A Bioluminescent Probe for Salivary Cortisol

• DOI: 10.1021/bc200220k
• Publication Date: 2011-08-22T12:09:34Z
• Authors: Sung Bae Kim; Yasuhiro Takenaka; Masaki Torimura
• Keywords: 0303 health sciences; Binding Sites; Hydrocortisone; Enzyme-Linked Immunosorbent Assay; Sensitivity and Specificity; 3. Good health; 03 medical and health sciences; Receptors, Glucocorticoid; Genes, Reporter; COS Cells; Chlorocebus aethiops; Luminescent Measurements; Animals; Humans; Luciferases; Promoter Regions, Genetic; Saliva; Glucocorticoids

Cortisol is a classical biomarker for the stress levels of human beings. We fabricated highly sensitive bioluminescent probes for salivary cortisol. The following strategies were contrived in the molecular design. Gaussia princeps luciferase (GLuc) was dissected into two fragments, between which an N-terminal-extended ligand binding domain of glucocorticoid receptor (GR HLBD), named Simgr4, was inserted. First, this unique single-chain probe was then situated downstream of a glucocorticoid response element (GRE) promoter in a reporter-gene system for constructing two ON-OFF switches for cortisol. Second, a circularly permutated (CP) variant of Simgr4 was formulated. The reporter-gene system exerted an improved signal-to-background (S/B) ratio of 8.5 to cortisol. Furthermore, a circularly permutated (CP) variant of Simgr4 exerted a 10× enhanced detection limit to cortisol and a long dynamic range from 10(-9) to 10(-6) M cortisol, covering all of the normal clinical ranges of serum, urine, and saliva. This optimized probe successfully determined daily fluctuations of salivary cortisol and the correlations with those by ELISA. This study is the first to investigate the contribution of the HLBD of a nuclear receptor and multiple ON-OFF switches for molecular probes and salivary cortisols.