De Novo Design of High-Performance Cortisol Luminescent Biosensors.

Frequent, reliable cortisol measurement is critical for diagnosing and managing adrenal disorders, stress responses, and circadian rhythm disruptions. However, current cortisol assays or detection methods remain confined to laboratory settings, limiting on-site testing. Protein-based biosensors provide a promising point-of-care (POC) solution, yet no robust, field-ready protein-based cortisol biosensor is available. Here, we de novo design cortisol-inducible dimerization modules and systematically sample their fusions with split luciferase reporters by using a protein structure prediction pipeline. The resulting biosensor, designed straight from the computer, yields over 300-fold luminescent response with picomolar sensitivity and can be rapidly imaged by a standard camera or smartphone. This work highlights the power of computational protein design for developing next-generation protein-based biosensors.