Abstract
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.
Highlights
Optical waveguide lightmode spectroscopy (OWLS) is a surface-sensitive biosensor for probing the surface adhesion and binding of biological and chemical species in a realtime and label-free manner
From among the large variety of repellent coatings, we focused on two traditional coatings, namely, bovine serum albumin (BSA) and I-block, and two novel polymer coatings, PLL-g-PEG (PP) (PLL is poly(Llysine)-graft-poly(ethylene glycol)) and PAcrAM-g-(PMOXA, NH2, Si) (PAcrAM-P)
optical waveguide lightmode spectroscopy (OWLS) chips were coated with BSA, I-block, PAcrAM-P and PP, and subsequent E. coli adhesion was monitored in situ
Summary
Optical waveguide lightmode spectroscopy (OWLS) is a surface-sensitive biosensor for probing the surface adhesion and binding of biological and chemical species in a realtime and label-free manner. OWLS detects refractive index changes 100–200 nm above the sensor’s surface, providing quantitative information on near-surface kinetic and structural processes [1,2]. Bacteria repellent surfaces and antibody (Ab)-based coatings for bacterial assays have crucial importance in the field of biosensors, and are highly relevant in the design and development of novel biomedical devices [3,4,5,6]. In-depth investigations and comparisons of possible surface coating solutions for this specific application are still missing. Nowadays there is a rapidly growing demand to develop bacteria repellent coatings. Bacteria can often adhere to surfaces, where they can subsequently form biofilms [7]
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