Rapid ultrasensitive measurement of salivary cortisol using nano-linker chemistry coupled with surface plasmon resonance detection

Cortisol detection in saliva is of great interest for the diagnosis of various disease states and the monitoring of stress in humans. Currently, measurements are performed predominantly by radioimmunoassay (RIA) which is expensive, labour intensive, uses hazardous radioisotopes and involves extensive delays in obtaining results. A rationally designed cortisol-linker conjugate allowing high assay sensitivity was employed as a coating antigen in a microfluidic surface plasmon resonance (SPR) biosensor immunoassay for the ultrasensitive and rapid detection of salivary cortisol. Detection of cortisol is by competitive immunoassay using a secondary antibody for signal enhancement. The method requires no chemical extraction or complex sample pre-treatment despite high saliva viscosity. The cortisol assay was optimized for maximum sensitivity in buffer before being adapted for the salivary matrix, where it showed a limit of detection of 49 pg/mL. The results showed good correlation to RIA (r = 0.94). The biosensor assays showed an inter-assay coefficient of variation (CV) of 13.5% and recoveries close to 100%. The covalently immobilized sensor surface provided stable responses for more than 140 binding and regeneration cycles, enabling re-use. Cortisol in saliva was detected across the physiologically relevant range using the SPR immunobiosensor by employing a rationally designed assay format including signal enhancement for maximum sensitivity. The system can handle saliva matrix effects by use of chemical treatment during the assay to reduce non-specific binding to sensor surfaces. This sensor system provides an automated, high sensitivity analytical tool capable of yielding results in approximately 15 min. This biosensor could potentially be used for active stress-monitoring and in the diagnosis of disease.