikos and Koppel, 2009; Sunkara et al., 2007) have been reported. Moreover, array-based systems generally rely on molecular diffu- sion to transport target DNA to the immobilised capture probe, which results in long hybridisation times of usually several hours (Rampal, 2007). These shortcomings have prevented DNA microar- rays from being widely utilised for true point-of-care testing. ∗ Corresponding author. Tel.: +44 1482 465027. E-mail address: n.pamme@hull.ac.uk (N. Pamme). Microfluidic chip based approaches for DNA analysis can over- come some of the limitations of microarray based systems (Auroux et al., 2004; Tegenfeldt et al., 2004). The drawback of slow hybridi- sation can be obviated by flowing liquids through microfludic channels featuring short diffusion distances of only a few tens or hundreds of micrometers and thus diffusion times of typically sev- eral minutes. Microfluidic DNA hybridisation can be performed in solution where probe molecules are not immobilised; the perfor- mance of such systems however is highly dependent on the mixing efficiency and DNA–dye interactions and so far, the ultimate sensi- tivity of such systems has been much lower than for immobilised systems (Benninger et al., 2007; Chen et al., 2008; Heule and Manz, 2004). DNA can also be immobilised on the channel walls (J.H.S. Kim et al., 2006; Watterson et al., 2004) or on the surface of micropar- ticles that are packed into microchannels (Ali et al., 2003; Fan et al., 1999; J. Kim et al., 2006; Ng et al., 2007; Wen et al., 2007; Zhang et al., 2008). Such particles offer high surface-to-volume ratios and thus high densities of immobilised probe, leading to higher sensitivities. Nonetheless, such particle based approaches are essentially batch procedures, requiring several sequential reac- tion and washing steps and thus rendering the analysis labour and time intensive. Here we propose a simple microfluidic system for rapid contin- uous flow DNA hybridisation (Fig. 1a). Parallel laminar flow streams containing reagents and buffer solutions are generated over a flow 0956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.01.034