Electrochemiluminescence of luminol for 2,4-D optical immunosensing in a flow injection analysis system

Abstract

Luminol-labelled antibodies have been prepared using glutaraldehyde as a cross-linking agent and used in a 2,4-dichlorophenoxyacetic acid (2,4-D) competitive electrochemiluminescent immunosensor. 2,4-D was covalently immobilized at a glassy carbon electrode surface, via a six-carbon spacer arm, by a novel procedure which allowed us to obtain stable immobilized antigens that could be then stored dry, used and regenerated 50 times without loss of binding capacity. The luminol electrochemiluminescence detection was performed in a flow injection analysis system (FIA). The optimum conditions were found to be an oxidation potential of +500 mV versus a platinum pseudo-reference electrode, in the presence of 600 μM H 2O 2. In these conditions, luminol could be detected in the range 5.5 fmol–55 nmol. Luminol-labelled anti-2,4-D antibodies or peroxidase-labelled secondary antibodies were tested for the 2,4-D immunodetection. With both the corresponding electrochemiluminescent and chemiluminescent immunoassays it was possible to detect 0.2 μg l −1 of free 2,4-D. The overall time of experiment was 50 min and a linear range from 0.2 μg l −1 to 200 mg l −1 was obtained with the peroxidase format whereas the range was 0.2–200 μg l −1 with the luminol format.