Homogeneous Immunoassay for Detection of TNT and Its Analogues on a Microfabricated Capillary Electrophoresis Chip

Avraham Bromberg† and Richard A. Mathies*
Department of Chemistry, University of California, Berkeley, California 94720
Anal. Chem., 2003, 75 (5), pp 1188–1195
DOI: 10.1021/ac020599g
Publication Date (Web): January 24, 2003
Copyright © 2003 American Chemical Society
†

 Permanent address:  Department of Physical Chemistry, IIBR, P.O. Box 19, Ness-Ziona 74100, Israel.

,
*

 Corresponding author. Phone:  (510) 642-4192. Fax:  (510) 642−3599. E-mail:  rich@zinc.cchem.berkeley.edu.

Abstract

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) × 109 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) × 107 M-1 s-1. Binding constants of TNT and five of its analogues are determined by competitive assays:  TNT (4.3 ± 2.6) × 108 M-1; 1,3,5-trinitrobenzene (5.1 ± 3.3) × 107 M-1; picric acid (7.5 ± 4.4) × 106 M-1; 2,4-dinitrotoluene (7.9 ± 4.0) × 106 M-1; 1,3-dinitrobenzene (1.0 ± 0.7) × 106 M-1; and 2,4-dinitrophenol (5.1 ± 3.0) × 104 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.

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History

  • Published In Issue March 01, 2003
  • Received for review September 30, 2002. Accepted December 13, 2002.

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