Immunochemical Technology for Environmental Applications

Chapter 16

Downloaded by NORTH CAROLINA STATE UNIV on October 4, 2012 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1997-0657.ch016

Transition from Laboratory to On-Site Environmental Monitoring of 2,4,6Trinitrotoluene Using a Portable Fiber Optic Biosensor 1

2

2

Brian L. Donner , Lisa C. Shriver-Lake , Alfonza McCollum Jr. , and Frances S. Ligler 2,3

1

Geo-Centers

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

2

The detection of the explosive TNT using a fiber optic biosensor has successfully undergone the transition from laboratory analysis to on -site, real time sample monitoring due to advances in miniaturization and portability. A larger "breadboard" system capable of monitoring a single probe was replaced by a lightweight, portable sensor that can monitor 4 optical probes simultaneously. This new fiber optic biosensor was taken to two contaminated military bases for on-site testing for TNT in groundwater. Prior to on-site testing, assay variables, including buffers and fluorescence analog concentration, were optimized to permit TNT detection in the 5-20μg/Lrange. In addition, several cross reactivity studies were performed demonstrating interference from TNB, a degradation product of TNT but not the explosive R D X or its degradation product, H M X . A fiber optic biosensor, which employs antibodies as the recognition element, has been developed (7,2). Antibodies have been immobilized on the surface of a fiber optic probe in order to detect various analytes (proteins, explosives, bacteria). A competitive immunoassay has been developed for the explosive 2,4,6trinitrotoluene (TNT)(3). Due to problems from munition storage and/or ordnance demilitarization, high levels of TNT have been found in soil at several military bases. The mobility of TNT in soil has permitted these explosives in the soil to migrate into the groundwater (4). As TNT and several of its degradation products are toxic, clean-up and monitoring are essential, especially in these days of base closure (5). On-site monitoring would greatly reduce time and cost involved with remediation relative to the current method which involves analysis off-site in a laboratory. Transitioning the fiber optic biosensor from the laboratory to the field was envisioned. •^Corresponding author © 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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The TNT assay was initially performed using a fiber optic biosensor which weighed 150 pounds with dimensions 2' x 3', and for all intents and purposes, limited the operator to the confines of a laboratory (Figure 1, top). This sensor monitored a single 200 um fused silica optical fiber laden with antibodies to detect the analyte. A miniaturized, portable version of this sensor, called Analyte 2000, was developed by Research International (Woodinville, WA) in collaboration with Naval Research Laboratory (Golden, J., et al., A portable multichannel fiber optic biosensor for field detection, submitted). This new sensor is based on the same principles of detection as its predecessor, but has been reduced to 6.5" x 4.5" x 3.5", and weighs 2.5 pounds (Figure 1 bottom). It can monitor four 600 urn fused silica optical fibers probes simultaneously. In addition, the sensor and the laptop computer required to operate it are both fully battery operable, resulting in a system that is highly portable. The on-site detection capabilities of this sensor were tested at two military bases in the Northwest for the detection of TNT in groundwater. The sites, Umatilla Army Weapons Depot (Hermiston, OR) and Naval Submarine Base Bangor (Bangor, WA), are but two of the more than 50 sites on the US Environmental Protection Agency's (EPA) Superfund list slated for hazardous waste characterization and subsequent remediation. The field trials were sponsored to test alternative methods of site characterization in order to minimize the cost and duration of the characterization/remediation project. The current method of characterization is high-performance liquid chromatography (HPLC) (6) at an offsite laboratory, which takes 2-4 weeks and costs hundreds of dollars per sample. Logically, an inexpensive on-site means of sample analysis would address both the cost and time issues. The portable fiber optic biosensor and 3 commercially available test kits were tested for their correlation with the standard HPLC analysis, and were compared by their efficacy, speed, cost, accuracy, and simplicity. This article will concentrate on the fiber optic biosensor. Use of this new fiber optic biosensor for the detection of TNT required adapting the protocols and techniques of the TNT assay from the previous system. In addition, new variables were identified for on-site field analysis and testing of real water samples. Several questions were examined, including: a) what conditions of the TNT assay on the breadboard system still hold for the portable system?, b) how do real groundwater samples affect an assay compared to laboratory buffer?, c) what is the ideal buffer for detecting TNT with this system?, d) what is the ideal concentration of our competitor compound for this competitive immunoassay?, and e) to what extent do related compounds cross-react with TNT for binding to the antibody? Portable Fiber Optic Sensor System The Fiber Optic Biosensor. The Analyte 2000 (Figure 1, bottom) fiber optic biosensor, developed as a compact, portable update to the breadboard device, consists of four identical computer cards, each supporting a diode laser (635) and a photodiode detector (7, Golden, J., et al, submitted). Signals are transmitted to and

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

IMMUNOCHEMICAL TECHNOLOGY FOR ENVIRONMENTAL APPLICATIONS


200

Figure 1. Versions of the fiber optic biosensor. The top picture is the original breadboard FOB which monitored 1 optical probe. The bottom picture is the portable Analyte 2000 FOB which monitors 4 independent optic probes simultaneously.



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DONNERETAL.


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from the fiber via a specially designed jumper (7). The jumper consists of a fiber bundle, with a single central fiber providing the fluorescence excitation and the surrounding fibers in the bundle collecting the fluorescence emission from the fiber probe. Data from the Analyte 2000 is read in pico-Amperes (pA) and displayed graphically in real time on a laptop computer screen. The principle of detection in both the Analyte 2000 and the breadboard fiber optic biosensor is the same (1, Golden, J., et al, submitted, 8). A region of energy known as the evanescent wave extends approximately 100 nm from the surface of the fiber when light is propagated along the fiber via total internal reflection. While, by Snell's law, light striking the core/cladding or core/solution interface reflects within the core, an electromagnetic component of the light traverses the core forming the evanescent wave. The power of this wave decays exponentially with distance from the core, resulting in a sensing region virtually unaffected by fluorescing molecules outside the confines of the evanescent wave. By immobilizing antibodies onto the core surface, one can perform an immunoassay using either a fluorescently tagged antigen or antibody. By using a light source above 600 nm and a fluorescent label that absorbs at this wavelength, one greatly reduces the effect of background fluorescence found in many natural samples (9). Fiber Tapering and Chemistry. The antibody immobilization chemistry used on the fiber probes was first described for glass cover slips (10,11). It was then adapted to prepare 200 urn fibers in conjunction with a tapering step (12). Preparing 600 um fibers for use in the portable fiber optic biosensor required only slight modifications from the 200 umfibertechnique. Briefly, the distal 12.5 cm portions of fused silica optical fibers are stripped of their protective cladding and tapered in hydrofluoric acid. The continuously tapered geometry has been shown to help overcome losses of the return signal from the sensing (declad) region back into the transmitting (clad) region of the fiber without loss of excitation light (13). The unclad region of the fibers are then silanized, resulting in exposed thiol groups which are then bound to the maleimide reactive site of a heterobifunctional crosslinker. The other functionality of the crosslinker (succinimide) binds to an amine group on the antibody. Competitive Immunoassays for TNT. Detection of TNT in this assay was based on the competition between unlabeled TNT and a fluorescently labeled analog of TNT for a limited number of antibody binding sites (3). A predetermined concentration of the labeled analog, referred to as the Reference or 100% solution, is introduced to the sensing region and allowed to bind. After 4 minutes the fiber is exposed to laser light, exciting any fluorescently labeled antigen within the evanescent wave, which then emits higher wavelength light that couples back into the fiber core. The bound TNT can be removed, thus "regenerating" occupied binding sites, by 1-minute exposure to 50% ethanol (3). Then, the same Reference solution is introduced along with the sample containing unlabeled TNT. A reduction in signal proportional to the concentration of TNT is observed (Figure 2), since both compounds are competing for the same number of antibody binding


202

IMMUNOCHEMICAL TECHNOLOGY FOR ENVIRONMENTAL APPLICATIONS 5000 .85 ng/mL TNT

4000

8.5 ng/mL TNT

50 ng/mL TNT i

c[ 3000 500 ng/mL TNT

15 c Downloaded by NORTH CAROLINA STATE UNIV on October 4, 2012 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1997-0657.ch016

O)

55 2000

/

850 ng/mL TNT

3000 ng/mL TNT

1000

-

1

—i

1

20

40

60

80

100

1

8500 ng/mL TNT

1—

140

120

Time (minutes) Figure 2. Multiple TNT detections on a single fiber optic probe. Measurement of reference signals and 7 concentrations of TNT were performed on a single fiber.

q S

1

1

1

i—>—>—i—'—i—'————r 15 20 25

TNT(,ig/L) Figure 3. TNT standard curves (± standard deviations) using various concentrations of Cy5-EDA-TNB (• - 3 ug/L, • - 5 ug/L, • -10 ug/L).


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sites. This signal reduction, referred to as the % Inhibition, is calculated by using the Reference trials before and after the TNT sample trials. The mean of these two Reference trials is used to account for any reduction of antibody activity due to repeated tests and regenerations. The percent inhibition is calculated by: Test signal

% Inhibition =


(

Reference

pre

*100

+Reference^

(1)

Using a fixed concentration of the fluorescently labeled analog along with known concentrations of TNT, one can establish standard curves which can be used to quantitate unknowns. Optimization of TNT Assay for On-Site Monitoring The use of the portable fiber optic biosensor in the field required both adapting the assay for TNT from the breadboard device (5) to the portable device, as well optimizing several variables one could expect to encounter in the field. The following experiments were performed prior to on-site monitoring. Labeled Analog of TNT. The compound used to compete with TNT in these assays was a labeled version of trinitrobenzene (TNB) (5). This compound, cyanine 5-ethylendiamine-trinitrobenzene (Cy5-EDA-TNB), was chosen not only because of the structural similarity between the TNT and TNB, but also because the monoclonal antibodies used in this assay (NRL clone 11B3)(74) were raised against TNB. The fluorescent label Cy5 was chosen because it absorbs at 650 nm, virtually eliminating any interference from naturally fluorescing compounds in real samples. Several concentrations of Cy5-EDA-TNB were tested to determine the optimal concentration for a competitive assay with TNT on the portable unit by determining the lowest limit of detection of TNT, the best signal-to-noise ratio, and the magnitude of the maximum signal. For each concentration of Cy5-EDA-TNB, a competitive immunoassay using increasing concentrations of TNT (described above) was performed. A minimum of 10% inhibition was arbitrarily picked as the cut-off for the detection of TNT in a sample as this level of inhibition was just above 2 standard deviations from the mean blank samples values. The inhibition curves using 3 ng/mL, 5 ng/mL and 10 ng/mL as the concentration of Cy5-EDATNB in the Reference solution are shown in Figure 3. As evidenced from Figure 3, a Reference solution containing 10 ng/mL Cy5-EDA-TNB detected TNT at a concentration of 10 ng/ml or greater. While both 3 ng/mL and 5 ng/mL Cy5-EDA-TNB appeared to detect TNT at 5 ng/mL, the maximum fluorescence signal when using 5 ng/mL Cy5-EDA-TNB was, logically, greater than that for 3 ng/mL Cy5-EDA-TNB. Low fluorescence signals from the Reference solution are undesirable because small inhibitions are not easily


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observed. For these reasons, 5 ng/mL was chosen as the concentration of Cy5E D A - T N B in the Reference solution and the TNT standards and unknown samples tested. Buffer Components. Several buffer components were tested to determine the optimal buffer for this assay using field samples. Variables tested consisted of the exclusion of salts or the inclusion of detergents, blocking proteins, or organic solvents into the phosphate buffer. The need for an organic solvent directly relates to the hydrophobicity of TNT and similar compounds. The solubility of TNT is 0.02 g/100 g in water, whereas it is 1.5 g/100 g in ethanol, and as high as 132 g/100 g in acetone, at 25° C (5). These solubility characteristics are also why a 50% ethanol solution was previously found to effectively dissociate the antibody-antigen interaction and regenerate the fiber in 1 minute (5). Detergents and blocking proteins were included to minimize nonspecific adsorption. The buffer components tested included acetone (1%, 5%, and 10%), methanol (5%), ethanol (10%), bovine serum albumin (BSA) (2 mg/ml), Triton X 100 (0.1%), Tween 20 (0.1%), and phosphate buffered saline (PBS). Several Reference buffers were made using combinations of the above components along with 5 ng/mL Cy5-EDA-TNB, with the remaining volume of the samples occupied by reverse osmosis (RO) deionized water. These Reference solutions were compared by their relative signal stability when the RO water was replaced with real water samples that contained no TNT or related compounds. After testing well water, bilge water, salt water, and Potomac River Water, two conclusions were made. The first is that the most stable buffer was composed of PBS (lx), B S A (2 mg/mL), acetone (5%), and Tween 20 (0.1%), which is referred to as PBAT. The second is that all of the real waters samples tested affected the signal to a noticeable extent. Thus Reference solutions were made to include an uncontaminated water blank at the same concentration as the samples. The water blank was obtained from water near the the contaminated site. Protocol for Sample Analysis. The analysis of unknowns or standards begins with a 2-minute incubation of the buffer PBAT void of any Cy5-EDA-TNB. After the incubation, the fibers are exposed to laser light and a reading is taken. This value is considered the background noise, and is subtracted from all the data. Following the background determination, either a Reference, standard or unknown sample is introduced to the fiber. A reading is taken after a 4-minute incubation period, and the background is subtracted. The Reference, standard and unknown samples all have the same concentration of Cy5-EDA-TNB (5 ng/mL), the same buffer components (PBAT), and the same volumetric concentration of either deionized water (for Reference and standard samples) or blank uncontaminated groundwater (for References and samples). These solutions should differ only by the amount of (unlabeled) explosives and/or explosive degradation products they contain. To regenerate the fiber, a 50% ethanol solution in PBS is incubated with the fiber for 1 minute. Fibers are typically regenerated over 20 times. The average total time for sample analysis, including two Reference solutions, is 18 minutes for four fibers


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run in parallel. As long as the samples and Reference solutions can be analyzed in an alternating mode, a new set of 4 samples can be tested every 12 min. Field Trials. Two field trials were conducted at the Umatilla Army Weapons Depot (Hermiston, OR) and Naval Submarine Base Bangor (Bangor, WA) during the summer of 1995. Both sites are on the EPA's Superfund list. In the case of Umatilla and Bangor, the environmental pollution is from contamination of soil and groundwater by explosives, a result of storage, recycling, disposal, and manufacturing of munitions. While a detection protocol exists (6), a thorough characterization of a site must be performed so that only contaminated sections are remediated. The current EPA-approved method for this characterization is S W-846 Method 8330, which is an HPLC analysis that takes place off-site at a certified laboratory. While accurate, the method is time consuming (2-4 weeks/sample) and expensive ($250-$1000/sample). In addition, the groundwater is remediated by filtration through granulated activated charcoal (GAC). Saturation of the filters needs to be determined with minimal turnaround time, and an on-site method would be better suited for this application than analysis in a central laboratory. In search of an on-site method of sample analysis that addresses both the cost and time concerns, the EPA sponsored field trials of on-site methods using both commercially available test kits as well as biosensors under development. Over 100 fibers were prepared at the N R L for the field trials at Umatilla and Bangor using the protocol described above. The antibody-coated fibers were each then secured in a 200 uL glass capillary tube with a "T" connector at each end, which serve as an input and output for the samples. Fibers were stored either in PBS at 4°C, or dried down in a 100 mM trehalose solution and stored at -20°C until the field trials. The protocol for sample analysis described above was used for all standard and unknown samples during the field trials at both sites. At Umatilla, all assays were performed on-site in an indoor storage area. Assays at Bangor were performed in a water treatment facility on the site partially open to the outdoors. While the purpose of the field trials was to demonstrate a fully portable sensor, electricity, available at both sites, was used for power. At both Umatilla and Bangor, all samples, standards and their dilutions were provided by Black and Veatch Waste Science, and were stored cool in amber glass bottles. Several standards containing 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5trinitro-l,3,5-triazine (RDX) 1,3,5-trinitrobenzene (TNB), octahydro-l,3,5,7-tetranitro-l,3,5,7-tetrazocine (HMX), or a combination thereof were tested at concentrations ranging from 1-10,000 ug/L for standard curves and cross-reactivity information. A blank water sample void of explosives, but representative of the water sample, was provided at each site and was used in the Reference solution, occupying a volume identical to that of the sample being tested in the unknown. Nineteen unknown groundwater samples and twenty-three unknown groundwater and leachate samples were analyzed with the portable fiber optic


206


biosensor at Umatilla and Bangor, respectively. A l l samples and standards were analyzed with 85% of the total volume being groundwater (blank or test), using a minimum of three fibers per sample. Many samples and standards from both sites were later reanalyzed at the N R L with the fiber optic biosensor and HPLC. The limit of detection of TNT using the portable fiber optic biosensor was 20 fig/L for the field samples, compared to a limit of detection of 5 ug/L in spiked laboratory buffer previously reported (5). The limit of TNT detection for the H P L C method SW-846 Method 8330 using a direct injection protocol was 20 ug/L. Qualitative comparison between the fiber optic biosensor and SW-846 Method 8330 using linear regression gave a slope of 1.23 and an r of 0.91. This indicates that on the whole the fiber optic biosensor assay produced slightly higher TNT values than Method 8330. This may be due to cross-reactivity of the antibody with trinitrobenzene since this same trend is seen with other immunoassays for TNT. There were 11.4% false positive and 2.9 % false negative identifications. A sampling of quantitative data from the trials is shown in Table I.


2

Table I. TNT Detection with Method 8330 and Fiber Optic Biosensor Sample

Method 8330 («5/L)

Fiber Optic Biosensor (Ug/L)

Umatilla Well47-4

1000*

octhydro-l,3,5,7-tetranitro-l,3,5,7-tetrazocine (HMX)

>1000

* These explosives leveled off at or prior to the I G

Conclusion The fiber optic biosensor has been successfully transitioned from a laboratory breadboard system to a field portable instrument. Optimization of the competitive fluorescent immunoassay for TNT reduced variability due to differences in different groundwater samples. Field portability and sensitvity of the biosensor was demonstrated in two successful field trials. In addition, further characterization of possible cross-reactants have shown that only explosives that are degradation products of TNT seem to have any effect on the assay. The portable fiber optic biosensor is able to detect TNT on-site which will result in a reduction in time and cost over the time frame of site characterization and remediation.

Acknowledgements The authors thank Anne Kusterbeck, Linda Judd, John Bart, George Anderson and Joel Golden for their assistance and suggestions in preparing for the field demonstrations. This work was funded by Office of the Undersecretary of Defense Environmental Security Technology Certification Program (ESTCP). The views


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expressed here are those of the authors and do not represent those of the U.S. Navy, Department of Defense or the U.S. Goverment. Literature Cited 1.


2.

3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14.

Ligler, F.S.; Golden, J.P.; Shriver-Lake, L.C.; Ogert, R.A.; Wijesuria, D.J.; Anderson, G.P. Immunomethods.1993, 3(2), 122. Anderson, G.P.; Golden, J.P.; Cao, L.K.; Wijesuriya, D.; Shriver-Lake, L.C.; Ligler, F.S. IEEE Engineering in Medicine and Biology. 1994, 13(3), 358. Shriver-Lake, L.C.; Breslin, K.A.; Charles, P.T.; Conrad, D.W.; Golden, J.P.; Ligler, F.S. Anal Chem. 1995, 67(14), 2431. Jenkins, T.F.;Walsh, M.E. Talanta. 1992, 39(4), 419. Yinon, J. and Zitrin, S. Modern Methods and Applications in Analysis of Explosives, John Wiley & Sons: Chichester England, 1993. U.S. Environmental Protection Agency , SW-846 Method 8330. Saaski, E.; Bizak, M.; Yeatts, J. SPIE Procceedings. 1995, 2574, 56. Golden, J.P.; Shriver-Lake, L.C.; Anderson, G.P.; Thompson, R.B.; Ligler, F.S. Opt Eng. 1992, 31(7), 1458. R.P. Haugland, in Biosensors with Fiber Optics; D. Wise, L . Wingard, (Eds.); Humana Press: Clifton, N.J. 1991; 85-110. Bhatia, S.K.; Shriver-Lake, L.C.; Georger, J.; Calvert, J.; Prior, K.; Bredehorst, R.; Liger, F.S. Anal Biochem. 1989, 178, 408. Ligler (misspelled Eigler), F.S.; Georger, J.H.; Bhatia, S.K.; Calvert, J.; Shriver-Lake, L.C.; Bredehorst, R. U.S. Patent 5,077,210, December 31, 1991. Shriver-Lake, L.C., Anderson, G.P., Golden, J.P., Ligler, F.S. Anal Letters. 1992, 25(7), 1183. Anderson, G.A.; Golden, J.P.; Ligler, F.S. Biosensors & Bioelectronics. 1993, 8, 249. Whelan, J.P.;Kusterbeck, A.W.; Wemhoff, G.A.; Bredehorst, R.; Ligler, F.S. Anal Chem. 1993, 65, 3561.


Immunochemical Technology for Environmental Applications

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