Bacteria repellent surfaces and antibody-based coatings for bacterial assays have
shown a growing demand in the field of biosensors, and have crucial importance in
the design of biomedical devices. However, in-depth investigations and comparisons
of possible solutions are still missing. The optical waveguide lightmode spectroscopy
(OWLS) technique offers label-free, non-invasive, in situ characterization of protein
and bacterial adsorption. Moreover, it has excellent flexibility for testing various
surface coatings. Here, we describe an OWLS-based method supporting the development
of bacteria repellent surfaces and characterize the layer structures and affinities
of different antibody-based coatings for bacterial assays. In order to test nonspecific
binding blocking agents against bacteria, OWLS chips were coated with bovine serum
albumin (BSA), I-block, PAcrAM-g-(PMOXA, NH2, Si), (PAcrAM-P) and PLL-g-PEG (PP) (with
different coating temperatures), and subsequent Escherichia coli adhesion was monitored.
We found that the best performing blocking agents could inhibit bacterial adhesion
from samples with bacteria concentrations of up to 107 cells/mL. Various immobilization
methods were applied to graft a wide range of selected antibodies onto the biosensor’s
surface. Simple physisorption, Mix&Go (AnteoBind) (MG) films, covalently immobilized
protein A and avidin–biotin based surface chemistries were all fabricated and tested.
The surface adsorbed mass densities of deposited antibodies were determined, and the
biosensor;s kinetic data were evaluated to divine the possible orientations of the
bacteria-capturing antibodies and determine the rate constants and footprints of the
binding events. The development of affinity layers was supported by enzyme-linked
immunosorbent assay (ELISA) measurements in order to test the bacteria binding capabilities
of the antibodies. The best performance in the biosensor measurements was achieved
by employing a polyclonal antibody in combination with protein A-based immobilization
and PAcrAM-P blocking of nonspecific binding. Using this setting, a surface sensitivity
of 70 cells/mm2 was demonstrated.
