Two-photon fluorescent immunosensor for androgenic hormones using resonant grating waveguide structures

An immunosensor approach based on non-linear two-photon fluorescence spectroscopy (TPF) coupled to a resonant double grating waveguide structure (DGWS) has been used for the detection of methylboldenone (MB), an androgenic anabolic steroid used illegally as growth promoter. This synthetic steroid is detected by the biosensor down to 0.1 μg l−1, two orders of magnitude lower than the minimum required performance limit (MRPL) required by the World Anti-Doping Agency (WADA). Nevertheless, we have focused this work on the synthesis and characterization of small fluorescent-labeled conjugates, as well as on the modification of the waveguide surfaces. Detection relies on a direct competitive format, using a boldenone–rhodamine conjugate as fluorescent competitor and a specific anti-MB antibody. The immunoreagents are immobilized onto a resonant Ta2O5 sensing chip after being activated with phosphonohexanoic acid spacers, a novel methodology for covalently surface immobilization of biomolecules, which shows better stability and homogeneity than classical silane chemistries. The developed immunosensor presents great potential as a robust device for its implementation on the detection of small illegal and toxic contaminants.