A bioorthogonal capture-and-release platform for isolation of nanoparticle protein coronas

The protein corona formed at the nanoparticle (NP)-biofluid interface critically influences the biological identity of nanomaterials, yet its characterization remains highly dependent on isolation procedures that may perturb weakly associated proteins or introduce contaminants. In this study, we developed a bioorthogonal capture-release platform for protein corona isolation by integrating protein-resistant tetrazine (Tz)-functionalized substrates with photoreleasable trans-cyclooctene (TCO)-functionalized poly(ethylene glycol) (PEG)-modified poly(lactic-co-glycolic acid) (PLGA) NPs. Tz-TCO conjugation enabled selective NP immobilization prior to in situ corona formation in biological media, followed by light-triggered recovery of NP-protein corona complexes through cleavage of a photocleavable linker at 365 nm. Surface optimization identified 30% Tz-EG₅ as providing a favorable balance between low nonspecific protein adsorption and retained bioorthogonal reactivity, while optimization of NP formulation and experimental conditions enabled efficient capture and release. Integration into a 24-well plate format further enabled parallel sample processing and provides scope for increased analytical throughput. Compared with conventional centrifugation, the capture-release approach recovered NP-corona complexes with lower size heterogeneity and yielded a greater amount and broader range of corona-associated proteins. Proteomic analysis further revealed differences in the molecular-weight and isoelectric-point distributions of the recovered proteins between the two isolation methods. These findings demonstrate that the isolation workflow can substantially influence the experimentally observed protein corona and establish bioorthogonal capture coupled with light-triggered release as a complementary approach for protein corona isolation and nano-bio interface characterization.