Advanced Homogeneous−Heterogeneous Immunosensing Format Employing Restricted Access Supports

A rapid immunosensing methodology that employs the so-called homogeneous-heterogeneous assay mode is presented. The immunosensor is based on the homogeneous competition among the analyte, a fluorescent tracer, and the antibody, followed by separation of free and bound species by means of a restricted access alkyl-diol silica C18 reversed-phase chromatographic support. In order to develop a general labeling methodology, fluorescent tracers are synthesized from oligonucleotides covalently bound to the hapten in 3' position and the marker in 5'. The immunosensor principle is demonstrated by determining atrazine in a completely automated manner at 2 min/sample without regeneration of the support and a limit of detection of 1.0 microg/L with the optimized system. Preliminary assays employing multilabeled tracers indicate that sensitivity can be improved. Organic solvents 2-propanol and acetonitrile up to 15% (v/v) are well tolerated, while methanol can be added to 50%. The sensor capabilities are demonstrated through the analysis of natural waters.