Quantification of Low Affinity Binding Interactions between Natural Killer Cell Inhibitory Receptors and Targeting Ligands with a Self-Induced Back-Action Actuated Nanopore Electrophoresis (SANE) Sensor.

A plasmonic nanopore sensor enabling detection of bimodal optical and electrical molecular signatures was fabricated and tested for its ability to characterize low affinity ligand-receptor interactions. This plasmonic nanosensor uses Self-Induced Back-Action (SIBA) for optical trapping to enable SIBA-Actuated Nanopore Electrophoresis (SANE) through a nanopore located immediately below the optical trap volume. A Natural Killer (NK) cell inhibitory receptor heterodimer molecule CD94/NKG2A was synthesized to target a specific peptide-presenting Qa-1bQdm ligand as a simplified model of low-affinity interactions between immune cells and peptide-presenting cancer cells that occurs during cancer immunotherapy. A cancer-irrelevant Qa-1bGroEL ligand was also targeted by the same receptor as a control experiment to test for non-specific binding. The analysis of different pairs of bimodal SANE sensor signatures enabled discrimination of ligand, receptor and their complexes and enabled differentiating between specific and non-specific ligand interactions. We were able to detect ligand-receptor complex binding at concentrations over 500 times lower than the free solution equilibrium binding constant (KD). Additionally, SANE sensor measurements enabled estimation of the fast dissociation rate (koff)for this low-affinity specific ligand-receptor system, previously shown to be challenging to quantify with commercial technologies. Thekoffvalue of targeted peptide-presenting ligands is known to correlate with the subsequent activation of immune cellsin vivo, suggesting the potential utility of the SANE senor as a screening tool in cancer immunotherapy.