Chromatographic Purification of Bacteriophages for Improved Surface Capture of Bacteria – Towards a General Approach R. Naidoo , A. Singh, N. Glass, S. Arya, S. Evoy* * Department of Electrical and Computer Engineering and National Institute for Nanotechnonology, University of Alberta, Edmonton, T6G 2V4 Canada, naidoo@ualberta.ca ABSTRACT We report in this study the largest surface capture density of E Coli using the wild-type T4 bacteriophage; whereby higher surface capture density can enable higher sensitivities for surface-based biosensors. This was possible due to our purification of the phage lysate, which significantly improved phage surface density, achieving maximum (jamming) surface coverage. Our methods could be generalized - to be applicable to a large set of phage biodiversity. We can develop a new screening method to select for the best bacteriophage, for bacterial biosensor application, out a set of candidate phages. Keywords: bacterial biosensor, wild-type bacteriophage, biointerfaces, chromatography, biodiversity. 1 INTRODUCTION The capture of bacteria to a surface is a critical aspect in the development of rapid biosensor platforms for their detection. Our objective in this study is to find a general method to achieve the highest surface capture density of bacteria, using wild-type bacteriophages. Most of the current literature presenting the use of bacteriophages for bacterial biosensor development implements M13 filamentous phage display technology; the M13 phage requires genetic modification of a high-repeat coat protein to typically display an antibody fragment. This study employs wild-type bacteriophages to quickly develop surfaces that exhibit highly specific affinity to pathogens of interest, as has been demonstrated recently [1]. Bacteriophages are probably the most widely distributed biological entity in the biosphere, with an estimated population density of ~10 million per cubic centimeter in any environmental niche where prokaryotes reside [2]. We believe this incredible biodiversity is a major strength of the wild-type phage approach - necessitating general methods applicable to a large set of phage diversity. We report in this study the largest surface capture density of E Coli using the model T4 bacteriophage; whereby higher surface capture density can enable higher sensitivities for surface-based biosensors. This was possible due to our purification of the phage lysate, which significantly improved phage surface density, achieving maximum (jamming) surface coverage. To our knowledge this has never been reported before. Additionally we purified 2 other bacteriophages: P22 and Campy P1, and also show that their phage surface densities improved significantly relative to unpurified suspensions. Previous work with unpurified T4 suspensions and Au surfaces demonstrates a poor phage surface density by physisorption at (0.49 ± 0.15) phages/µm 2 ; covalent attachment by cysteamine-glutaraldehyde improves this to reach (18 ± 0.15) phages/µm 2 [1]. One would expect that by attaining the jamming coverage of phages on the surface that this would correlate with the highest possible bacterial capture density – however it is not the case. We have instead determined an optimal phage surface density for the model T4 system. We also apply these improvements to demonstrate the real-time detection of E. Coli using surface plasmon resonance along a T4 immobilized surface. 2 EXPERIMENTAL METHODS 2.1 Bacterial Culture and Bacteriophage Amplification Bacterial enumeration was done by plate count method and was expressed in cfu/ml while the phage count was performed using the soft agar overlay technique and expressed in pfu/ml (Sambrook and Russell, 1989). For growing P22, the Salmonella enterica serovar Typhimurium culture was prepared by streaking onto a Nutrient agar plate and incubated overnight at 37°C. Two single colonies from the plate were inoculated into 3 ml Nutrient broth (NB) and were grown overnight at 37°C in a shaker to obtain an overnight bacterial culture. Then, 900 µl of 10 7 pfu/ml P22 phages were mixed with 3.6 ml of Salmonella enterica serovar Typhimurium culture and incubated at room temperature for 10 min. This mixture was added to 630 ml of LB and was incubated at 37°C, while shaking at 150 rpm for 15h. The amplified phages were then centrifuged at 2500 rpm for 20 min to remove bacterial cells. The supernatant was vacuum filtered and ultracentrifuged at 55,000 rpm for 1 h. The pellet was resuspended in 1ml SM buffer, vacuum filtered and titrated. The phage solution was purified to remove bacterial contaminant proteins. The phages were used for NSTI-Nanotech 2010, www.nsti.org, ISBN 978-1-4398-3415-2 Vol. 3, 2010 456