Bacteriophages represent a remarkably versatile probe for biosensing and a key component of a new class of bioactive surfaces. Chemical immobilization of bacteriophages is a key operation enabling such applications, yet despite this, rarely is a comparison made between immobilization chemistries or for multiple phages with the same parameters. Here, we report the immobilization of bacteriophages 44AHJD, P68, Remus, and gh-1 by physisorption and covalent cross-linking via a series of thiolated reagents: 11-mercaptoundecanoic acid (11-MUA), l-cysteine with 11-MUA, l-cysteine with glutaraldehyde, and dithiobis(succinimidyl propionate). Surprisingly, phage purification protocols showed significant impact on the phage immobilization efficiency. Indeed, purification of phages by density gradient (CsCl) ultracentrifugation and centrifugal ultrafiltration was found to have a dramatic determinant effect on the quality of the immobilized layer. Surface densities of 160 ± 13.9 phage/μm2 were observed when careful phage purification was combined with 11-MUA self-assembled monolayer functionalization of the surface. High-resolution scanning electron microscopy enabled direct confirmation of immobilization, along with calculation of phage densities on the surface, and even resolution of phage capsid substructures.
Keywords: bacteriophage; bioactive surfaces; biomaterials; biosensor; electron microscopy; immobilization; physisorption.