Regenerable immunobiosensor for the chemiluminescent flow injection analysis of the herbicide 2,4-D

A semi-automated chemiluminescent competitive immunosensor for the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is presented. Anti-2,4-D polyclonal antibodies are directly labelled with horseradish peroxidase allowing a p-iodophenol enhanced chemiluminescent detection. Using antigen immobilised on UltraBind type pre-activated membranes, the 2,4-D immunosensor exhibits low non-specific/specific binding ratio (maximum ratio: 5%) of the labelled antibodies. The quantification of free 2,4-D in water is performed by co-injecting the sample and the labelled antibodies in the flow system, incubating this solution with the antigen immobilised membrane and measuring the amount of specifically bound labelled antibodies. Such an analytical system enables the detection of 4 μg l−1 of free antigen in 20 min, and the 2,4-D detection is possible in the range 4 μg l−1–160 mg l−1. The immunosensor can be regenerated by simply flowing a chaotropic solution (0.1 M HCl, 0.1 M NaCl, 0.1 M glycine) in the system. This regeneration ability enables the achievement of more than 30 measurement cycles of free 2,4-D with the same antigen immobilised membrane with a good reproducibility (RSD=12.5%).