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.

Publication date: May 17, 2026

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.

Semantics

Original Article