Quantitative Analysis of Protein-Receptor Binding Using Solid-State Nanopores: Accurate Measurement of Dissociation Constants for KREMEN1 and ASGR1 with SARS-CoV-2 Spike RBD Protein

Accurately quantifying protein–protein binding at the single-molecule level is essential for understanding the mechanisms of viral infection and therapeutic targeting. Here, we use solid-state nanopores (SSNs) to detect complex formation between the SARS-CoV-2 Spike receptor-binding domain (Spike RBD) and the alternative host receptors KREMEN1 and Asialoglycoprotein Preceptor 1 (ASGR1). Single-molecule translocation events were analyzed using unsupervised Gaussian mixture modeling and a control-anchored semisupervised classification framework to resolve overlapping free-protein and complex populations. This approach enabled direct identification of receptor-Spike RBD complexes and calculation of apparent dissociation constants under experimental conditions. The inferred affinities were 261.1 nM for ASGR1 and 56.6 nM for KREMEN1, in good agreement with reported literature values and can be used as rough estimates on Spike and its receptor affinities. A negative control using human serum transferrin and Spike RBD showed no emergent high-ΔI population, supporting the specificity of the observed interactions. These results establish SSNs as a scalable and quantitative platform for single-molecule affinity measurements.