A modular platform for surface-bound biosensing: SpyCatcher-Mediated functionalization of antifouling polypeptide...

Precise molecular engineering of surfaces is critical for advancing biosensing, antifouling technologies, and smart material interfaces, yet current methods often suffer from uncontrolled orientation or require complex surface chemical modifications. Here, we report a modular protein-based platform that combines two key elements: (1) self-assembling B-M-E protein antifouling brushes composed of a solid-binding peptide (B), a multimerization domain (M), and an antifouling polypeptide (E). (2) Bioorthogonal SpyCatcher/SpyTag chemistry for precise post-assembly covalent immobilization of target molecules with site-specific control. We demonstrate high-efficiency conjugation on both gold and polystyrene surfaces using quartz crystal microbalance with dissipation (QCM-D) and fluorescence assays. This bioorthogonal strategy offers one-step surface coating without complex chemical modifications, tunable and stable protein immobilization, and universal substrate compatibility. Our post-assembly functionalization platform provides a versatile toolbox for creating functional protein coatings by suppressing non-specific binding, which minimizes background interference and improves detection sensitivity and specificity. This approach holds significant potential for applications such as point-of-care diagnostics and continuous monitoring devices.