Development of a non-labeled immunosensor for the herbicide trifluralin via optical waveguide lightmode spectroscopic detection

A highly sensitive immunosensor using optical waveguide lightmode spectroscopy (OWLS) was developed for the detection of the herbicide trifluralin. OWLS as an in situ and label free method of detection, based on the measurement of the diffraction of a linearly polarized laser beam (He-Ne laser, 632.8 nm) on a diffraction grating in a thin waveguide layer (SiO₂-TiO₂), offered means to produce immunosensors utilizing immobilized antibodies raised against trifluralin allowing a non-competitive biosensor, or immobilized trifluralin conjugate allowing a competitive biosensor for this analyte. Immobilization of molecules sensitizing the sensor was undertaken on amino silanized waveguide surfaces in a two-step procedure using glutaraldehyde. Within the immobilized antibody (Ab) based immunosensor the signal measured was proportional to the trifluralin content in the samples, but the method allowed detection of trifluralin only above 100 ng ml⁻¹ due to the small molecular size of the antigen (Ag). In the immobilized antigen based immunosensor, a trifluralin-bovine serum albumin (BSA) conjugate was covalently linked to the waveguide surface. During measurements the standard solutions and samples were mixed in 1:1 ratio with antiserum, containing constant amounts of antibodies. The amount of free antibodies bound to the surface was inversely proportional to the trifluralin content of the solutions measured. The immobilized antigen based method allowed detection of trifluralin in the concentration range of 2×10⁻⁷ to 3×10⁻⁵ ng ml⁻¹. Results of trifluralin determinations were compared to those obtained in parallel enzyme-linked immunosorbent assay (ELISA) tests and in gas chromatorgraphic-mass spectrometric (GC-MS) analyses, and indicated an increase of six orders of magnitude in the limit of detection (LOD).