An Improved Method for TNT Analysis in Ground Water, Seawater, and Soil

E.R. Goldman, G.P. Anderson, and J.M. Mauro
Center for Bio/Molecular Science and Engineering

Introduction: The Navy is interested in TNT (2,4,6-trinitritoluene) detection for monitoring the clean up of soil and ground water at former munitions manufacturing and storage facilities. On-site test methods are useful for assessing the nature and extent of contamination as well as for monitoring clean up processes. Current on-site tests involve multiple steps and can require up to 2 hours to complete. We have invented a rapid, simple, and sensitive test method that is amenable to high throughput screening for the presence of TNT in water and soil samples.

The new assay was made possible by our discovery that binding a fluorescent TNT analog to an anti-TNT antibody increases the fluorescence emission of the analog. Free TNT added to a solution containing the antibody-bound fluorescent TNT analog competes with the analog for binding to the antibody. When fluorescent TNT analog releases from the antibody as TNT binds in its place, fluorescent emission from the sample decreases (Fig. 10), providing the basis for the analytical method we have developed. As more TNT is added, the signal decreases until TNT has replaced all the fluorescent TNT analog.

FIGURE 10
Fluorescent emission decreases as the antibody binds TNT in place of the fluorescent TNT analog.

The Assay: We have developed two configurations of the new TNT assay: a solution-based assay format in which all reagents are in solution, and a solid-phase assay format in which the anti-TNT antibody is immobilized on a surface.¹ We routinely perform these assays in 96-well microtiter plates, allowing us to simultaneously process many samples.

The solution-based assay format has the advantage of having no washing or incubation steps. We add the antibody and fluorescent TNT analog to wells, then add the TNT-containing liquid sample to each test well, and read the fluorescence output in a standard fluorescent microtiter plate reader. A 96-well microtiter plate filled with samples can be processed in approximately 5 minutes. We have been able to detect TNT at a level of 0.5 ng/ml (0.5 ppb) in laboratory buffer (Fig. 11). TNT quantitation can be achieved by constructing standard curves in the appropriate concentration range using TNT standards of known concentration.

In the solid-phase assay format, anti-TNT antibody is pre-bound by adsorption within the wells of polystyrene microtiter plates and then loaded with the fluorescent TNT analog. Excess unbound fluorescent analog is washed away, TNT-containing samples are added to the wells, and fluorescence output is measured for each sample. This format allows us to detect TNT down to a level of 0.5 ng/ml (0.5 ppb) in buffer (Fig. 11). This analysis format requires more setup than the solution-based assay. Antibody-coated plates are prepared a day before use, and about 15 minutes are needed to saturate the immobilized antibody with fluorescent TNT analog. We believe it should be possible to prepare and store plates loaded with antibody and fluorescent TNT analog to allow an assay time of less than 5 minutes per 96-well plate.

The limits of detection we have obtained using these assay formats are competitive with other on-site test methods for monitoring TNT in water. Comparing six on-site analysis methods for detecting explosives in water, only the RaPID Assay Kit had a lower reported level of detection (0.07 ppb TNT in water samples); however, it takes approximately 70 minutes to run 51 samples using the RaPID method.² In comparison, the solution-based format of the new assay can perform triplicate analysis of 51 samples in less than 10 minutes.

Our new test method also performs well for analyzing TNT present in artificial seawater. In this case, we can detect TNT at levels of 0.5 ng/ml (0.5 ppb) using the solution-based method and 0.05 ng/ml (0.05 ppb) with the solid-phase format (Fig. 11). This is an important finding, as some methods for detection of TNT do not work well in high saltwater concentrations like seawater, and often require additional dilution or extraction steps. Finally, we have tested methods for extracting TNT from contaminated soil samples followed by rapid screening or quantitative analysis using the new assay.

Conclusions: Based on our observation that binding of a fluorescent TNT analog to an anti-TNT antibody increases the fluorescence emission of the TNT analog, we have developed a test method that has great potential for use in efficient on-site detection and quantitation of TNT levels in ground water and soil. The formats we have tested are simple, rapid, and should be readily adaptable for high-throughput, parallel sample processing and automation. Our next goal is to use the new methodology to test for TNT in the field and to evaluate its sensitivity and reliability directly compared to other available detection and standard high-performance liquid chromatography-based methods.

[Sponsored by ONR]

References

¹ E.R. Goldman, G.P. Anderson, N. Lebedev, B.M. Lingerfelt, P.T. Winter, C.H. Patterson, and J.M. Mauro, "Analysis of Aqueous 2,4,6-Trinitrotoluene (TNT) Using a Fluorescent Displacement Immunoassay," Anal. Bioanal. Chem, in press.
² A.B. Crockett, H.D. Craig, and T.F. Jenkins, "Field Sampling and Selecting On-Site Analytical Methods for Explosives in Water," EPA/600/S-99/002; http://www.epa.gov/swertio1/tsp/download/water.pdf.