Surface-immobilization of chromatographically purified bacteriophages for the optimized capture of bacteria

Abstract

Bacteriophages offer interesting alternatives to antibodies for the specific capture and detection of pathogenic bacteria onto biosensing surfaces. Procedures for the optimal chemical immobilization of lytic bacteriophages onto surfaces are presented. More specifically, the removal of lysate contaminants from bacteriophage suspensions by size exclusion chromatography significantly increases the resultant planar surface density of immobilized bacteriophages. E. coli T4 and Salmonella enterica serovar Typhimurium P22 phage systems seem to undergo highly heterogeneous adsorption to the surface, possibly explaining the observed phage clustering at higher surface densities. The T4 phage and its E. coli host were initially employed as a model system where we discovered an optimal planar surface density of phages for best bacterial capture: 18.9 ± 0.8 phages/μm2 capturing 18.0 ± 0.3 bacteria/100 μm2. Phage surface clustering ultimately limits the T4 phage-immobilized surface’s ability to specifically capture its host bacteria. Nevertheless, this is to our knowledge the largest surface capture density of E. coli reported using intact T4 bacteriophages. Two additional purified bacteriophage systems (P22 and Campylobacter jejuni phage NCTC 12673) were then similarly studied for their ability to capture their corresponding host bacteria (Salmonella enterica serovar Typhimurium and Campylobacter jejuni respectively) on a surface.

Recent Advances in Bacteriophage Based Biosensors for Food-Borne Pathogen Detection

“…Phages are amplified in their host bacterial culture to achieve high titers; and despite repeated centrifugation, the contamination of bacterial protein, lipids and carbohydrates could severely affect the efficiency of immobilization and binding ability of the phages. Phage lysate have therefore been purified by a host of methods such as ultra-high speed centrifugation [25], ultra-filtration [32], poly(ethylene glycol) precipitation-gradient centrifugation [33], chromatofocusing [34] and size exclusion chromatography [35]. An interesting study demonstrates that purified phage lysate can be essentially used to systematically study the binding kinetics of the phages on to an activated surface for chemical immobilization.…”

“…An interesting study demonstrates that purified phage lysate can be essentially used to systematically study the binding kinetics of the phages on to an activated surface for chemical immobilization. Study with T4, P22 and NCTC 12673 phages model systems revealed that the phage binding kinetics does not follow the idealized and homogenous Langmuir adsorption isotherm but is governed by heterogenous adsorption closely related to Brouers-Sotolongo isotherm [35]. Such rigorous surface binding studies are extremely important for understanding of phage immobilization on a surface and cannot be realized in the presence of contaminations in the lysate.…”

Bacteriophage based self-assembled monolayer (SAM) on gold surface used for detection of Escherichia coli by electrochemical analysis

“…Again washing was done using deionized water (DI) and dried in steam of nitrogen. Incubation of Gold surface immersed deep in 1 ml of isolated bacteriophage was done at room temperature for 1-2 h. Washing of electrode surface was carried out 3 times with PBS buffer (Hi Media) (pH 7.4), then with 10 µl of Bovine serum albumin (BSA) (Sigma-Aldrich) solution (1%) and incubation done for 20 minto prevent non-specific adsorption of non-targeted bio-components (Lucarelli et al, 2005;Geng et al, 2008;Tolba et al, 2010;Naidoo et al, 2012;Tlili et al, 2013).…”

“…Thus, there is an immediate need for developing sensitive, selective, reliable and quick bacterial sensing platforms which ensure pathogen detection at low concentration, and are affordable to use (Chemburu et al, 2005). In this regard, researchers are now-a-days focusing towards development of rapid detection methods (Petty et al, 2006;Alocilja and Radke, 2003;Naidoo et al, 2012).…”

Bacteriophage Receptor Binding Protein Based Assays for the Simultaneous Detection of Campylobacter jejuni and Campylobacter coli

“…Unfortunately, these schemes demand the laborious and time consuming preparation of bacterial cultures and may be limited by the problems associated with the production and storage of these phages. We have also shown that phages immobilized on solid surfaces can be utilized for the detection of bacterial pathogens without the necessity for traditional culturing [19], [20], [21]. However, like other detection methods, immobilized phage-based assays have their shortcomings.…”