Environ. Sci. Technol. 2000, 34, 4641-4650
On-Site Immunoanalysis of Nitrate and Nitroaromatic Compounds in Groundwater PAUL T. CHARLES,† PAUL R. GAUGER,‡ CHARLES H. PATTERSON JR.,† AND A N N E W . K U S T E R B E C K * ,† Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, D.C. 20375-5348, and Geo-Centers, Inc., 1801 Rockville Pike, Suite 405, Rockville, Maryland 20852
A miniaturized field-portable immunosensor (FAST 2000) was field tested at former military munitions sites for the detection and quantitation of TNT and RDX in groundwater. Developed by the Naval Research Laboratory (NRL) and engineered by Research International, Inc. (Woodinville, WA), the immunosensor performed on-site analysis of contaminated groundwater at three military bases identified by the U.S. Environmental Protection Agency (EPA) as priority Superfund cleanup sites. The locations were Naval Submarine Base, SUBASE Bangor, Bangor, WA; Umatilla Army Depot Activity, Umatilla, OR; and Naval Surface Weapons Center, Crane, IN. The immunosensor uses an antibody as the recognition element for the explosive molecule in a displacement assay format. Detection of the explosive molecule contained in groundwater is confirmed by an increase in fluorescence over background from the displaced cyanine-labeled explosive analogue. At each site, groundwater samples containing unknown concentrations of explosives were collected from monitoring wells and analyzed on-site by the portable immunosensor with no sample pretreatment or concentration. Groundwater samples were split and shipped to an independent certified U.S. EPA laboratory for analysis using SW-846 Method 8330 (HPLC). Statistical analysis based on linear regression curves comparing the immunosensor and the HPLC method showed good correlation, although site-dependent problems were encountered with some samples. The immunosensor required small sample volumes (150 µL/test) and less than 3 min to analyze samples with a method detection limit of 10 µg/L in the laboratory-spiked samples. The results from this field demonstration show the viability of the immunosensor for on-site analysis of explosives to assist in environmental remediation and monitoring efforts.
Introduction Subsurface contamination in groundwater and soil from environmental pollutants such as pesticides, heavy metals, and explosives continue to concern Federal and State environmental protection agencies. A list prepared by the U.S. Environmental Protection Agency (EPA) shows a number * Corresponding author phone: (202)404-6042; fax: (202)404-8897; e-mail:
[email protected]. † Naval Research Laboratory. ‡ Geo-Centers, Inc. 10.1021/es001099c CCC: $19.00 Published on Web 09/22/2000
2000 American Chemical Society
of Superfund sites that are former military-munitions manufacturing, storage, and demilitarized zones and are heavily saturated with high levels of explosive compounds. The most common explosives at these sites are 2,4,6trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and dinitrotoluene (DNT) as well as other nitrosubstituted breakdown products that result from microbial and photochemical degradation. Explosive compounds in the groundwater have presented an intriguing problem for environmental scientists and remediation work crews. Explosives are hydrophobic molecules that aggregate in the soil and have unpredictable migration patterns in the subsurface. Regional concentration mapping of explosive-laden soil and groundwater have shown highly contaminated regions often referred to as “ hot spots” or “hot zones” (1) miles away from the initial washout plant. Given this unpredictable nature of the explosives and fluctuations in the water table, complete characterization and cleanup would require sampling that could cover hundreds of acres resulting in more than 1000 samples. Sampling and cleanup of this magnitude has been estimated to take up to 30 years (2-4), which is far beyond anticipated plans of the EPA. However, the greatest concern to the EPA and the Department of Defense (DoD) was the potential spreading of the explosives and the toxic effects they pose to humans, animals, and agricultural produce on the periphery of these former munitions sites. As a result, imposed health advisory limits have been instituted through the Resource Conservation and Recovery Act (RCRA) and the Clean Water Act (5, 6) prompting cleanup and remediation efforts at the contaminated sites. In recent years, the EPA has placed precedence on research and development of analytical sensors that provide a method to characterize and monitor the presence of explosive contaminants in groundwater and soil. Progression of this research has produced sophisticated, accurate, and sensitive instrumentation with the capability to detect explosives at trace levels. However, environmental sampling at some locations has been a problem. The locations of the contaminated sites and groundwater monitoring wells were sometimes remote, which made transportation of samples to the laboratory or movement of instrumentation to the field difficult. As a result, research and development efforts have not only focused on trace level detection but also on design improvements to sensors for field portability in an attempt to provide on-site analysis in both an efficient and a cost-effective manner. Current methods to characterize and remediate explosives are often time-consuming and costly. Cleanup procedures to assist in these efforts employ a number of techniques [e.g., granular activated charcoal filtration systems (GAC), composting, natural attenuation (7), or biodegradation of the explosive with microbes (8-10)]. Since these methods are long term, additional analytical methods are required to confirm the concentration level of the remaining explosives. A number of sophisticated techniques have been utilized for explosive analysis [e.g., GC mass spectrometers (11, 12), amperometric gas sensors (1), high-performance liquid chromatography (HPLC)/liquid chromatography (13-17), and proton nuclear magnetic resonance (18)]. However, the most commonly used field systems are colorimetric or immunoassay kits (19-24). Immunoassay test kits can be convenient but often require timed reagent addition, involve multiple steps (24), and are not easily adapted to online monitoring. Colorimetric methods, which are commercially available, have similar limitations to immunoassay kits but VOL. 34, NO. 21, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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also require large quantities of solvents and disposable materials (21). Estimated cost per test for on-site test analysis range from $3 to $38 per test based on test kit chemical reagents, which is well below the cost for sample collection and sample shipment from the field site to the central laboratory (5, 23). The most frequently used laboratory analytical method for explosive analysis has been HPLC (25, 26). Current research in the field of immunotechnology has led to a number of other systems that employ biomolecules to detect and quantify explosives. These systems exist as fiber optic probes, which employ evanescent wave technology as a means to detect and quantify (27, 28) or immunosensors (biosensors) that employ biomolecules and fluorescence as a method of detection (29-35). Recent evidence has demonstrated limits of detection for TNT and RDX at femtamole levels using a microcapillary design (36-38). Research and development of immunosensor technology at the Naval Research Laboratory to detect explosives in groundwater and soil has progressed from a benchtop prototype immunosensor (39, 40) to the miniaturized fieldportable flow immunosensor (FAST 2000). Presented in this report is data collected from field trials conducted at three military installations that demonstrate the efficacy of the immunosensor for on-site analysis and characterization of areas contaminated with explosives. The three field trials were conducted at SUBASE Bangor (Bangor, WA), Umatilla Army Depot (Hermiston, OR), and Naval Surface Weapons Center (Crane, IN). Field samples were analyzed on-site by the immunosensor and shipped to an independent certified laboratory for analysis using U.S. EPA SW-846 Method 8330. Quantitative results from the field demonstration for TNT and RDX detection by the immunosensor were compared to the HPLC method for accuracy and precision.
Experimental Section Preparation of Antibody-Coated Membrane. Immobilization of the antibody-antigen dye complex for use in the immunosensor was performed on the Immunodyne ABC microporous membrane (Pall-Gellman Sciences). The microporous membrane contains a reactive moiety for covalent linkage of proteins to the nylon membrane. The membrane has a pore size of 5.0 µm that allows ease of fluid flow through the membrane. Immobilization of the monoclonal anti-TNT antibody (clone 11B3; custom synthesized Georgetown University) or the anti-RDX antibody (Strategic Diagnostics, Inc.) to the membrane was achieved through a series of steps. First, the membrane was cut with a hole punch to approximately 1 cm for a precise fit into the immunosensors disposable coupon. Each membrane is placed in a Petri dish, and the antibody (10 µL; 0.7 mg/mL) was applied in a solution of phosphate-buffered saline (PBS), pH 7.4. The antibodycoated membrane was allowed to incubate at room temperature for 1.5 h. After the 1.5-h incubation period, the antibody-coated membrane was placed in a casein (0.5%) solution for 1.0 h to block all unreacted sites on the membrane. The membrane was placed on a mechanical swivel platform to provide an even distribution of the casein solution. Upon completion of the casein blocking step, the membrane was washed three times (10 mL each wash for 5 min) with the following series of buffers: (i) PBS, pH 7.4; (ii) PBS-Tween 20 (0.01%); and (iii) PBS, pH 7.4. After the wash steps were completed, the membrane was placed in a dry Petri dish. Next, the fluorescent-labeled analogue (15 µL; 30 µM), cyaninecadaverine-trinitrobenzene (CY5-Cad-TNB) or cyaninecadaverine-RDX (CY5-Cad-RDX) (30), was applied to the respective antibody-coated membrane to saturate antibody binding sites. Each membrane was allowed to incubate for 1.0 h at room temperature before being stored at 4 °C overnight. Petri dishes containing the membranes were 4642
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wrapped in Parafilm and covered with foil until required for sample analysis. Immunosensor Operation. The immunosensor combines the displacement assay technology developed at NRL with micro-optics and micro-fluidics designed by Research International, Inc. in a field-portable unit. The key components of the immunosensor are (i) antibodies specific for the analyte, (ii) fluorescent signal molecules that are similar to the analyte but labeled with a fluorophore (a cyanine-5 dye) so they are highly visible to a fluorescence detector, and (iii) a fluorescence detector. The antibody was immobilized on an activated porous nylon membrane support, and the fluorophore-labeled signal molecule was bound to the antibody, creating an antibody/signal molecule complex. The derivatized antibody/fluorescent-labeled membrane was then placed in a disposable coupon that contains independent flow channels, a membrane and filter pocket in a removable plug, pneumatically controlled valves, and a septum seal area for injecting fluids into the coupon. The immunosensor was controlled in a PCMCIA-based PC application mode connected to a laptop computer. The laptop computer was a 486/80 MHZ, 16 MB of RAM unit for Windows 95 operation. A data acquisition card, DAQCard1200 (National Instruments, Inc.), was used to acquire data from the control unit. The optical source was a 5 mW solid 21-state diode laser, preset for 635 nm wavelength for excitation of the fluorescent analogue. The power requirement to operate the immunosensor was 300 mA at 5 V for an 8-h period. The computer supplied all power necessary to operate the sensor pumps, valves, and fluorometer. System parameters, assay conditions, and data analysis were set using a Windows-based custom software program. Prior to sample analysis, a double-bladder PVC [poly(vinyl chloride)] bag (200 mL capacity) was pressurized with an internal air pump. As the immunosensor reached operating pressure, valves were systematically controlled and opened to circulate the flow of buffer [sodium monophosphate (10 mM), ethanol (2.5%), and Tween 20 (0.01%)] through the channels and then to the antibody-fluorescent antigen membrane complex. Through the system software, immunoassays were performed by a sequence of valve controls that metered the assay fluid through the coupon and into the membrane. The buffer exited the membrane and traveled into the fluorometer, where an increase in fluorescence above background was recorded. Fluorescence signals result from the displacement of the fluorescence analogue from the antibody in the presence of explosive material (analyte) (schematic shown in Figure 1). Analyte concentration in the sample was determined by the system software, which compares fluorescence intensity of the unknown sample to that of a reference standard. An entire unit, including the computer, can be easily carried into the field in a computer carrying case. The immunosensor provides a rapid and convenient system for doing displacement assays, with a detection limit of approximately 10 µg/L or parts per billion (ppb) for explosives in laboratory-prepared flow buffer. Field Test Site Locations. Selection of the sites was based on several criteria: (i) contamination with explosives, (ii) accessibility to the site and the groundwater monitoring wells, and (iii) U.S. EPA interest (i.e., Superfund). Two of the three facilities (SUBASE Bangor and Umatilla) are currently undergoing extensive remediation for groundwater contamination with TNT and RDX using pump-and-treat technology. As a result, these sites provided a number of platforms for effective testing of the immunosensor: (a) direct measurement of contamination levels in monitoring wells, (b) analysis of samples from the treatment system (pre- and post-filtration), and (c) direct comparisons with current field and lab measurements using SW846 Method 8330.
FIGURE 1. Schematic of FAST 2000 immunosensor operation. Included is a diagram of the displacement assay format for TNT, where a sample containing explosive (TNT and/or RDX) flows through microchannels to a microporous membrane containing anti-TNT antibody and fluorescent TNT analogue. As the TNT molecule interacts with the antibody-fluorescence complex, the fluorescence analogue is displaced from the antibody and flows to the detector. Fluorescence intensity is measured, resulting in a peak profile and peak area units that are recorded and integrated. The sample is then directed to waste. Naval Submarine Base (SUBASE) Bangor is located northwest of Seattle, WA, and is currently the home port for Trident submarines. From 1942 to 1973, SUBASE Bangor was used as an ammunition depot. Two sites (sites A and F) on the base have been inactivated due to explosive contamination from ordnance that was disposed into an unlined lagoon. Currently this site is undergoing cleanup via a pump-andtreat method through granular activated charcoal filters. Sediment that accumulated at site F was also transported to site A for burning and disposal in a lined area. Water was flushed through the contaminated soil, collected as leachate, and processed through a granular activated charcoal (GAC) unit. The four major explosives identified are TNT, TNB, RDX, and HMX, ranging in concentration from 0 to 10 000 µg/L. Umatilla Army Depot Activity (UMDA) is located in eastern Oregon and is slated for closure. The base was established as an Army ordnance depot in 1941. From the 1950s until the mid-1960s, UMDA operated as an explosive washout facility to remove and recover explosives from munitions. The standard procedure at that time was to flush and drain the washout system into two unlined infiltration basins or lagoons. As a result, a 45-acre plume of RDX was identified in the shallow groundwater aquifer near the lagoons. Further investigation documented the presence of explosives in both soil and groundwater, ranging in concentration from 0 to 10 000 µg/L in the groundwater aquifer. These explosives included TNT, TNB, RDX, and HMX. Bioremediation of the soils from the lagoons is currently underway. Treatment of the groundwater also consists of pump-and-treat through granular activated charcoal filters, with re-injection of the polished water back into the aquifer. Naval Surface Weapons Center (NSWC), formerly known as Naval Ammunition Depot, located in Crane, IN, was primarily used to load, prepare, renovate, receive, store, and issue ammunition to the fleet. During its tenure, NSWC’s role included pyrotechnics production, mine filling, rocket assembly, torpedo storage, ordnance spare parts, and mobile equipment storage. Explosive contamination from these activities was located at three sites: (a) ammunition burning ground (ABG), (b) Rockeye, and (c) rifle range. Contamination was primarily due to the demilitarization and disposal of ammunition and pyrotechnics. Initially, solid explosive residues were spread out on burning pads or in flash pits and ignited. Today, clay-lined steel pans are employed. For the liquid explosive-contaminated material, three surface ponds were employed to remove the liquid from combustible sludge. In 1982, the ponds were modified to include a liner
and leachate collection system. Currently, sludge burn pads are used, and the ponds are closed. Leachate and runoff were initially stored in two underground tanks but have since been transferred to two aboveground tanks, resulting in the closure of the subsurface tanks. Demilitarization continues with more stringent requirements to prevent soil and water contamination. Groundwater Sample Collection. Groundwater samples were collected from monitoring and extraction wells at each location by facility managers or EPA-contracted personnel. Groundwater samples from the monitoring wells and the influent and effluent of the GAC units were analyzed at all three sites. Samples were collected into 20-L EPA-approved cleaned containers and sealed until on-site analysis or shipment to an independent laboratory. Groundwater samples were collected directly from all 10 of the extraction wells. In addition, groundwater samples were collected from the combined flow from the extraction wells at sampling ports before and after initial particulate filters, which are located upstream of the granular activated carbon (GAC) unit. After the samples were collected, they were refrigerated in EPA-approved amber bottles at 4 °C. Sample splits from the large sample container were used for the reference laboratory and field analysis. These aliquots (1 L for the reference laboratory and 40 mL for on-site analysis by the immunosensor) were stored in EPA-approved amber bottles at 4 °C. Because of TNT degradation in water, laboratory analysis for TNT was performed within 1 month of collection. Quantitation of Explosives in Groundwater. Analysis of groundwater samples containing explosives with the immunosensor was achieved by a set protocol. The first step required insertion of the coupon into the instrument followed by a computer command to start buffer circulation through the channels. Before assays were run, unbound fluorophorelabeled molecules were removed by the buffered flow stream until a stable background signal was reached (typically 1520 min). System flow buffer [phosphate buffer (10 mM) + Tween 20 (0.01%) + ethanol (2.5%)] was circulated through the network of valves and channels located in the coupon to the compartment containing the antibody-fluorescent analogue membrane. Groundwater samples (150 µL total volume) were injected into the sample port located on the coupon, and the fluorescence intensity was monitored. For each groundwater well, a total of seven injections was applied to the immunosensor. Prior to the seven injections of groundwater, a standard solution of known explosive concentration was injected into the immunosensor, and the VOL. 34, NO. 21, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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fluorescence signal response was measured. The fluorescence signal response from the standard solution was directly compared to the signal response of the groundwater sample to quantify the explosive concentration. The background fluorescence signal also served as a threshold for membrane replacement if the fluorescence signal was below a preset limit (50 pA). A positive response for the presence of explosives was indicated by an increase in fluorescence intensity over background. The magnitude of the fluorescence signal response was measured by the immunosensor within 2-5 min/sample. Results obtained from the analysis of groundwater samples by the immunosensor were then compared to the HPLC SW-846 Method 8330. Laboratory Validation/Confirmation. For validation of results, the EPA-approved method for explosive analysis in groundwater, EPA SW-846 Method 8330, was used. This method employs HPLC and a UV detector to determine explosive concentrations. Groundwater samples containing
