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

Abstract

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-1b Qdm 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-1b GroEL 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 (K D ). Additionally, SANE sensor measurements enabled estimation of the fast dissociation rate (k off) for this low-affinity specific ligand-receptor system, previously shown to be challenging to quantify with commercial technologies. The k off value of targeted peptide-presenting ligands is known to correlate with the subsequent activation of immune cells in vivo, suggesting the potential utility of the SANE senor as a screening tool in cancer immunotherapy.

Future Prospects for Biomolecular Trapping with Nanostructured Metals

“…Unlike biolayer interferometry, the aperture optical tweezer does not require surface tethers/binding, and it operates at the single molecule level. Fabrication complexity and cost has also been a challenge for nanotechnologies; however, colloidal-based techniques offer a facile way of creating high-quality DNHs for optical tweezing of proteins and other biomolecules. , Field-induced breakdown may allow for colocating the DNH with a nanopore, which would greatly simplify this dual method that has already shown remarkable application to immunotherapy targets. , Therefore, it is my perspective that recent advances have improved the prospects of this technique becoming a widely used technology.…”

“…Works combining aperture trapping with nanopores have a complementary ionic current signal, which only goes further to verify that this is “nano-optical trapping of single proteins”. , …”

“…Two groups have combined aperture tweezers with nanopores to obtain complementary ionic current and optical signals. ,,,, Figure shows a comparison between ionic current, ionic translocation time, optical trapping time, and optical step height used to distinguish between an antigen and an antibody. It was found that the optical step change “provided the clearest separation between antigen and antibody”, and so that was used in the subsequent analysis .…”

Localized Nanopore Fabrication via Controlled Breakdown

“…Optical nanopores [ 96 ] can benefit from the high bandwidth of optical measurements, as well as the ease of monitoring optical signals at high throughput [ 22 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 146 , 147 , 148 ]. On the other hand, nanopore techniques can actively deliver target molecules into the optical sensing area instead of relying on diffusion or tethering [ 95 , 105 , 110 , 112 , 149 ]. Given the advantages of CBD nanopore fabrication, it could be very attractive if CBD could generate nanopores at specific positions in a membrane.…”

“…In addition, the research field of plasmonic optical sensing, or fluorescence-based single-molecule sensing, could benefit from the electrical signal of nanopore sensing. Nanopores can assist the capture rate of biomolecules for nanophotonic sensing, while at the same time, the electrical signal from nanopore sensing can reinforce the single-molecule signal from optical measurements [ 95 , 100 , 109 , 110 , 111 , 112 ]. CBD, however, faces challenges in forming nanopores in a precise position for optical sensing.…”