Environmental Immunochemical Methods - American Chemical Society

Chapter 5

Environmental Immunosensing at the Naval Research Laboratory Lisa C. Shriver-Lake, Anne W. Kusterbeck, and Frances S. Ligler

Downloaded by PURDUE UNIV on August 29, 2014 | http://pubs.acs.org Publication Date: October 23, 1996 | doi: 10.1021/bk-1996-0646.ch005

Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Code 6910, 4555 Overlook Avenue Southwest, Washington, DC 20375-5348

In the Center for Bio/Molecular Science and Engineering at the Naval Research Laboratory (NRL), two different types of immunosensors are being developed for detection of environmental pollutants and monitoring of bacteria for bioremediation. Both biosensors are rapid, sensitive and easy to operate. The first sensor, the continuous flow immunosensor, is based on displacement of fluorescently-labeled antigen from antibodies immobilized on beads. Antigen is injected into a flow stream that passes over a 100 μL bed volume of antibody-coated beads saturated with fluorescently-labeled antigen. The displacement of the labeled antigen causes an increase in fluorescence, proportional to the antigen concentration, to be observed downstream. The other sensor, the fiber optic biosensor, utilizes long, partially clad optical fibers. Antibodies are immobilized onto the fiber core in the unclad region at the distal end of the fiber. Upon binding of antigen and a fluorescent molecule, a change in the fluorescence signal is observed. For small molecules, competitive immunoassays are performed in which a decrease in the fluorescence signal is observed which is proportional to the antigen concentration. For bacterial cells, sandwich or direct immunoassays are performed which generate an increase in the fluorescence signal proportional to the specific cell concentration.

Accurate monitoring of the environment for pollutants and other toxic chemicals has become increasingly important in recent years. Current technologies, such as gas chromatography/ mass spectrometry (GC/MS), are costly and timeconsuming. A single sample may require up to a week to analyze at a cost of $1000-$2000. The development of antibodies specific for over 50 pollutants and

This chapter not subject to U.S. copyright Published 1996 American Chemical Society In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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organic compounds within the last few years now makes it possible to exploit immunoassay technology for the detection of environmental contaminants.(1) To meet U.S. Navy and U.S. EPA guidelines for monitoring and controlling military/industrial pollution, NRL has developed two biosensors. The continuous flow immunosensor is a relatively simple device for repetitive analyses for small molecules. The fiber optic biosensor is technically more complex, but can be adapted to measure both small molecules and the bacteria capable of degrading them. Continuous flow immunosensor Continuous flow immunosensor detection of small molecules involves a sequence of events that takes place when the analyte of interest is injected into the system. The key elements of the sensor are: 1 ) an antibody specific for the analyte, 2) signal molecules similar to the analyte, but labeled so they are highly visible to a fluorescence detector, and 3) a fluorescence detector. A schematic of the immunosensor lab device is shown in Figure 1. To perform an analysis, the antibodies which specifically recognize the contaminants are immobilized on a solid support and the fluorescently-labeled signal molecules are bound to them, creating an antibody/signal molecule complex. The functionalized support is placed in a small column (typically 100 μ ί bed volume) and connected to a water stream. A sample is then introduced to the system. If the sample contains the target analyte, a proportional amount of the labeled signal molecule is displaced from the antibody and detected by a fluorometer downstream. Once the appropriate operating parameters have been determined, the displacement reaction is highly reproducible and predictable for a given antibody/analyte combination. As seen in Table I, the system has been developed to detect a wide range of compounds, including drugs, explosives, and pesticides.(2,3) Using mathematical equations derived to describe the behavior of the sensor over time(4,5), dose response curves and detection limits can be determined for each assay. The detection limit for the small molecular weight analytes is typically in the low ng/mL (ppb) range. Figure 2 illustrates a typical dose response curve for the explosive trinitrotoluene (TNT) generated using a single antibody column. A number of features make the continuous flow immunosensor wellsuited to in situ monitoring. The instrument can be used for measuring either discrete samples containing small molecules in under five minutes per test or monitoring process streams at selected intervals. Because there are no incubation periods or reagent additions required, the analysis time is minimal, making repeat field measurements of a single sample possible. Alternatively, multiple samples can be examined rapidly. Operational costs are also minimal. Samples collected in a water environment usually require no pretreatment or extractions. Unlike immunoassays using disposable kits, column reagents are not expended if the sample contains no target molecules. Over 50 positive samples (>500 ng/mL TNT) have been run on one column before all the fluorescent signal molecules were depleted. For less concentrated samples, or when fewer samples are

In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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ENVIRONMENTAL IMMUNOCHEMICAL METHODS

Figure 1 : Schematic of the continuous flow immunosensor. The system is comprised of a peristaltic pump, a low pressure sample injector, antibody/fluorescently-labeled analyte column, fluorometer, and signal processor. A continuous aqueous flow stream is established using phosphate buffered saline containing 0.1% Triton X-100 + 12.5% ethanol.



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Figure 2: Standard curve for TNT detection with the continuous flow immunosensor. Fluorescence intensity is in arbitrary units. A minimum of 3 assays was performed for each concentration of TNT tested. The r value for the linear region is 0.998. 2



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tested, a single column has been used over the course of four 8-hour days without changing. Coupled with the small size of the antibody-analyte column (approximately 1/2 in χ 1/4 in), this repetitive sampling capability means that the cost per test is significantly reduced from other technologies. Finally, the laboratory prototype is small enough to fit inside two standard carrying cases. The largest components associated with the system are the personal computer used for directing the system operations and collecting the data, the fluorescence detector and the fluidics needed to pump the water through the column. A research program is currently underway to design and build a fieldable device that is approximately 4 χ 3 χ 7 in. The new instrument will incorporate advances in fluidics, optics and electronics. Beyond instrumentation, other issues must be resolved before use of the continuous flow immunosensor is practical for all field samples. Environmental samples may contain oils, for example, that may significantly interfere with either the antibody columns or the fluorescent signal. We are looking at ways to overcome such matrix effects. Assay conditions, including appropriate antibody affinities, buffer conditions, flow rates, and cross-reactivity to other compounds must be determined for each analyte. We are also actively examining alternative methods for collecting and extracting samples from different environments, such as soil or oil/water mixtures. Fiber optic biosensor The fiber optic biosensor was developed as an ultrasensitive detection system utilizing the sensitivity and specificity of antibodies, the signal-to-noise discrimination of fluorescence measurements, and the rapid signal transduction capabilities of fiber-optic sensing. In this sensor, long optical fibers are employed to permit handling of toxic or hazardous materials away from the instrumentation and operator. The glass core is clad with silicone except for the last 12 centimeters where the cladding is removed to expose the fiber core, thereby creating a sensing region. Antibodies are attached to the fiber core in the sensing region, and the fiber is immersed in an aqueous sample. Detection of analyte occurs via a direct binding of fluorescently labeled analyte to the immobilized antibody (i.e., a competitive immunoassay between the sample and a fluorescently-labeled analyte) or a sandwich immunoassay. In the sandwich immunoassay, the antibody-coated fiber optic probe is exposed to the test sample, rinsed and then exposed to a fluorescently-labeled secondary antibody. All antibody-analyte binding with the fluorescent tag takes place on the surface of the optical fiber within the evanescent wave region (Figure 3). The evanescent wave region is generated when electromagnetic radiation from the light propagating within the core extends beyond the confines of the fiber core. Since the evanescent wave only penetrates about 150 nm into the sample, fluorescent moieties in the bulk solution have little effect on signal levels. Thus the biosensor is particularly well adapted to detection of analytes in heterogenous samples with minimal sample handling. To facilitate application of the sample to the sensing region of the fiber probe, a chamber was constructed from a 200 μ ί capillary tube which can be connected to either a peristaltic pump or a syringe. The volume in this chamber



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can be adapted to a variety of sample sizes by changing the capillary tube. The chamber is disposable, easy to sterilize, small, and protects the fiber from breakage during shipment as well as during use. It is also possible to keep the sample in a totally enclosed system-a feature important to handling of hazardous materials. The capability of the fiber optic biosensor for immunoassay detection of small molecules, toxins, proteins and bacteria has been demonstrated.(6) The selection of antibodies and protocol development involve many of the same types of decisions as other immunoassay procedures as far as specificity and avidity are concerned. A competitive immunoassay was developed for the detection of TNT.(7,8) In this assay, Cy5™-labeled analyte was tested to determine the fluorescent signal associated with the absence of TNT. This labeled analyte was then removed from the immobilized antibodies with 50% ethanol in phosphate buffered saline solution. Next, the test sample containing the labeled analyte and TNT were passed over the fiber. If TNT was present in concentrations > 10 ng/mL, a reduction in fluorescence signal compared to that obtained for the sample without TNT was observed (Figure 4). One method for monitoring bacterial cells is a sandwich immunoassay. In this type of assay, a sample containing bacterial cells is incubated with an antibody-coated fiber optic probe for 5-10 minutes. Next, the probe is exposed to a fluorescently-labeled antibody which also binds to the cells of interest forming a sandwich complex. The fluorescent label is excited when it is bound to the fiber, generating an increase in signal. This assay is being optimized for Pseudomonas cepacia, a bacteria used to degrade trichloroethylene. Another assay for whole cells has been developed which is unusual in that all cells in the sample are stained nonspecifically (i.e., Nile Red dye) and the capture antibody on the fiber specifically binds the cells of interest.(9) The staining procedure is simple and stained cells can be detected in 300 pL samples after 1-2 minutes. Bacillus anthracis concentrations as low as 3000 cells/mL were detected. The dose-response is linear for over two orders of magnitude. To be of practical use, assays must be conducted in real samples, not simply in phosphate buffered saline, to assess the effect of the sample matrix on sensitivity and signal generation. Results similar to buffer tests were obtained in the TNT competitive assay when 90% surface water (river and harbor) or bilge water were used in place of buffer. These 'water* samples were not prefiltered or treated prior to the test and contained particulate matter. From these and other studies (10), it has been demonstrated that the fiber optic biosensor immunoassays were not adversely affected by samples that were opaque, viscous, or contain particulates. Use of the fiber optic biosensor for analysis of environmental samples requires 1) shelf storage of the antibody-coated fiber probes for at least a year, 2) reusability of the coated fibers, and 3) automated sample handling. The first two items make the system more economically feasible. A storage stability test was performed using fibers coated with a polyclonal rabbit anti-goat IgG (specific) or goat IgG (control).(11) Compared with the activity of the



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Figure 3: Evanescent wave immunosensing. Total internally reflected light travels the length of the fiber core with a small portion of the power outside the core in an area referred to as the evanescent wave. The evanescent wave in the sensing region penetrates approximately 120 nm. Only fluorescent complexes bound to the antigen/antibody complex within the evanescent wave are excited, generating a signal.

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TNT Concentration (ng/mL) Figure 4: Standard curve for TNT detection with the fiber optic biosensor. The percent inhibition of the reference signal for various concentrations (1-1000 ng/mL) TNT are shown. A minimum of 3 assays was performed for each concentration with the exception of 200 ng/mL. The r value for the linear region is 0.96. 2



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immobilized antibody on day 0, over 80% of the activity was retained for a year, even when the fibers were stored at room temperature. Extended studies showed that activity in the antibody-coated optical fibers lyophilized in the presence of the sugar trehalose maintained over 60% of their activity when stored at room temperature and 80% of their activity when stored cold (4°C and -20°C) for over 12 months (Figure 5). There are two issues involved when discussing reusability of the antibody-coated fiber optic probe: 1) use after negative and low analyte samples and 2) removal of analyte from immobilized antibody (regeneration). Data indicate that as long as the samples contain low levels of the analyte to be detected, assays can be performed repeatedly on the same fiber.(5) Once the fiber is saturated with analyte, however, the analyte has to be removed prior to additional assays. In addition, the competitive assays are best performed when the maximum fluorescence signal is obtained prior to the test sample but on the same fiber probe. To accomplish this, the labeled analyte must first be removed from the immobilized antibody. The method for regeneration of the immobilized antibody varies depending on the antibody-antigen affinity and chemical properties of the analyte. In the competitive TNT assay, regeneration was achieved with the probe being exposed to 50% ethanol for 1 minute. Fifteen cycles of analyte exposure/regeneration using this protocol were accomplished with < 30% loss of immobilized antibody activity. The loss of activity was accounted for by running a reference sample after every test sample and correcting the % inhibition. The ultimate goal is to have an automated fiber optic biosensor that can be used for field screening. To reach this goal, a prototype of a portable fiber optic sensor that can monitor 4 fiber probes simultaneously has been constructed by Research International (Woodinville, WA) in collaboration with NRL. This sensor is 6 χ 4 χ 2 in. with all the optics, light sources and detectors for each probe on a single computer card. Studies indicated similar or slightly improved signal generation/recovery with this sensor. The improved signal may be due in part to the use of optical fibers to transmit light through the entire system as well as the QA/QC done by the manufacturer. Work is underway to automate sample and reagent delivery to the fiber probe. At least two important problems remain to be solved before the fiber optic biosensor becomes a commercially available, widely used detection system. First, manufacturing techniques for producing the tapered fiber probes inexpensively and in large quantities must be developed. Second, procedures for minimizing fouling and protease degradation of the antibody-coated fibers must be developed for specific applications that require extended periods of use. Once these problems are solved, the fiber optic biosensor will be used for environmental and clinical monitoring. Conclusion The continuous flow immunosensor and the fiber optic biosensor have demonstrated an ability for sensitive detection of environmentally relevant compounds within minutes and should, in the near future, provide viable alternatives for detection of groundwater contamination at designated EPA



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Figure 5: Immobilized antibody activity after storage. Antibodies were immobilized onto a piece of optical fiber and stored under various conditions. A portion of the fibers were stored in phosphate buffer saline (wet), others were air dried, and the last group was lyophilized in the presence of the cryoprotectant trehalose. These groups were then split into subsets for storage at three different temperatures. The percent of the original antibody activity is shown for various storage conditions after 1 year.

remediation sites. For site characterization and continuous monitoring of water effluents, the continuous flow immunosensor would be appropriate. The fiber optic biosensors could be adapted to remote monitoring of toxic agents, hazardous chemicals in storage or production facilities, and various other agents. The spectrum of possible analytes include hazards in closed environments such as engineering spaces or magazines, explosives and byproducts of explosive manufacture, pollutants, drugs or pathogenic organisms. Literature Cited 1. 2.

Van Emon, J.M. and Gerlach, C.L. (1995) Environ Sci Tech 29(7), 312A317A. Ogert, R.A., Kusterbeck, A.W., Wemhoff, G.A., Burke, R., Bredehorst, R., and Ligler, F.S. (1992) Anal. Letts. 25, 1999-2019.


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Whelan, J.P., Kusterbeck, A.W., Wemhoff, G.A., Bredehorst, R., and Ligler, F.S. (1993) Anal. Chem. 65, 3561-3565. Wemhoff, G.A., Rabbany, S.Y., Kusterbeck, A.W., Ogert, R.A., Bredehorst, R., and Ligler, F.S. (1992) J Immunol Methods, 156, 223230. Rabbany, S.Y., Kusterbeck, A.W., and Ligler, F.S. J Immunol. Methods,(1994) 168,227-234. Ligler, F.S., Golden, J.P., Shriver-Lake, L.C., Ogert, R.A., Wijesuria, D., and Anderson, G.P. Immunomethods, 3(2), 122-127, 1993. Shriver-Lake, L.C., Breslin, K.A., Golden, J.P., Judd, L.L., Choi, J.D., and Ligler, F.S. SPIE-Optical Sensing for Environmental and Process Monitoring, (1995) 2367, 52-58. Shriver-Lake, L.C., Breslin, K.A., Golden, J.P., and Ligler, F.S. Anal Chem (1995) 34, 2431-2435. Ligler, F.S., Shriver-Lake, L.C., and Wijesuriya, D.C. U.S. Patent # 5496700, issued March 5, 1996. Cao, L.K., Anderson, G.P., Ligler, F.S. and Ezzell, J. (1995) J Clin. Microbiol. 33(2), 336-341. Ligler, F.S., Shriver-Lake, L.C., and Ogert, R.A. (1992) in Proceedings of Biosensors'92 (Turner, A.P.F., Ed.) pp. 308-315, Elsevier, Oxford.


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Environmental Immunochemical Methods - American Chemical Society

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