Label-Free Analysis of Multivalent Protein Binding Using Bioresponsive Nanogels and Surface Plasmon Resonance (SPR)

Abstract

Precise identification of protein–protein interactions is required to improve our understanding of biochemical pathways for biology and medicine. In physiology, how proteins interact with other proteins or small molecules is crucial for maintaining biological functions. For instance, multivalent protein binding (MPB), in which a ligand concurrently interacts with two or more receptors, plays a key role in regulating complex but accurate biological functions, and its interference is related to many diseases. Therefore, determining MPB and its kinetics has long been sought, which currently requires complicated procedures and instruments to distinguish multivalent binding from monovalent binding. Here, we show a method for quickly evaluating the MPB over monovalent binding and its kinetic parameters in a label-free manner. Engaging pNIPAm-co-AAc nanogels with MPB-capable moieties (e.g., PD-1 antigen and biocytin) permits a surface plasmon resonance (SPR) instrument to evaluate the MPB events by amplifying signals from the specific target molecules. Using our MPB-based method, PD-1 antibody that forms a type of MPB by complexing with two PD-1 proteins, which are currently used for cancer immunotherapy, is detectable down to a level of 10 nM. In addition, small multivalent cations (e.g., Ca2+, Fe2+, and Fe3+) are distinguishably measurable over monovalent cations (e.g., Na+ and K+) with the pNIPAm-co-AAc nanogels.

Label-free methods for opticalin vitrocharacterization of protein–protein interactions

“…Given the large number of SPR publications, we refer here to reviews showcasing recent developments in SPR applications for protein–protein interaction quantification, 26–28 high-throughput with SPR imaging sensors (SPRi), 29 sensitivity and detection speed, 30,31 influence of capture surfaces, 32 and overcoming challenges with multi-valent binding. 33,34 …”

“…Given the large number of SPR publications, we refer here to reviews showcasing recent developments in SPR applications for protein-protein interaction quantification, [26][27][28] high-throughput with SPR imaging sensors (SPRi), 29 sensitivity and detection speed, 30,31 influence of capture surfaces, 32 and overcoming challenges with multi-valent binding. 33,34 Mamer et al, in their review, show SPR's convergence to cell-based protein-protein interaction measurements. 35 Recent research focusing on SARS-CoV-2 protein interactions highlights the applicability of SPR, as shown in Fig.…”

Plasmonic nanomaterials with responsive polymer hydrogels for sensing and actuation

“…18B ). 161 Small multivalent cations ( e.g. , Ca 2+ , Fe 2+ , and Fe 3+ ) can be distinguished by SPR over monovalent cations ( e.g.…”

Novel Regeneration Approach for Creating Reusable FO-SPR Probes with NTA Surface Chemistry

“…Surface plasmon resonance (SPR) technology has been used for biosensing in various fields, spanning from the pharmaceutical analysis [1,2], medical and food diagnostics [3][4][5][6], to environmental monitoring [7]. In addition to pure analyte quantification, SPR has become a powerful tool to determine binding specificity, affinity and kinetics, thanks to the ability to perform real-time label-free monitoring of biomolecular interactions in both buffer and complex matrices [8][9][10][11]. To date, plenty of effort has been made to improve sensitivity [12], specificity [13], portability [14] and reusability of the SPR sensing surface [15].…”