Detection and Quantitation of the Explosives 2,4,6-Trinitrotoluene and

Chapter 17

Downloaded by STANFORD UNIV GREEN LIBR on October 6, 2012 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1997-0657.ch017

Detection and Quantitation of the Explosives 2,4,6-Trinitrotoluene and Hexahydro-1,3,5trinitro-1,3,5-triazine in Groundwater Using a Continuous Flow Immunosensor 1

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John C. Bart , Linda L. Judd , Karen E. Hoffman , Angela M . Wilkins , Paul T. Charles , and Anne W. Kusterbeck 1

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Naval Research Laboratory, Code 6910, U.S. Department of the Navy, 4555 Overlook Avenue, Southwest, Washington, DC 20375 Geo-Centers Inc., 10903 Indian Head Highway, Fort Washington, MD 20744 2

Detection of contaminants in a fast, accurate, and cost-effective manner is the first step in the process of environmental clean-up of hazardous wastes. Processes which utilize the high degree of selectivity for a single analyte afforded by antibodies are called immunoassays, and are currently becoming accepted as alternatives to more traditional instrumental analytical techniques in environmental testing. One such immunoassay is described here, and data from two on-site field tests of the sensor are compared to the results obtained by high-performance liquid chromatography analysis. The accuracy and reliability of this sensor is clearly demonstrated.

The U.S. Department of Defense currently owns over 50 military sites which are on the U.S. Environmental Protection Agency's (EPA) Superfiind list, many of which are contaminated with explosive compounds. In recent years, immunoassays have become accepted as serious alternatives to more traditional instrumental methods of analysis in the field of environmental monitoring and remediation because they offer advantages in portability, cost per sample, and sample processing time (1). In the specific realm of monitoring explosives in contaminated water or soil samples, the EPA-approved technique (SW-846 Method 8330) is high-performance liquid chromatography (HPLC) coupled with ultraviolet (254 nm) detection (2). Recently, the E P A has been

© 1997 American Chemical Society In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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investigating various immunoassays as alternatives to HPLC for the on-site analysis of samples. Hand-held test kits or portable instruments are potentially superior to the standard HPLC test in that they can be used by non-technical personnel on-site to get accurate results in minutes at a fraction of the cost of off-site analysis. In the scenario where a large number of samples must be tested (to determine the extent of contamination at a polluted site, for example), an immunoassay performed on-site could easily afford savings of one order of magnitude in both time and money. During the summer of 1995, two separate tests of commercially available kits (both immunoassays and non-immunoassays) and prototypes of explosives detectors were performed at EPAmonitored sites to determine the accuracy of such on-site analyzers compared to HPLC. Site Characteristics for the Field Trials Field tests of the assay kits and prototypes were conducted at two military bases containing sites currently undergoing EPA-monitored remediation of groundwater and soil contamination by various explosive compounds. The health advisory values for 2,4,6-trinitrotoluene (TNT) and hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX) as determined by the EPA are 2 ppb (3,4), though most analytical techniques currently have limits of detection about an order of magnitude higher. TNT levels at these sites had a mean concentration of 915 ppb with a maximum of 3630 ppb. R D X levels had a mean concentration of 1132 ppb and a maximum value of4580 ppb. Umatilla Army Weapons Depot located outside of Hermiston, Oregon was the first site where analyses were performed. This base is situated in the arid northeast section of the state, where the soil is very sandy and dry during the summer months and contains relatively high levels (132 ppm) of inorganic nitrate salts. Operations at this location, dating back to the Second World War, included the dismantling of outdated or damaged munitions. Explosive charges were removed from the various weapons and collected for mass burnings on the open desert floor. This practice led to acres of soil becoming highly contaminated; currently these regions are undergoing composting treatment to remove the explosives from the soil. The empty shell casings were steam cleaned so that the metal could be recycled; a procedure which produced large quantities of explosive-contaminated water which was dumped into shallow, unlined waste lagoons. Over the past five decades, the toxic waste in these lagoons has not remained confined to the topsoil, but has leached into the groundwater aquifer which lies about 90 feet below the surface. While several explosives and their decomposition products are found at this site, the contamination is predominately caused by TNT and RDX. R D X is the more water soluble of the two explosives and can be found in a 45 acre plume in the groundwater. TNT, on the other hand, is less water soluble and is thus confined to a somewhat smaller contamination zone. Since the depot is bordered by several large farms which rely on wells for crop irrigation, remediation of the groundwater aquifer is of the utmost importance. Many monitoring wells have already been drilled throughout the contaminated area and it was from these wells that the samples which were analyzed by the field-portable sensors and test kits were taken. The second site where the sensors were tested was Naval Submarine Base Bangor, another former weapons demilitarization site which is located outside of Seattle, Washington. In contrast to the rather sparse terrain at Umatilla, SUBASE Bangor is

In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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IMMUNOCHEMICAL TECHNOLOGY FOR ENVIRONMENTAL APPLICATIONS

Phosphate buffer (0.01 M, pH = 7.4) + Tween 20 (0.01%) + Ethanol (2.5%)

WASTE

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Flow

Antibody-Antigen* Microcolumn

Stream

Fluorometer

wmmmm

I I I Signal

Output

Signal Integrator and Disk Drive

Figure 1. Components of the Continuous Flow Immunosensor (CFI).

CH

CH

3

3

.S0 pyH

0,S>

3

-CH N CH

H

3

3

C -

^

N CH 1 CH

2

1 CH

2

CH 1 CH | CH 1 o=c

2

3

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2l

0 N 2

N — (CH ) -N 2

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-NOo

0 N 9

Figure 2. Structure of the sulfoindocyanine dye-labeled TNT analog.


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Detection and Quantitation of TNT and RDX

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situated in the forests of northwest Washington. This location provided the opportunity to test the devices' capabilities to accurately measure the amount of explosives in aqueous samples which contained significant quantities of humic material. Samples from Bangor also tended to have significantly higher concentrations of 1,3,5trinitrobenzene (TNB), a photodecomposition product of TNT, and octahydro-1,3,5,7tetranitro-l,3,5,7-tetrazocine (HMX), a by-product of the synthesis of R D X and an explosive which is occasionally mixed with TNT or R D X to form plastic explosives (5). Contaminated soil has been isolated in plastic-lined pits and is currently being flushed of explosives with large quantities of water. This soil leachate solution is then processed in granular activated carbon (GAC) units. Groundwater which contains explosives is treated by pumping the water up from the aquifer, processing it in a G A C unit, and then returning it to the aquifer. Samples analyzed during this trial included groundwater, soil leachates, and post-treatment water samples. Operation of the Continuous Flow Immunosensor Work at the Naval Research Laboratory (NRL) has focused on trying to develop an automated sensor which couples the inherent high degree of selectivity provided by antibodies with the excellent sensitivity of fluorescence spectroscopy. The result is the Continuous Flow Immunosensor (CFI), a device which is capable of detecting explosives at low parts per billion (ppb) levels in aqueous samples (6). A pictorial representation of the CFI is shown in Figure 1. Although the sensor which was used to collect all of the data presented here was the laboratory device (occupying approx. 0.5 m of tabletop space) and not the more portable sensor currently under development, it could be completely unpacked and assembled in less than one hour. The microcolumn contains 100 uL of polyacrylamide beads (Emphaze AB1, 3M) to which monoclonal antibodies against the explosive of interest have been covalently attached. The beads average 60 urn in diameter and contain an azlactone group which is capable of forming a stable amide bond with any primary amine group on the antibody. The 11B3 strain of monoclonal anti-TNT antibody was generated specifically for use at N R L (6) and is not commercially available. The 50518 strain of monoclonal anti-RDX antibody was purchased from Strategic Diagnostics, Inc. (Newark, DE). Screening assays using standard ELISA (enzyme-linked immunosorbant assay) techniques were performed to determine the relative binding affinities of the monoclonal antibodies for labeled and unlabeled antigens prior to assay development. Crossreactivities were determined using the CFI to test a panel of explosives. 2

Once the antibodies are immobilized on the surface of the support, a solution of dye-labeled antigen (which we synthesized) is added and allowed to fill the available binding sites on the antibodies. An example of the dye-labeled antigen for TNT is shown in Figure 2. The particular sulfoindocyanine dye used to label the antigens was chosen because of its adventitious physical properties. Its multiple charges make for excellent solubility in aqueous solutions, and its large (2.5 x 10 M " cm ) extinction coefficient and high (>0.28) quantum yield ensure that even an extremely low concentration of the dye can be detected byfluorescencespectroscopy. Most importantly, this compound has an absorbance maximum at 650 nm and fluoresces at 667 nm, a region of the visible spectrum where there is virtually no interference from naturally-occurring species which 5

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IMMUNOCHEMICAL TECHNOLOGY FOR ENVIRONMENTAL APPLICATIONS

might be present in environmental samples. This provides the low fluorescence background needed to get a limit of detection in the low ppb concentration range. In order to prevent the relatively hydrophobic explosive compounds from aggregating or adsorbing to the walls of the tubing in the sensor, the solvent used in the CFI is a pH 7.4 phosphate buffer (0.01 M) to which has been added an organic cosolvent (2.5% v/v ethanol) and a surfactant (0.01% v/v Tween 20, Aldrich). As the buffer sweeps the sample into the column, the analyte of interest can come in close proximity to the antibody binding sites and there is the possibility that a dye-labeled antigen molecule will be displaced in favor of binding the native explosive. Thus the effluent from the column is a mixture of non-fluorescent analyte which did not bind to the antibodies within the column and fluorescent antigen which was replaced by the analyte present in the sample. Unlike other immunoassays, the best antibody for use in the CFI is not the one with the highest affinity for its antigen, rather it is the one which has the optimal binding coefficient. If the dissociation constant, k , is too large, the dye-labeled antigen can dissociate before being replaced by the analyte, thus increasing the background fluorescence level and, consequently, the limit of detection while shortening the column's useful lifetime. On the other hand, if k is too small, the analyte will have difficulty displacing the fluorescent antigen and the signal from the fluorometer will therefore be quite small. This property can be controlled two ways: via the strain of antibody used, but also to a lesser extent by the structure of the dye-labeled antigen. We have found that varying the length of the alkyl chain spacer between the fluorescent dye and the explosive analog has a significant effect on the affinity of the antibody for this compound. Detection of the dye-labeled species is achieved using a fluorometer (Jasco 821FP, equipped with a 16 uL flow cell) which is located downstream from the column. The species in the flow cell are excited at 632 nm while the fluorescence is recorded at 663 nm. When the analyte of interest is not present in the sample, the signal from the fluorometer vs. time is a flat line, indicating only the background fluorescence was detected. On the other hand, if the explosive being monitored is present in the sample aliquot, a peak in the fluorescence vs. time trace will be observed. There is a direct correlation between the peak area and the amount of analyte present in the sample. Each run was then stored on a 3.5" floppy disk for future reference. Unlike other commercially-available assays which achieve ppb sensitivity only via the concentration of liters of sample down to a few milliliters, the CFI is able to analyze the samples directly, which saves processing time and reduces the amount of sample required for analysis from several liters to less than 1 mL. d

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Sample Handling and Analysis. A l l groundwater samples were analyzed on-site shortly (

Detection and Quantitation of the Explosives 2,4,6-Trinitrotoluene and

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