Abstract: A continuous-flow immunoassay for the plastic explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was developed using anti-RDX antibodies and a sulfoindocyanine dye-labelled analog of RDX. Anti-RDX antibodies were immobilized onto Emphaze beads (50-80 µm diameter) and 100 µL of the coated beads was transferred to a microcolumn (2 cm x 5 mm i.d.). The column was incorporated into a FIA system consisting of a pump, 100 µL injection loop and a fluorimetric detector. A 100 µL 2.5-5 µM-RDX-dye conjugate in PBS was loaded onto the column and incubated overnight at room temperature to allow the dye-labelled RDX to bind with the immobilized antibody. The column was then washed with a carrier stream (2 ml/min) containing 12% ethanol/0.1% Triton X-100 in PBS to remove unbound dye and produce a stable baseline. Solutions containing RDX (samples or standards) were then injected into the carrier stream. The displacement of the RDX-dye conjugate with RDX was monitored by measuring the change in fluorescence at 663 nm (excitation at 632 nm). The calibration graph for 15-1200 ng/ml RDX is presented. The linear range was 18-300 ng/ml. Fifty analyzes were performed with each column.

Abstract: Groundwater was mixed with phosphate buffer of pH 7.4, ethanol and surfactant and analyzed by continuous-flow immunosensors CFI), combining the highly selective antibody-antigen binding interaction with the sensitivity of fluorescence spectroscopy as the signal generation mechanism. The CFI was a semi-automated system in which the groundwater was pumped through a column containing antibodies raised against the analyte which were covalently attached to small plastic beads. As the analyte passed through the antibody matrix, some of the fluorescent dye-labelled explosive analogue was displaced from the antibody in favour of binding the analyte. The two most commonly used military explosives: 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) were detected by the method and quantified fluorimetrically. Field testing of the immunosensor proved its usefulness as a faster, less expensive and portable alternative to HPLC.