Secretory Bioluminescent Reporter Sensor for Functional Assessment of Glucocorticoids In Vitro and In Vivo

A reliable assessment of glucocorticoid activity is critical for understanding endocrine regulation and stress responses. Conventional structure-based detection methods, including immunoassays and electrochemical sensors, often exhibit limited capacity to discriminate structurally similar glucocorticoids and their metabolites. Although mass spectrometry offers high specificity, it generally yields endpoint measurements and requires sophisticated instrumentation and extensive sample processing. To address these limitations, we developed a secretion-based genetically encoded biosensor for glucocorticoid detection that employs intein-mediated protein splicing triggered by Glucocorticoid Receptor (GR) activation. Ligand binding induces GR nuclear translocation, bringing split intein domains into proximity and initiating protein trans-splicing. This process reconstitutes a split secretory peptide, resulting in the secretion of a luciferase reporter into the extracellular space and enabling real-time quantification of receptor-mediated glucocorticoid activity. Our sensor demonstrated dose-dependent luminescent responses to glucocorticoids across a dynamic range of 0.1 nM-10 μM, with a detection limit of 10 nM, and a rapid response detectable within 5 min. The sensor selectively distinguished functional GR agonists from structurally related inactive analogues. When encapsulated sensor cells were implanted in mice, extracellular luminescent readouts of systemic corticosterone were obtained, enabling detection of endogenous glucocorticoid fluctuations under both basal and restraint-stress conditions. This platform enables dynamic and functionally relevant assessment of glucocorticoid activity and supports ligand screening, mechanistic investigation, and real-time monitoring of stress responses in preclinical animal models. Furthermore, the system holds potential for ex vivo analysis of human biofluids, with prospective applications in clinical diagnostics, therapeutic monitoring, and drug development for endocrine disorders.