Ultrasensitive detection of TNT in soil, water, using enhanced electrogenerated chemiluminescence

Tommie L. Pittman, University of Southern Mississippi
Bethtrice Thomson, University of Southern Mississippi
Wujian Miao, University of Southern Mississippi


The ultrasensitive detection of 2,4,6-trinitrotoluene (TNT) was accomplished on the basis of sandwich-type TNT immunoassay combined with electrogenerated chemiluminescence (ECL) technology. Biotinylated anti-TNT species were attached to the surface of 1-mu m diameter streptavidin-coated magnetic beads (MB) and 10-mu m diameter avidin-coated polystyrene microspheres/beads (PSB) preloaded with ECL labels (similar to 7 billion hydrophobic ruthenium(II) tris(2,2'-bipyridine) (Ru(II)) molecules per bead) to form anti-TNT <-> MB and anti-TNT <-> PSB(Ru(II)) conjugates, respectively. Sandwich-type PSB(Ru(II)) <-> anti-TNT < TNT > anti-TNT <-> MB aggregates were formed when PSB(Ru(II)) <-> anti-TNT was mixed with anti-TNT <-> MB conjugates in the presence of analyte TNT and 2.0% bovine serum albumin blocking agent. The newly formed aggregates were magnetically separated from the aqueous reaction media and dissolved in acetonitrile containing 0.10 M tri-n-propylamine ECL coreactant-0.055 M trifluoroacetic acid-0.10M tetrabutylammonium tetrafluoroborate electrolyte. ECL as well as cyclic voltammetric measurements were carried out with a potential scan from 0 to 2.8 V vs Ag/Ag(+), and the integrated ECL intensity was found to be linearly proportional to the analyte TNT concentration over the range of 0.10-1000ppt (pgmL(-1)). The limit of detection (<= 0.10 +/- 0.01 ppb) is about 600x lower as compared with the most sensitive TNT detection method in the literature, and the absolute detection limit in mass (similar to 0.1 pg) is only similar to 0.5% of that from mass spectroscopy. The approach coupled with the standard addition method was applied to measure the TNT contaminations in soil and creek water samples collected from a military training base. (C) 2008 Elsevier B.V. All rights reserved.