Microfluidic biosensor for the detection of DNA by fluorescence enhancement and the following streptavidin detection by fluorescence quenching Jun Wang a,b,c,n , Michihiko Aki d , Daisuke Onoshima a,b,e , Kenji Arinaga d , Noritada Kaji a,b,d , Manabu Tokeshi a,b,f , Shozo Fujita d , Naoki Yokoyama d , Yoshinobu Baba a,b,e,g a Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Nagoya, Japan b MEXT Innovative Research Center for Preventive Medical Engineering, Nagoya University, Nagoya, Japan c Department of Chemical and Biological Engineering, Chalmers University of Technology, Göteborg, Sweden d Fujitsu Laboratories Ltd., Tokyo, Japan e FIRST Research Center for Innovative Nanobiodevice, Nagoya University, Nagoya 464-8603, Japan f Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, Sapporo, Japan g Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Japan article info Article history: Received 15 May 2013 Received in revised form 16 July 2013 Accepted 30 July 2013 Available online 6 August 2013 Keywords: Microfluidic biosensor Fluorescence switching Complementary ssDNA Streptavidin abstract We reported an optical DNA/protein microfluidic sensor which consists of single stranded (ss) DNA-Cy3 probes on gold surface and simple line-shape microfluidic channel. These ssDNA-Cy3 probes with random sequence in bulk solution or on gold surface exhibits fluorescence enhancement after binding with complementary ssDNA (cssDNA) targets. Particularly it did not require complicated design or hairpin-like stem-loop conformation, which made it easier to be made and applied in analytes detection by fluorescence switching techniques. Using ssDNA-cy3 probes attached on gold surface in a microfluidic channel, strong fluorescence enhancement was measured by ssDNA with cssDNA binding or ssDNA with cssDNA–biotin binding. The following introduction of streptavidin resulted in fluorescence quenching (fluorescence decrease) because of the binding of hybridized DNA–biotin with streptavidin. This sensor showed strong affinity and high sensitivity toward the streptavidin, the minimum detectable concentration for streptavidin was 1 pM, equating to an absolute detection limit of 60 amol in this microfluidic channel. Microfluidic channel height and flow rate is optimized to increase surface reaction efficiency and fluorescence switching efficiency. In contrast to previously reported optical molecular beacon approach, this sensor can be used not only for the detection of cssDNA target, but also for the detection of streptavidin. This microfluidic sensor offers the promise of analyzing kinds of molecular targets or immunoreactions. & 2013 Elsevier B.V. All rights reserved. 1. Introduction In recent years, the interest for DNA or protein-based diagnos- tic tests has been growing. The development of sensors allowing DNA or protein detection is motivated by applications in many fields: DNA diagnostics, gene analysis, fast detection of biological warfare agents, and forensic applications (Csako, 2006; Han et al., 2013; Tsongalis and Coleman, 2006; Epstein et al., 2002; Yeung et al., 2008). Numerous DNA detection systems based on the hybridization between a DNA target and its complementary probe, which is present either in solution or on a solid support, have been described (Heller, 2002; Bowtell, 1999; Winzeler et al., 1999; Cooper et al., 2001; Park et al., 2002; Livak, 1999; Lipshutz et al., 1999; Albert et al., 2009). Now the high importance is the need for techniques that do not require labeling of the target sample, because that increases the time, cost, and potential for error inherent in the analysis. In the context of solution-phase assays, the molecular beacon concept based fluorescence switching has proven itself to be both sensitive and reliable for their use as sensors for DNA sequencing (Broude, 2002; Dai et al., 1997; Heyduk and Heyduk, 2002; Tyagi and Kramer, 1996). Besides solution phase use, DNA hairpin probes have been immobilized onto solid substrates (Piestert et al., 2003; Liu and Tan, 1999; Fang et al., 1999). However, most of these employ an attached single molecule as quencher, while the material on which the hairpin is immobilized serves only a passive role, the substrates served only as an anchor: the function of the probe was unchanged from the solution assays. Several groups have used gold substrate as a more efficient quenching agent instead of a quencher dye molecule for a surface- immobilized hairpin stem-loop single stranded DNA (ssDNA) Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/bios Biosensors and Bioelectronics 0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.07.058 n Corresponding author at: Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan. Fax: +81 52 789 5499. E-mail address: wjzyy988@gmail.com (J. Wang). Biosensors and Bioelectronics 51 (2014) 280–285