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.
Bromberg
Avraham
A
Department of Chemistry, University of California, Berkeley, California 94720, USA.
Mathies
Richard A
RA
D000465:Algorithms; D019075:Electrophoresis, Capillary; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes; D007118:Immunoassay; D007202:Indicators and Reagents; D008838:Microcomputers; D014303:Trinitrotoluene / Q000031:analogs & derivatives / Q000032:analysis*
D005452:Fluoresceins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D014303:Trinitrotoluene