We describe the development of an easy-to-use sensor with widespread applications for detecting small molecules. The flow immunosensor can analyze discrete samples in under one minute or continuously monitor a flowing stream for the presence of specific analytes. This detection system is extremely specific, and achieves a level of sensitivity which meets or exceeds the detection limits reported for rival assays. Because the system is also compact, transportable, and automated, it has the potential to impact diverse areas. For example, the flow immunosensor has successfully detected drugs of abuse and explosives, and may well address many of the needs of the environmental community with respect to continuous monitoring for pollutants. Efforts are underway to engineer a portable device in the field

Kusterbeck, Anne W.

Ligler, Frances S.

English

NASA Technical Reports Server

N92-22698
DETECTION OF SMALL MOLECULES WITH A FLOW IMMUNOSENSOR
AnneW. KBtm¢m_ & Frucm S. Ligler
Center for Bio/Molecular Science and Eugineerin&
Naval Research Laboratory,
4,555 Overlook Avenue, Washlulgtom, DC 20375-5000
ABSTRACT
We describe the development of an easy-to-use sensor with widespread applications for detecting small
molecules. The flow immunoseusor can analyze discrete samples in under 1 minute or continuously monitor a
flowing stream for the presence of specific analytes. This detection system is extremely specific, and achieves a
level of sensitivity which meets or exceeds the detection limits reported for rival assays. Because the system is
also compact, transportable, and automated, it has the potential to impact diverse areas. For example, the flow
hnmunosensor has successfully detected drugs of abuse and explosives, and may well address many of the needs
of the environmental community with respect to continuous monitoring for pollutants. Efforts are underway to
engineer a portable device for use in the field.
INTRODUCTION
Market demand for a rapid means of detecting small molecules in diverse environments has increased
dramatically in recent years. Monitoring for environmental contaminants, screening for drugs of abuse, and
controlling fermentation processes all require accurate and sensitive determinations of what compounds are
present in a complex sample. Current methods rely on optical seusins, which does not provide specificity, or
on more complex analytical techniques, which are time consuming and labor intensive. The Center for
Bio/molecular Science and Engineering at the Naval Research Laboratory has developed a sensor which operates
in flow and is capable of detecting small molecular weight compounds in the parts per billion range in minutes
(1,2).
BACKGROUND
The flow immunoseusor capitalizes on the specific recognition between an antibody and its selected
target molecule, the antigen. With the current technologies, large quantifies of antibody with a defined specificity
can be readily produced in the laboratory. This confers a great degree of flexibility on this sensor with respect
to the compounds it can be designed to detect.
Though other antibody assay systems are in use, they rely on either direct binding of antigen (i.e.
sandwich immunoassays) or a competitive binding of antigen versus labeled antigen. Only the latter configuration
has been widely used for the detection of small molecules. The flow immunoseusor relies on a distinctly different
type of antibody-antigen interaction. This device measures antigen displacement events rather than binding
events.
TECHNICAL APPROACH
The key elements of the sensor are: 1) the antibody specific for the target, 2) signal molecules similar
to the target, but labeled so they arc highly visible to a detector, and 3) a detector. Antibodies arc chemically
immobilized on a solid support, for exmnpic glass beads. All the antibody binding sites arc saturated with a
fluorescently-labeled analog of the antigen, creating an antibody/labeled antigen complex. The support is packed
into a 1/2 inch column and counected to a water stream. The target molecule is introduced via a continuous
or pulsed stream under noneqnillbrium conditions. Only the target molecules in a complex sample are
recognized. When the target molecule is present in the sample, the labeled antigen is displaced from the
antibody and moves into the flow stream. The displaced labeled antigen enters the flow cell of a simple
fluorimeter and triggers a response.
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The signal from the detector will be proportional to the number of target molecules in the sample. No reagent
addition is required throughout the assay and analysis time is minimal. A laptop computer is used to control
system hardware, as well as handle data acquisition and analysis. A schematic of the system is shown in Figure 1.
EXAMPLE OF FLOW IMMUNOSENSOR USE
The flow immunosensor developed at NRL has successhdly detected both drugs of abuse and explosives
present at low levels in water samples. For example, to detect cocaine, an antigen to the immobilized antibody
was reacted with fluorescein-labeiled benzoylecgonine, the major metabolite of cocaine. Samples containing
cocaine were introduced into the water stream. Analysis time was rapid, as detection of the cocaine was
accomplished within I minute. Multiple samples could also be detected on the same column. Figure 2 illustrates
the application of 750 ng/mi samples of cocaine to a single column. Over 50 positive samples were detected.
The explosive molecule TNT has also been tested using the flow immunosensor. As shown in Figure 3,
when TNT was introduced into the flow stream, the magnitude of the signal was WoPortioual to the amount of
TNT introduced into the system. The response was specific for TNT, as a compound with a similar ring structure
did not generate a signal.
ADVANTAGES OF THIS APPROACH
The flow immunosensor has many advantages over emting technologies. Operation of the sensor is
straightforward and fast, and does not require a skilled operator or extensive training. The prototype now in use
requires only two computer keystrokes. In its simplest version, the user introduces the sample at the beginning
of the system and records the results within I minute of sample introduction. Again, this is in contrast to the
user intensive and time consuming operation of currently available detection devices. The widely used methods
often require addition of different reagents throughout the assay and lengthy incubation times, or demand the
use of large, sophisticated instruments. In the NRL sensor, all the components required to recognize the target
and release a signal are contained within a mall column.
The flow immunosensor is also well-characterized. Experimental parameters, including column size and
flow rate, have been studied extensively. Usin 8 equations derived at NRL, we are able to predict the behavior
of the sensor for a given antibody-analyte pair. In addition, becanse the immunosensor is antibody-based,
detection is extremely specific for the target molecule.
System manufacturing costs and portability are also important considerations. The components of the
current system are inexpensive and off-the-shelf. Cost for the laboratory prototype is under $10,000, and the
sensor can be engineered to fit into a sinsle briefcase with microprocessor control.
An additional strength of the NRL detector is that it can be used in a variety of environments. It is
readily adaptable for use with individual samples injected by hand, air samplers that extract vapors into water,
or super sipper systems that rapidly inject samples from hundreds of vials.
Finally, the detection limit of the flow immunosensor is already comparable to established, more
complicated systems. Using the NRL sensor, cocaine and TNT in water have been detected at levels of less than
5 parts per billion (equivalent to 5 ng/ml). This level of sensitivity is well-below that obtained nsiag
precipitation, dip stick, ELISA and fluorescence polarization methods, and is comparable to radioimmunoassays.
CONCLUSION
A flow immunosensor has been developed and tested at NRL which will successhdly detect both drugs
of abuse and explosives present at low levels in water samples. This detection system relies on the ability of
sample antigen to displace labeled antigen from antibody under flow conditions. The laboratory prototype is
currently being used to detect cocaine, heroin, and TNT. The advantages of the NRL sensor include its speed,
spedfidty, and versatility with regard to measurement of a range of analytes and sampling conditions.
192
NOTES AND ACKNOWLEDGEMENTS
Two patents (3,4) have been fded covering this technology and are available for license. This work was
supported by the Federal Aviation Administration and the U.S. Customs Service. The authors thank Reinhard
Bredehorst, Robert Ogert, Paul Charles, Jeffrey Chilla, and James Whelan for their scientific contributions. The
views expressed here are the authors' own and do not reflect the policy of the U.S. Navy, Department of Defense
or United States Government.
REFERENCES
. A. W. Knsterbeck, G. A. Wemhoff, P. Charles, R. Bredehorst, F. S. Ligler, A Continuous Flow
Immunoassay for Rapid and Sensitive Detection of Small Molecules, YYmmuno Meth 135, 191-
197 (1990).
, Bredehorst, R., Wemhoff, G. A., Knsterbeck, W. E., Charles, P. T., Thompson, R. B., Ligler,
F. S., & Vogel, C.-W., Novel Trifunctional Carrier Molecule for the Fluorescent Labeling of
Haptens. Anal Biochem 193, 272-279 (1991).
, Ligand-Label Conjugates which Contain Polyoxoanions of Sulfur or Phosphorus, (submitted
4/90).
4. Flow Immunosensor Method and Apparatus, Patent Appfication # 07-486024 (submitted 2/90)
FIGURES
Figure 1.
Figure 2.
Figure 3.
Flow immunosensor operation
Repetitive detection of cocaine samples.
Detection of TNT with the flow immunosensor
193
Sample
Antibod__y__.
__Labeled antigen
eter "I
tCornputer I
Figure I - Flow immunosensor operation. A sample is added to a small column
containing immobilized antibodies saturated with labeled antigen molecules. A
proportional amount of labeled antigen is displaced by the sample and is moved by the
flow stream to the fluorimeter. Signal output from the fluorimeter is transmitted to the
computer and analyzed.
194
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Figure 2 Repetitive detection of cocaine samples. Consecutive samples of cocaine,
200 _ti at 750 ng/ml, were applied to the flow immunosensor. The integrated peak area
of fluorescence intensity was calculated for each sample and plotted.
195
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Figure 3 Detection of TNT with the flow immunosensor
196


Detection of small molecules with a flow immunosensor

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