Ultrasensitive detection of testosterone using conjugate linker technology in a nanoparticle-enhanced surface plasmon resonance biosensor.

A rationally designed oligoethylene glycol linker conjugate to testosterone was synthesised and covalently immobilized on a surface plasmon resonance (SPR) biosensor surface. The sensing surface was stable for more than 330 binding and regeneration cycles allowing a high degree of re-use. This surface was then used in the development of an ultrasensitive immunobiosensor system for testosterone in buffer utilizing both secondary antibody and gold nanoparticle signal enhancement. The mechanism for the increased sensitivity results from increased binding mass and a gold plasmon coupling effect. The addition of a secondary antibody with an attached gold nanoparticle increased the signal sensitivity of the assay 12.5-fold compared with primary antibody alone. In the enhanced format the assay had limits of detection (LOD) of 3.7 pgml(-1) with standard in running buffer, and 15.4 pgml(-1) in a stripped human saliva matrix. This immunobiosensor system has sufficient sensitivity to measure testosterone across the broad physiologically relevant range in male saliva (29-290 pgml(-1)) in under 13 min allowing monitoring of testosterone in near real-time.