Homogeneous immunoassay for detection of TNT and its analogues on a microfabricated capillary electrophoresis chip

A homogeneous immunoassay for TNT and its analogues is developed using a microfabricated capillary electrophoresis chip. The assay is based on the rapid electrophoretic separation of an equilibrated mixture of an anti-TNT antibody, fluorescein-labeled TNT, and unlabeled TNT or its analogue. The band intensities of the free fluorescein-labeled TNT and of the antibody-antigen complex reveal the relative equilibrated concentrations. Titration of the anti-TNT antibody with a fluorescein-labeled TNT derivative yields a binding constant of (3.9 +/- 1.3) x 10(9) M-1. The dissociation rate constant of the complex is determined by kinetic capillary electrophoresis using a folded channel and a rotary scanner to interrogate the separation at multiple time points. The dissociation rate constant is found to be 0.035 +/- 0.005 s(-1), and the resulting binding rate constant is (1.4 +/- 0.7) x 10(7) M-1 s(-1). Binding constants of TNT and five of its analogues are determined by competitive assays: TNT (4.3 +/- 2.6) x 10(8) M-1; 1,3,5-trinitrobenzene (5.1 +/- 3.3) x 10(7) M-1; picric acid (7.5 +/- 4.4) x 10(6) M-1; 2,4-dinitrotoluene (7.9 +/- 4.0) x 10(6) M-1; 1,3-dinitrobenzene (1.0 +/- 0.7) x 10(6) M-1; and 2,4-dinitrophenol (5.1 +/- 3.0) x 10(4) M-1. TNT and its analogues can be assayed with high sensitivity (LOD 1 ng/mL) and with a wide dynamic range (1-300 ng/mL) using this chip-based method.