Surface plasmon resonance (SPR) biosensors utilize refractive index changes to sensitively detect mass changes at noble metal sensor surface interfaces. As such, they have been extensively applied to immunoassays of large molecules, where their high mass and use of sandwich immunoassay formats can result in excellent sensitivity. Small molecule immunosensing using SPR is more challenging. It requires antibodies or high-mass or noble metal labels to provide the required signal for ultrasensitive assays. Also, it can suffer from steric hindrance between the small antigen and large antibodies. However, new studies are increasingly meeting these and other challenges to offer highly sensitive small molecule immunosensor technologies through careful consideration of sensor interface design and signal enhancement. This review examines the application of SPR transduction technologies to small molecule immunoassays directed to different classes of small molecule antigens, including the steroid hormones, toxins, drugs and explosives residues. Also considered are the matrix effects resulting from measurement in chemically complex samples, the construction of stable sensor surfaces and the development of multiplexed assays capable of detecting several compounds at once. Assay design approaches are discussed and related to the sensitivities obtained.

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.