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, fluoresceinlabeled TNT, and unlabeled TNT or its analogue. The band intensities of the free fluorescein-labeled TNT and of the antibodyantigen 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) × 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) × 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) × 10^8 M^-1; 1,3,5-trinitrobenzene (5.1 +- 3.3) × 10^7 M^-1; picric acid (7.5 +- 4.4) × 10^6 M^-1; 2,4dinitrotoluene (7.9 +- 4.0) × 10^6 M-1; 1,3-dinitrobenzene (1.0 +- 0.7) × 10^6 M^-1; and 2,4-dinitrophenol (5.1 +- 3.0) × 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.