Upon exposure to biological fluids, nanomaterials rapidly acquire dynamic layers of adsorbed proteins called the protein corona, redefining the biological identity of nanomaterials and governing their in vivo fate. While corona composition has been extensively profiled, two aspects remain poorly understood: dynamic evolution and the receptor-mediated mechanisms underlying its cellular recognition. Here we present a broadly applicable, real-time biosensor-based protocol. This workflow characterizes nanomaterial–protein interactions and identifies cell membrane receptors involved in corona recognition. The protocol distinguishes between soft corona, hard corona, and total corona layers. It can not only quantify dynamic and competitive interactions but also map receptor–corona and receptor–plasma protein interactions using a label-free biosensor-based platform. This method combines biolayer interferometry, or alternatively surface plasmon resonance, or magnetic isolation, with downstream proteomics. It enables functional dissection of the nano–bio interface and provides insights into how corona dynamics govern cellular uptake. The method is compatible with diverse nanomaterial types and biofluids, depending on the experimental scope. The protocol is designed for users with basic experience in areas such as nanotechnology, molecular interaction analysis, proteomic profiling, cell culture, and biofluid sample preparation. The entire process takes ~10 days to complete.

Nat. Protoc. 2026, https://doi.org/10.1038/s41596-026-01358-6