Grating-coupled interferometry reveals binding kinetics and affinities of Ni ions to genetically engineered protein layers

Abstract

Reliable measurement of the binding kinetics of low molecular weight analytes to their targets is still a challenging task. Often, the introduction of labels is simply impossible in such measurements, and the application of label-free methods is the only reliable choice. By measuring the binding kinetics of Ni(II) ions to genetically modified flagellin layers, we demonstrate that: (1) Grating-Coupled Interferometry (GCI) is well suited to resolve the binding of ions, even at very low protein immobilization levels; (2) it supplies high quality kinetic data from which the number and strength of available binding sites can be determined, and (3) the rate constants of the binding events can also be obtained with high accuracy. Experiments were performed using a flagellin variant incorporating the C-terminal domain of the nickel-responsive transcription factor NikR. GCI results were compared to affinity data from titration calorimetry. We found that besides the low-affinity binding sites characterized by a micromolar dissociation constant (Kd), tetrameric FliC-NikRC molecules possess high-affinity binding sites with Kd values in the nanomolar range. GCI enabled us to obtain real-time kinetic data for the specific binding of an analyte with molar mass as low as 59 Da, even at signals lower than 1 pg/mm2.

Developing an Optical Interferometric Detection Method based biosensor for detecting specific SARS-CoV-2 immunoglobulins in Serum and Saliva, and their corresponding ELISA correlation

“…However, the significant advantages of optical biosensing systems based on combined interferometry and resonance, used with or without optical waveguides, are worth mentioning [ 17 , 18 ]. The applications of surface plasmon resonance-based biosensors [ [19] , [20] , [21] ], diffraction grating coupled interferometry [ 22 ], photonic crystals [ 23 , 24 ], ring resonators [ [25] , [26] , [27] ], Mach–Zehnder [ 28 , 29 ], guided-mode resonance biosensors [ 30 , 31 ], Young interferometers [ 32 ], and resonant nanopillars [ 33 ], among others, have been widely reported.…”

Bacterial Flagellar Filament: A Supramolecular Multifunctional Nanostructure

“…Flagellin monomers and flagellin–protein fusions readily adsorb to hydrophobic surfaces with their D3 domains oriented towards the liquid phase [ 235 ], a feature that can be utilized in different ways. For example, Jankovics et al [ 236 ] designed a biosensor by immobilizing a flagellin fusion in which D3 was replaced by the nickel-binding domain of the transcription factor NikR, and successfully used it in binding kinetics studies. Different variants of flagellin can be used to create surface coatings that have either anti-adhesive (in case of the wild type flagellin) or highly adhesive (D3 replaced with a RGD motif) properties [ 237 ].…”

All-Optical Switching Demonstrated with Photoactive Yellow Protein Films

“…By adsorbing a transparent NLO material on top of the arms, adlayers are formed that can alter the effective refractive index, thus creating a phase difference between the arms joining at the output of the device, manifesting in intensity changes at the MZI's output. This principle is often used for biosensing applications, such as sensing bacteria or proteins in action, as well [17,18]. Hence, we performed all-optical switching experiments using an IO MZI, with a PYP adlayer as an active NLO component, for the first time.…”