Oriented Immobilization of Bacteriophages for Biosensor Applications

Abstract

A method was developed for oriented immobilization of bacteriophage T4 through introduction of specific binding ligands into the phage head using a phage display technique. Fusion of the biotin carboxyl carrier protein gene (bccp) or the cellulose binding module gene (cbm) with the small outer capsid protein gene (soc) of T4 resulted in expression of the respective ligand on the phage head. Recombinant bacteriophages were characterized in terms of infectivity. It was shown that both recombinant phages retain their lytic activity and host range. However, phage head modification resulted in a decreased burst size and an increased latent period. The efficiency of bacteriophage immobilization with streptavidin-coated magnetic beads and cellulosebased materials was investigated. It was shown that recombinant bacteriophages form specific and strong bonds with their respective solid support and are able to specifically capture and infect the host bacterium. Thus, the use of immobilized BCCP-T4 bacteriophage for an Escherichia coli B assay using a phage multiplication approach and real-time PCR allowed detection of as few as 800 cells within 2 h.

Modified Bacteriophage S16 Long Tail Fiber Proteins for Rapid and Specific Immobilization and Detection of Salmonella Cells

“…The use of affinity-based magnetic separation can simultaneously tackle two critical parameters to reduce detection assay time and increase sensitivity, by (i) enriching viable cell numbers to a detectable level and (ii) isolating cells from contaminants and debris found in food samples for more accurate downstream identification and characterization (51). Toward our aim to develop a sensitive and specific assay for bacterial enrichment that can outcompete the conventional application of antibodies for biomolecular recognition, the bacteriophage S16 LTF joins other approaches employing either whole phages (25,32,33,58,59) or phage-encoded proteins, such as endolysin cell wall-binding domains (CBDs) (36)(37)(38) and tail spike RBPs (39)(40)(41).…”

Biocontrol of Listeria monocytogenes and Escherichia coli O157:H7 in Meat by Using Phages Immobilized on Modified Cellulose Membranes

“…The site-specific immobilization of phages was suggested as a way to orientate the immobilized phages in such a way as to have tail fibers free and, thereby, increase the efficiency of the capture of the target bacteria. The genetic modification of wild-type T4 phage through a phage display technique has been used to give well-oriented T4 phage particles when immobilized on cellulose membranes or streptavidin-coated magnetic beads, which resulted in a high capture efficiency for Escherichia coli cells (38). However, this protocol was initially laborious and costly even with T4 phage, which is one of the better-characterized phages with a fully sequenced and annotated genome.…”

“…However, this protocol was initially laborious and costly even with T4 phage, which is one of the better-characterized phages with a fully sequenced and annotated genome. In addition, these modified phages exhibited a lower burst size and less infectivity than wild-type T4 (24,38). Therefore, alternative simple protocols are needed to immobilize phages in the right orientation that can be applied to newly isolated phages with known structures but that have not been fully sequenced.…”

Real-time analysis of dual-display phage immobilization and autoantibody screening using quartz crystal microbalance with dissipation monitoring

“…The phage could be used to display different types of peptides, proteins, and antibodies to be employed in affinity selection of target-specific phage particles. 1,2,[5][6][7][8] Current advances in biosensors are providing the possibility for immobilizing bioreceptor molecules in a site-directed pattern while retaining their intact…”

“…9 As recently reported, the oriented immobilization of bacteriophages on a sensing platform resulted in a higher sensitivity in the detection of autoantibodies in serum samples. 6 A main drawback of bacteriophages-based diagnostic assays is the nonspecific adsorption of proteins, leading to a high background signal and decreasing sensitivity. Due to the lack of a specific strategy to bring the phage onto a sensing platform, most of the phage-based assays are limited to an ELISA format.…”