Biosensors and Bioelectronics 26 (2010) 131–138 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios Bacteriophage tailspike proteins as molecular probes for sensitive and selective bacterial detection Amit Singh a , Sunil K. Arya a , Nick Glass a , Pejman Hanifi-Moghaddam b , Ravendra Naidoo a , Christine M. Szymanski c , Jamshid Tanha b , Stephane Evoy a,∗ a Department of Electrical and Computer Engineering, National Institute for Nanotechnology, University of Alberta, Edmonton T6G 2V4, Canada b Institute for Biological Sciences, National Research Council, Ottawa, ON K1A 0R6, Canada c Department of Biological Sciences, Alberta Ingenuity Centre for Carbohydrate Science, University of Alberta, Edmonton, AB T6G 2E9, Canada article info Article history: Received 5 March 2010 Received in revised form 14 May 2010 Accepted 17 May 2010 Available online 24 May 2010 Keywords: Tailspike protein (TSP) Bacteriophage Salmonella Biosensor Surface plasmon resonance abstract We report the use of genetically engineered tailspike proteins (TSPs) from the P22 bacteriophage for the sensitive and selective detection of Salmonella enterica serovar Typhimurium. High yields of two mutant TSPs, one with an N-terminal cysteine (N-Cys) and another with a C-terminal cysteine (C-Cys), have been obtained using recombinant protein expression and purification in Escherichia coli. The mutant TSPs did not have the native endorhamnosidase enzymatic activity of intact P22 phage as well as wild type TSPs (wtTSPs). We have used the Cys-tag to immobilize these TSPs onto gold coated surfaces using thiol-chemistry. Our results demonstrate that the N-Cys configuration of TSPs gives a bacte- rial capture density of 25.87 ± 0.61 bacteria/100 m 2 while the C-Cys configuration shows a density of 8.57 ± 0.19 bacteria/100 m 2 . This confirms that the appropriate orientation of the TSPs on the surface is important for efficient capture of the host bacteria. The bacterial capture density of the mutant N-Cys TSP was also 6-fold better than that obtained for intact P22 phage as well as wtTSPs. Bovine-serum albumin was used as a protective layer to prevent any non-specific binding of the bacteria onto the gold substrate. The recognition specificity was confirmed using 3 strains of E. coli which showed negligible binding. In addition, the host bacteria did not show any binding in the absence of the TSPs on the surface. We fur- ther show a selective real-time analytical detection of Salmonella by N-Cys mTSP-immobilized on gold coated SF-10 glass plates using surface plasmon resonance. The sensitivity of detection was found to be 10 3 cfu/ml of bacteria. © 2010 Elsevier B.V. All rights reserved. 1. Introduction A large variety of bacteria, viruses and other microorganisms exist in the environment and many of these organisms cause severe illness in humans and animals. Detection and identifica- tion of pathogenic bacteria have become an area of tremendous interest in the field of food and water safety, bioterrorism preven- tion and public health. The conventional microbiological methods are highly selective and sensitive towards pathogen identification. They, however, rely on culture-based biochemical and serological assays which are time consuming, laborious and cost-ineffective. Biosensors have been regarded as an attractive alternative for the detection of bacterial cells and their toxins compared to these con- ventional approaches (Ivnitski et al., 1999; Lazcka et al., 2007). A variety of techniques such as the quartz crystal microbalance (QCM) (Minunni et al., 1996; Fung and Wong, 2001), micromechanical res- ∗ Corresponding author. Fax: +1 780 492 1811.. E-mail address: evoy@ece.ualberta.ca (S. Evoy). onators (Ilic et al., 2001, 2004), flow cytometry (Abdel-Hamid et al., 1998, 1999), amperometry (Gau et al., 2001), and surface plasmon resonance (SPR) (Taylor et al., 2005; Oh et al., 2005), have been extensively researched. Among them, SPR has been used exten- sively for label-free detection of bacteria, their toxins and spores. Different biological probes such as DNA (Liao and Ho, 2009), RNA (Joung et al., 2008), monoclonal (Taylor et al., 2005; Oh et al., 2005) and polyclonal antibodies (Su and Li, 2005; Taylor et al., 2006) have been used for detection of bacteria using SPR-based platform. Recent attempts have focused on the development of bacteriophage-based detection technologies for pathogen detec- tion. Bacteriophages are the viruses that bind to specific receptors on the bacterial surface. Many phages recognize and bind the host bacterial receptors using their tailspike proteins (Kutter and Sulakvelidze, 2004). The recognition offered by the tailspike protein is highly specific and thus phages have been employed for the typ- ing of bacteria. High levels of specificity and selectivity also make bacteriophages a lucrative probe for the development of pathogen detection technologies. Whole phages have been exploited for developing different detection platforms (Balasubramanian et al., 0956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.05.024