Development and In-Depth Characterization of Bacteria Repellent and Bacteria Adhesive Antibody-Coated Surfaces Using Optical Waveguide Biosensing

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.

Peptide-based direct electrochemical detection of receptor binding domains of SARS-CoV-2 spike protein in pristine samples

“…The CV of bare SPE and GO/SPE does not have redox activity, however the MB-GO/SPE exhibit a redox peak (quasi-reversible) due to the transformation of MB ↔ LMB. The redox behaviour of MB-GO is maintained after activation of the AnteoBind ( b ), N-terminal h ACE-2 peptide (NP), and bovine serum albumin (BSA) layers, with only a minor reduction in current sensitivity [33] , [34] , [35] . The NP is well retained by the interface of b on the electrode surface.…”

“…The surplus electrode materials on the surface were gently removed by immersing for 2 seconds in a PBS solution [28] , [29] , [30] , [31] , [32] To avoid non-specific bindings, the electrode was treated with bovine serum albumin (BSA) solution (4 μL, 0.05 mg/mL). After rinsing with PBS buffer for 2 s, the prepared BSA/NP/ b /MB-GO/SPE sensor platform was demonstrated for probing SARS-CoV-2 SP antigen [33] , [34] , [35] …”

Review of Label-Free Monitoring of Bacteria: From Challenging Practical Applications to Basic Research Perspectives

“…The advantages of electrochemical methods are the lower manufacturing costs and ease of system miniaturization and integration, and the ease of use in integrated pointof-care devices [23]. In general, the disadvantages of some systems, which should be critically considered, are the irreproducibility, high limits of detection in real-life assays, and problems with nonspecific response [23,171].…”

Functional blood cell analysis by label-free biosensors and single-cell technologies

“…Oriented layers show optical anisotropy, which is missing for a randomly oriented antibody layer. A. PLL-g-PEG-biotin avidin B. Mix&Go (also called AnteoBind) and C. protein A based coupling [147].…”