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

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-μm diameter streptavidin-coated magnetic beads (MB) and 10-μm diameter avidin-coated polystyrene microspheres/beads (PSB) pre-loaded with ECL labels (∼7 billion hydrophobic ruthenium(II) tris(2,2′-bipyridine) (RuII) molecules per bead) to form anti-TNT ↔ MB and anti-TNT ↔ PSB(RuII) conjugates, respectively. Sandwich-type PSB(RuII) ↔ anti-TNT < TNT > anti-TNT ↔ MB aggregates were formed when PSB(RuII) ↔ 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–0.10 M 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 0.10–1000 ppt (pg mL−1). The limit of detection (≤0.10 ± 0.01 ppb) is about 600× lower as compared with the most sensitive TNT detection method in the literature, and the absolute detection limit in mass (∼0.1 pg) is only ∼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.