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Anal. Chem. 1999, 65, 3561-3565
Continuous-Flow Immunosensor for Detection of Explosives James P. Whelan; Anne W. Kusterbeck, Gregory A. Wemhoff? Reinhard Bredehorst,s and Frances S. Ligler’ Center for BiolMolecular Science and Engineering, Code 6900, Naval Research Laboratory, Washington, D.C. 20375-5348
A continuous-flow immunosensorfor detection of trinitrotoluene (TNT) in aqueous solution is described. The sensor utilizes an immobilized monoclonal antibody specific for TNT and saturated with fluorescein-labeled antigen. When introduced into the flow stream through the column, samples containing unlabeled antigen displacethe bound labeled antigen. The displaced labeled antigen is detected downstream using a fluorometer. The displacement of labeled antigen is directly proportional to the concentration of unlabeled antigen appliedto the column over a range of 20 to 1200 ng/mL (ppb). The sensitivity is increased when solvents (ethanol or 2-propanol) are included in the flow buffer at concentrations up to 25% (v/v). By use of a cyclone air sampler and a TNT vapor generator,TNT could be detected in collected samples. The flow immunosensor provides the means to detect trace levels of TNT with high specificity in times of less than 1 min. Such qualities make the flow immunosensor a promising low-cost alternative to explosive detection devices currently available to transportation and security industries. INTRODUCT10N The urgent need in the air transportation industry for an inexpensive, rapid, sensitive, and selective detector for explosives was the major impetus for development of an antibody-based biosensor (immunosensor). Dogs trained to detect explosives provide both sensitivity and selectivity. However, dogs are unpredictable and require specialized training and maintenance. The currently used manmade explosive detection devices fall into two categories: penetrating emission devices, such as thermal neutron analysis (TNA) and X-ray backscattering, or collection and analysis devices that screen for the illicit substance directly, such as the Egis system (Thermedics, Inc., Woburn, MA) (see ref 1 for review). Although all of these systems have apparent sensitivities sufficient for detection of explosives in a real environment, the cost for these systems range from $150,000 for the Egis System to over $1 million for a TNA unit. Portability is also a concern since these systems can be quite large; the TNA system from Science Applications, Inc. weighs close to 14 tons. Low-cost “electronic dogs” or biosensors are an attractive alternative to the high cost and cumbersome “state-of-the-
* Author to whom correspondence should be addressed.
+ Current addreas: U.S.DrugTesting,Inc., 10410TrademarkS.,Ramho
Cucamonga, CA 91730. t Current address: Immunochemicals,Department59,Sigma Chemical Co. St. Louis, MO 63178. Current address: Department of Biochemistryand MolecularBiology, University of Hamburg, Martin Luther King Platz.6, 2000 Hamburg, Germany; (1) Fainberg, A. Science 1992,255, 1531-1537. 0003-2700/93/0365-3561$04.00/0
art” systems. Immunosensors in particular have been employed in a variety of devices against a diverse group of specific target molecules or antigens.24 Antibodies serve as selective binding molecules. When incorporated into a simple electronic monitoring device, immunological detection methods provide the strengths of both trained dogs and man-made sensors without many of the inherent weaknesses. A prototype of the continuous-flow immunosensor for detection of small molecular weight compounds has been described previously for detection of dinitrophenol7 and cocaines . An original specification in the design of the flow sensor was to produce a positive signal in less than 1 min. Additional requirements were for a low-cost device that was user-friendly; i.e., amenable to use by nontechnical personnel. Although available state-of-the-art technologies fail to meet one or more of these requirements, all of these characteristics are incorporated into the current prototype flow immunosensor. Here we describe the use of the flow immunosensor for the detection of T N T and report preliminary results from using it in the field in conjunction with a cyclone air sampler. EXPERIMENTAL SECTION TrinitrobenzeneConjugatePreparation. Trinitrobenzene (TNT)-carrier conjugates were formed by coupling trinitrobenzenesulfonate (TNBS) (Aldrich Chemicals, Milwaukee, WI)) to ovalbumin (TNB-OVA) or bovine serum albumin (TNB-BSA) according to the procedure of Chesebro and Metzger? Monoclonal Antibody Generation and Selection. Monoclonal antibodies with specificity for TNT were generated by immunizing pristane-primed mice with TNB-OVA. The procedure for selecting appropriate antibodies involved screening hybridoma supernatants first for binding to TNB-BSA and second for displacement of the antibody after binding to the same immobilized antigen. Monoclonal antibodies specific for TNT were initially selected and titered by use of an ELISA assay againstTNB-BSA bound to the microtiter plate. Briefly, clones were screened by adsorbing bovine TNB-BSA (2 pg/mL in phosphate-bufferedsaline, pH 7.4 (PBS)) to Immulon-1 microtiter plates (Dynatech Laboratories,Chantilly, VA) overnight at 4 “C. The plates were subsequently blocked with BSA in PBS (2 mg/mL) and then reacted with a serial dilution of test supernatant for 1h at 37 O C . After washing, each well was then incubated with peroxidase-conjugated anti-mouse IgG (Sigma, St. Louis, MO) (1/5000dilutionin PBS) for 1h. Specificbinding was visualized by adding o-phenylaminediamine (Sigma) at 0.3 (2) Barnard, G. J.; Collins,W. P. Ann. Chern. Biochern. 1983,20,294301. (3) Nolan, J. P.; DiBenedetto, G.; Tarsa, N. J. Clin. Chem. 1981,27, 738-741. (4) Hayashi, S.; Kurooka, S.; Arisue, K.; Kohda, K.; Hayashi, C. Clin. Chem. 1983,29, 179C-1792. ( 5 ) Ikariyama, T.; Kunoh, H.; Aizawa, M. Biochem. Biophys. Res. Commun. 1988,128,987-992. (6) Jenkins, S. H.; Heineman, W. R.; Halsall, H. B. Anal. Biochem. 1988,168,292-299. (7) Kusterbeck, A. W.; Wemhoff, G. A.; Charles, P. T.; Yeager, D. A.; Bredehorst, R., Vogel, C.-W.; and Ligler, F. S. J . Immunol. Methods 1990,135, 191-197. (8) Ogert, R. A.; Kusterbeck, A. W.; Burke, R.; Ligler, F. S. Anal. Lett. 1992,245, 1999-2019. (9) Chesebro, B.; Metzger, H. Biochemistry 1972,11, 766-771. 0 l W 3 Amerlcan Chemical Society
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mg/mL with 0.025% HzOz in 0.1 M citrate in a 0.1 M phosphate buffer, pH 5.0, for 20-30 min. Optical density was read at 465 nm. The test dilution employed for the displacement assay was adjusted to the concentration of monoclonal antibody supernatant at which positive binding began to decrease from a maximal value along a serial dilution. At this concentration, antibody was no longer in excess for a given immobilized antigen concentration. In the displacement assay, selected clones were screened for their displaceability by first adsorbing TNB-BSA as before. The plates were then blocked with BSA in PBS (2 mg/mL) and reacted with a limiting dilution of test supernatant for 1 h at 37 "C. A TNT stock solution (10 mg/mL), prepared by dissolving crystalline TNT (a generous gift from Dr. Lou Wasserzug at the Naval Explosive Ordinance Disposal Technology Center, Indian Head, MD) in 100% ethanol, was used to make serial dilutions of the TNT in PBS. Following, plates were incubated for 2 h with the various concentrations of TNT. Each well was then incubated with peroxidase-conjugated anti-mouse IgG and developed as described above. Synthesis of Fluorescently Labeled Trinitrobenzene. TWBS and fluorescein-coupled cadaverine (FL-CV) (Molecular Probes, Eugene, OR) were dissolved separately to 17 and 1.89 mM, respectively, in 0.17 M borate buffer, pH 8.6-9.0, containing 0.12 M sodium chloride. A l-mL aliquot of the TNBS solution was added dropwise with gentle mixing into 10 mL of the FL-CV solution. Under these conditions, a final molar ratio was achieved of 0.9:l for TNBS:FL-CV. The mixture was rotated gently overnight at 4 "C. Under these conditions, the majority of TNBS reacted with FL-CV, leaving negligible levels of free TNBS in solution, as determined by analytical thin-layer chromatography (TLC). This solution of fluorescein-cadaverine-trinitrobenzene (TNB-CV-FL) was neutralized to pH 7.2 with 1M HCl and used directly to saturate the immobilized antibody. Antibody Immobilization and Saturation with Analyte. Monoclonal antibodies were coupled to tresyl chloride-activated Sepharose 4B (Sigma) according to manufacturer's standard procedures. Briefly, preweighed dried Sepharose powder (1g of powder gives -4 mL of swollen gel) was suspended and washed for 1h with 1mM HC1onasinteredglassfilter. Purified antibody was dissolved into coupling buffer (0.1 M NaHC03, pH 8.6,0.5 M NaCl) at a concentration of 0.2-0.5 mg/mL and mixed with washed Sepharose at the ratio of 5 mL/g of preweighed gel. The mixture was rotated end over end overnight at 4 OC. Unbound antibody was removed by washing with coupling buffer until the optical density at 280 nm was zero. The remainingtresyl chlorideactivated sites were blocked by incubation with 0.1 M Tris-HC1 buffer, pH 8.0, for 4 h at room temperature while rotating end over end. The product was washed with three cycles of alternating pH, consisting of a wash with 0.1 M acetate buffer, pH 4.0, containing 0.5 M NaC1, followed by 0.1 M Tris-HC1 buffer, pH 8.0, containing 0.5 M NaC1. The coupled gel was stored in PBS containing 0.01% NaN3 at 4 "C. Estimation of coupled antibody was determined using a Coomassie blue assay for immobilized proteins.10 Under these conditions, 2-8 pmol of antibody were coupled per milligram of dry Sepharose. Antibody-coupled gel was saturated with TNB-CV-FL in borate buffer, pH 7.2, by incubating the gel with a 100-fold molar excess of labeled antigen to antibody binding sites for at least 7 days. Sensor Design. The flow immunosensor (Figure 1) is comprised currently of a buffer reservoir, a Rabbit-Plus peristaltic pump (Rainin Instruments, Emeryville, CA), a Rheodyne fiveway valve (Rainin Instruments, Emeryville, CA) employed as a low-pressure sample injector, a 4.6-mm (i.d.) Biocompatible Guard column (Upchurch, Oak Harbor, WA) containing the antibodycoated Sepharose, a Model 821-FP spectrofluorometer (Jasco, Inc., Easton, MD), and a HP3396B Series I1 integrator (HewlettPackard, Inc., Rockville, MD). A Model HP-9114B disc drive (Hewlett-Packard, Inc.) was utilized to store analog output from the fluorometer for subsequent analysis. Connecting tubing was 0.6-mm-i.d. Teflon (Upchurch). For sample testing, a continuous-flow buffer stream was established through the column at a flow rate of either 0.4 or 1.0 mlimin. Standard flow buffer was PBS, pH 7.4, containing 0.13'% Triton X-100 (Fisher Scientific, Pittsburgh, PA). Four solvents (10)Ahmad, H.; Saleemuddin, M. Anal. Biochern. 1992, 248,533-541.
i-L: 0.1% Triton
I
P
Perlrtaltlc
flow stream
Sepharow AntlbodyAntlgen' Column
HP 3396-6 Integrator
I I
I Fluorimeter Model 821
WASTE
Flgure 1. Components for the NRL continuous-flow irnmunosensor. (ethanol, 2-propanol, dimethylformamide, dimethyl sulfoxide) were tested individually as additives in the flow buffer. Columns were washed of excess labeled antigen until a steady baseline signal was established. At both flow rates, baselines were established in less than 30 min. Stock solutions of T N T and dinitrotoluene (DNT; Aldrich, Milwaukee, WI) were prepared in 100% ethanol by slow rotation at room temperature in glass vials. Phenylalanine (5 pg/mL), which like TNT has a six-carbon ring, and either glysine or lysine (8 pg/mL) were routinely employed as negative controls. Samples were diluted in the flow buffer and introduced into the flow stream using the low-pressure sample injector. Results are given in arbitrary area units from each sample peak as determined by signal integration. Air Sample Tests. Field tests were conducted under the auspices of the Federal Aviation Administration onboard a sealed Boeing 727 on the ground. Air samples were collected by Midwest Research Institute (MRI, Kansas City, MO) using an MRI Spincon air sampler. The Spincon air sampler was run for 5 min adjacent to a TNT vapor generator at air flow rates of either 50 or 100 mL/min. Air was drawn through the sampler at a rate of 1000Limin. Samples were collected into distilled water in the Spincon, and aliquots (200 pL) were tested on the plane using the flow immunosensor. The immunosensor employed a 500-pL bed volume column and a 0.5 mL/min flow rate. Flow buffer was PBS containing 0.1% Triton-X-100. For independent confirmation of the immunosensor results, duplicate aliquots were collected, frozen, and analyzed at a later time by MRI using gas chromatography with electron capture detection (GC-ECD)." Aqueous samples were extracted using toluene prior to GC-ECD analysis.
-
RESULTS Screening of Monoclonal Antibodies. Ten subclones producing antibody specific for TNB-BSA were first tested for their ability to bind and then be displaced by T N T (Table I). Displacement of bound antibody was measured by adding T N T at two concentrations to TNB-BSA-coated wells preincubated with monoclonal cell supernatants at the test dilution. For use in the flow immunosensor, an antibody that could be displaced at the lowest T N T concentration was chosen since displacement of TNB-CV-FL by T N T is required for sensor function. Given the high titer of the l l B 3 subclone and its efficient displaceable binding, it was selected for subsequent use in the immunosensor. Solvent Effects. The low solubility of T N T in aqueous solutions led to a study of the effect of various organic solvents (11) Douse, J. M. F. J . Chrornatogr. 1981, 208, 83-88
ANALYTICAL CHEMISTRY, VOL. 65, NO. 24, DECEMBER 15, 1993
Table I. Hybridoma Screening for Displacement of Antibody by Free TNT. % displacement (r/mLfree TNT) clone titer 45 22 2H3
1:16
2C3
1:4
6B6
1:8
7Ell
1:4
7H7
neat
8E5
neat
9H8
1:8
llB2
1:lOoo
llB3
1:1m
llB3
1:2m
96.8 96.8 47.8 44.2 78.4 76.0 100 100 100 91.2 93.0 95.6 67.1 71.6 94.0 94.0 100 98.9 98.0 94.2
11
1.2
18.5 6.4 6.2 13.4 9.0 22.4 18.3
6.8 10.5 24.7 17.4 22.4 18.3
0 Positive monoclonal antibodies selected from ELISA assays against TNT-BSA were screened for the ability of the antibody to be displaced from immobilized TNT-BSA by free TNT as described under the Experimental Section. Values are duplicatedeterminations of relative displacementsby free TNT given as a percentage of control absorbances in the absence of free TNT.
on the flow sensor. Different solvents, including ethanol and 2-propanol, were tested at concentrations of 1.5,3.0,6.0,12.5, and 25% (v/v) in PBS containing 0.1 % Triton X-100 as the flow buffer. T N T standards injected onto each column were diluted in the flow buffer containing the solvent to determine the lowest concentration of T N T producing a signal above background. Control columns had no solvent included in the flow buffer. Dimethylformamide and dimethyl sulfoxide were both tested at the lowest concentration only (1.5 % ,v/v) since these solvents proved detrimental to the ability of the immobilized antibody to bind antigen even at this low concentration (Table 11). Results with both ethanol and 2-propanol show improved detection sensitivity. Ethanol a t a concentration of 12.5% demonstrated the greatest improvement in sensitivity, giving a positive signal a t 2.5 ng/ mL compared to 20 ng/mL in controls with no solvent included. A concentration of 12.5 % 2-propanol improved sensitivity of the system to T N T as well, giving a positive signal down to 5 ng/mL. At concentrations of 25 % for ethanol and 2-propanol, no additional increase in sensitivity was observed. TNT and DNT Assays. Sepharose coated with monoclonal antibody l l B 3 and saturated with TNB-CV-FL was used in the flow immunosensor for detection of either T N T or DNT introduced into the buffer flow stream containing 12.5 % ethanol. Integrated areas from the fluorimeter analog output were plotted against antigen concentration (Figure 2). At a flow rate of 0.4 mL/min, the flow sensor gave a positive signal above background at concentrations as low as 0.25 ng of TNT. Linear signals, however, were produced for T N T between 10 and 40 ng/mL and for DNT between 20 and 1200 ng/mL. Over the concentration range tested, DNT gave an average displacement signal 84 f 2 % (mean f SEM) of the displacement by TNT. This is in accordance with the crossreactivity of monoclonal antibody l l B 3 with DNT as measured by competition inhibition assays. Phenylalanine (500 ng) and glycine (800 ng) controls produced no fluorescent displacement signal when introduced into the flow stream.
950
Flow Rate Effects. The effects of flow rata were examined on Sepharose coated with l l B 3 monoclonal antibody and saturated with TNB-CV-FL. The maximal flow rate recommended by the manufacture for Sepharose is 1.0 mL/min. A standard curve was run for T N T a t two different flow rates (0.4 and 1.0 mL/min) to examine the effects on magnitude of total displacement, system sensitivity,effective linear range, and column life. Results indicate that the absolute displacement signal at a flow rate of 1.0 mL/min is significantly less than a t 0.4 mL/min (Figure 3). Furthermore, the larger displacements produced by the slower flow rate ultimately resulted in a 10-fold better sensitivity than when the faster rate was used (2 ng/mL as compared to 20 ng/mL). This increased sensitivity is directly related to the increased displacement obtained by the slower flow rate without an increase in background fluorescence. However, use of the faster flow rate did exhibit some advantages over use of the slower flow rate. First, the linear range for the quantitation of T N T was significantly broader a t the faster flow rate (20 to >1200 ng/mL) as compared to the slower rate (10-40 ng/mL). Second, though the flow rates differed by only 2.5-fold, the total amount of TNB-CV-FL displaced during the experiment shown in Figure 3 (sum of integrated signals from all displacement envents) was 10fold higher a t the 0.4 mL/min flow rate than that observed for 1.0 mL/min (4.0 X lo6vs 40 X lo6fluorescence area units, respectively). These results suggest that column lifetime can be extended by increasing flow rate. Air Sample Tests. Samples collected from air with a cyclone air sampler were tested for T N T using both the flow immunosensor and GC-ECD. All seven samples that gave positive responses in the flow immunosensor were verified by GC-ECD to contain T N T or some percentage of DNT. The 13 negative responses reported using the flow immunosensor were shown by the GC-ECD analysis either to be negative for T N T and DNT or to contain these explosives at levels below the detection sensitivity of the immunueensor. The detection sensitivity of the GC-ECD analysis was 0.1 ng/mL. Control samples collected for 10min without the T N T vapor generator operating gave no signal above baseline in the immunosensor.
DISCUSSION The flow immunosensor technology relies upon detection of labeled antigen molecules displaced by unlabeled antigen in a continuous-flow stream. This approach achieves detection sensitivities in the same range as traditional radioimmunoassays or the sensitive ELISAs without the need for time-consuming incubations. For use in the flow immunosensor, an antibody must possess an affinity which is sufficient to bind the labeled antigen but not so high as to preclude displacement by unlabeled antigen. Samples are dissolved in the flow buffer and introduced into the flow stream. Multiple samples may be analyzed on a single column, and the displaced fluorescent signals that are obtained are linear with respect to antigen concentration over an adjustable range. By varying flow rate and column size, it is possible to customize the system for both sensitivity and useful column lifetime. Slower flow rates increase the residence time of sample antigen on the column and result in increased displacement efficiency a t a cost of decreased column life. In practice, columns with a 0.2-mL bed volume run a t 0.4mL/min can produce multiple positive signals. In the case where real samples are tested that may contain high levels of background interferents, a slower flow rate is an effective means for increasing signal. In contrast, when background interferents are low or ultimate sensitivity is not required, faster flow rates can be employed to increase column lifetimes, thereby decreasing the cost and
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 24, DECEMBER 15, 1993
Table 11. Effects of Solvents on Lower Detection Limitsa
[TNTI (ng/mL)
additives to PBS-Triton
1.25
2.5
no solvent 12.5% ethanol
-
-
-
12.5%2-propanol 1.5 % dimethylformamide 1.5 % dimethyl sulfoxide
-
+-
-
10
-
-
+ +
-
-
5 -
-
+ +
20
40
I5
150
300
+ + +-
+ + +-
+ + +-
+ +
-
-
-
+ + ++
TNT was injected into the flow stream (100 pL) at the concentrations indicated using a flow rate of 0.4 mLimin: no signal.
/
'**1
~
0.04 0
'
I
100
'
1
200
"
300
"
400
"
500
1
600
'
' 700
[Antigen] ng/mL Flgure 2. Displacement of TNB-CV-FL by TNT arid DNT. Displacement of labeled antigen by either TNT (0)or DNT (m) was conducted on 200-~Lbed volumes of monoclonal antibody 11B3-coupledSepharose saturated with TNB-CV-FL. Flow rate was 0.4 mL/min, sample size was 100 kL, and flow buffer was PBS, pH 7.4. containing 12.5% ethanol and 0.1 % Triton X-100. Each value represents the mean integrated area of fluorescence & SEM for at least six displacement events. For both linear fits, P > 0.99.
"z 1
jl/ I
Q
2.0
0
1
0
200
400
500
800
lo00
1200
1400
[TNTIng/mL Effects of flow rate on TNB-CV-FL displacement. Columns prepared as in Figure 2 were tested for displacement of TNB-CV-FL by TNT at flow rates of 0.4 (0) and 1.0 (W) mL/min. Triplicate determinationswere performed at each concentration. All points were included in the curve fit, which produced correlation coefficients of = 0.89 for TNT and = 0.99 for DNT. Figure 3.
the frequency of column replacement. Column size can be increased for proportionately longer column lifetime. We have run between 10 and 50 positive samples over various columns.
++
+, positive signal; -,
The use of solvents in the flow system increased the sensitivity. Both ethanol and 2-propanol increased sensitivity of the flow immunosensor for T N T by 1 order of magnitude when included in the flow buffer a t concentrations between 12.5 and 25 % . This effect may be due to increasing both the solubility of T N T and the quantum yield of fluorescein. Increasing the solubility of the T N T helps minimize adsorption to the sample container and tubing before the displacement reaction can occur. However, the measureable increase in quantum yield of the fluorescein in ethanolic solutions is probably the more important factor. In our system, TNBCV-FL a t concentrations of lo-" M in 12.5 9% ethanol in PBS gives a signal 36.7 & 8.7 % higher than in PBS containing no alcohol. This effect may relate to the stabilizing effect of ethanol on the fluorophore itself since Soper et a1.12 found that, in the case of rhodamine-6G, the fluorescence photodestruction was 2 orders of magnitude lower in pure ethanol than in aqueous solutions. We have found that we can utilize ethanol at concentrations up to 25 % (v/v) with no detrimental effect on antibody binding in ELISA assays (data not shown). Fortuitously, at concentrations of 25 % , ethanol and 2-propan01 serve an additional role as a preservative, facilitating long-term storage and use of the columns. The ability of the T N T flow immunosensor to detect DNT is reflective of the specificity of the antibody employed. Increased selectivity theoretically can be attained by screening antibodies in a displacement ELISA for the specificity required. However, cross-reactivities can often be used to advantage. Since DNT is a major contaminant of industrial grade T N T and the vapor pressure of DNT is significantly greater than that of T N T (145 ppb compared to 6 ppb), crossreactivity to DNT serves to make the flow immunosensor more effective as an explosives detector for TNT. Detection of T N T in samples collected by the MRI Sp'incon device indicates that the prototype flow immunosensor for T N T detection is a viable alternative to current methods of explosive detection. However, other methods of sample collection for injection into the flow immunosensor are currently being investigated. The low vapor pressures of nitrous group-based explosives, especially the standard 'plastic" explosive compounds, are most likely too low (ppb to ppt) to be collected efficiently by currently available air sampling methods. Particle collection devices are employed currently in several systems on the market (Egis, Thermedics, Woburn, MA)13 and IT1 85 (Ion Track, Wilmington, MA)14 and may provide samples with explosive concentrations within levels detectable using the flow immunosensor (low ng/mL). The NRL flow immunosensor prototype was assembled with off-the-shelf components a t a cost of less than $15,000. (12) Soper, S. A.; Shera, E. B.; Martin, J. C.; Jett, J. H.; Hahn, J. H.; Nutter, H. L.; Keller, R. A. Anal. Chem. 1991, 63, 432-437. (13) MacDonald, S. J.; Rounbehler, D. P. Proceedings of t h e First International Symposium o n Explosiue Detection Technology, Nou. 1315, 1991; FAA Technical Center, Atlantic City, NJ, 1992; pp 584-588. (14) Jenkins, A,; McGann, W.; Ribeiro, K. Proceedings First International Symposium o n Explosive Detection Technology, Nou. 13-15. 1991; FAA Technical Center, Atlantic City, NJ, 1992; pp 532-551.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 24, DECEMBER 15, 1993
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Significant displacement signals above background have been obtained after more than 24 h of continuous buffer flow. In general, the chemistry necessary to immobilize antibody is standard technology, available as low-cost kits in most cases. In the system prototype, the Sepharose minicolumns containing the antibody and fluorescent antigen can be changed within minutes. These features of long periods of constant utility with little down time make the flow immunosensor attractive as a continuous-monitoring system. Finally, the flow immunosensor is readily adaptable to other explosives by employing antibodies to the target molecule. A competitive ELISA-based assay has been described that detects T N T residue on hands at levels of 50 pg.l6 However, ELISA-based systems normally require assay times of over 90 min. In addition, although competitive immunoassays are quantitative, such assays rely upon a decrease in signal relative to a standard signal to indicate the presence of the target molecule. In such systems, risk of false positives is high since any nonspecific conditions or additive that interferes with antibody binding will be construed as a positive signal. There are two potential sources of false negative results using the flow immunosensor, but controls for both conditions are simple. First, an increase in background due to sample fluorescence could obscure the fluorescence from a displacement reaction. Running a parallel control column reveals the level of fluorescence in the sample itself. Sample fluorescence was not a problem with samples from the air
sampler, even when the samples were darkened by collection of jet fuel fumes. Second, inactivation of the antibody would result in the fluorescent antigen being washed off prior to the analysis. This problem is easily addressed by running a lowconcentration standard prior to each set of analyses. We have demonstrated rapid and selective detection of both T N T and DNT to the 2.5 ng/mL level (11nM or 2.5 ppb) using the flow immunosensor. The displacement signal was linear over 2 orders of magnitude. This level of sensitivity is sufficient for measuring the T N T collected in currently existing samplers. However, for the detection of plastic explosives, which have much lower vapor pressures, sensitivity must be increased even further. Such increases in sensitivity may be achievable by utilizing more efficient fluorophores and/or solvent systems. Antigen carriers with multiple fluorophores16 have also been shown to increase signal in the flow immunosensor. Current work focuseson alternative dyes with higher extinction coefficients or quantum yields to increase the signal-to-noise ratios.
(15) Fetteroff, D.D.;Mudd, J. L.; Teten, K. J. Forensic Sci. 1991,36, 343-349. (16) Bredehorst, R.;Wemhoff, G. A.; Kusterbeck, W. E.; Charles, P. T.;Thompson,R.B.;Ligler,F.S.;Vogel.,C.-W.Anal.Biochem. 1991,193, 272-279.
RECEIVEDfor review June 30, 1993. Accepted October 8, 1993.'
ACKNOWLEDGMENT This work was supported by the Federal Aviation Administration Technical Center, Atlantic City International Airport, NJ. We thank MRI for collecting and analyzing T N T field samples. The opinions presented here are those of the authors and not those of the Navy, Department of Defense, or US. Government.
Abstract published in Aduance ACS Abstracts, November 15,1993.
