Detection of microorganisms and toxins with evanescent wave fiber

Detection of Microorganisms and Toxins with
Evanescent Wave Fiber-Optic Biosensors
Invited Paper
Conventional procedures to detect microorganisms and toxins
in food, water, and human specimens can take hours or days to
perform and may provide inconclusive identification. The complex
nature of many sample matrices as well as the presence of particulate matter in samples often severely reduces the sensitivity and
specificity of conventional bacterial detection systems, especially
those that rely on immunological reactions for capture or detection.
Evanescent wave fiber-optic biosensors can identify such target analytes in minutes directly from complex matrix samples using robust antibody-based assays, significantly improving the detection
sensitivity, selectivity, and speed. In addition, live microbial targets
can be recovered from fiber-optic waveguides to determine microorganism viability, confirm identification, and preserve as evidence.
Keywords—Evanescent wave, fiber optic, biosensor, waveguide.
The detection of microbial pathogens and biological
toxins in food, water, and human specimens is a difficult
problem, characterized by the need for sensitivity, specificity, and speed. High sensitivity is especially important for
the rapid detection of microbial pathogens where the presence of even a few microbial pathogens or toxin molecules
can sometimes be enough to cause disease. High specificity
is also important to minimize background signals and
false-positive results from samples that are often complex,
uncharacterized mixtures of organic and inorganic materials.
Speed is clearly important when dealing with diagnosis,
treatment, and prevention of human infection.
Conventional laboratory methods are often time consuming, require extensive training in microbiology, and
Manuscript received January 9, 2003; revised February 14, 2003. This
work was supported in part by the U.S. Army Soldier and Biological Chemical Command under Grant DAAD13-00-C-0037 and in part by the U.S.
EPA Assistance Agreement GR828138-01-0.
The author is with the Department of Biology and Center for Biological
Defense, University of South Florida, Tampa, FL 33620-5200 USA.
Digital Object Identifier 10.1109/JPROC.2003.813574
give delayed results. Typically, the targeted microorganism
must first be separated from other, background microorganisms in the sample by streaking the sample onto an agar
medium. After 24–48 h of incubation, separated colonies
of microorganisms on the agar plate are visually examined
and further processed by stains, biochemical tests, and/or
immunodiagnostic tests to identify the targeted microbe. In
some instances, the original sample must be homogenized
or incubated in a nutrient enrichment medium prior to
streaking onto an agar medium to aid in isolation of the
desired microorganism.
The situation is somewhat improved with automated
systems, which use similar tests as conventional, manual
procedures to identify microorganisms, but perform these
tests with minimal human involvement [1]–[3]. Automated
systems can identify microorganisms in a few hours and
are easy to use, but require pure cultures and utilize large,
expensive pieces of equipment. Furthermore, automated
systems have been used principally by hospital laboratories; their databases are therefore designed primarily
for human clinical pathogens and not for identification of
microorganisms found in food, water, or the natural environment. Immunofluorescence [4], slide agglutination [5],
and enzyme-linked immunosorbent assays (ELISAs) [6] are
examples of immnodiagnostic tests that utilize antibodies
to identify microorganisms by their antigenic composition.
These tests are moderately sensitive and specific, can often
be performed in a few hours, but typically require at least
10 organisms for detection. If such numbers of microorganisms are not present in the sample, they must first be
grown on agar or in nutrient enrichment broth for at least
one or two days to reach the required levels for testing. Bioluminescent analyzers detect the presence of viable bacteria
by adenosine triphosphate (ATP) bioluminescent assays, but
require moderate to high levels of bacteria for detection and
do not identify specific microorganisms [7]. Nucleic acid
probes, which recognize specific nucleic acid sequences,
and the polymerase chain reaction (PCR), a technique that
0018-9219/03$17.00 © 2003 IEEE
Fig. 1. Biosensor sandwich assay. The target antigen is bound by a capture antibody on the
fiber-optic waveguide. A fluorophore (Cy5)-labeled detection antibody is then attached to form a
sandwich assay. The fluorophore is excited by a laser to generate a detectable signal.
rapidly amplifies microbial DNA or RNA for identification,
are available for identification of some microorganisms
[8], [9]. While such procedures are highly specific, they
require trained personnel and ultraclean facilities, cannot
ascertain pathogen viability, and are difficult to develop
without isolation and extensive characterization of genes
unique to the target organism. In addition, extensive sample
processing is often required due to the presence of inhibitory
substances and particulate matter within sample matrices.
Various other rapid methods for the detection of pathogens
in food and other sample matrices have also been attempted,
such as immunomagnetic-electrochemiluminescence and
immunomagnetic separation with flow cytometry [10],
[11]. These methods, while rapid (total processing time of
a few hours), require sophisticated, expensive, nonportable
equipment, thus limiting their usefulness as real-world
detection systems. Their sensitivities also are often limited.
The development of biosensors has greatly improved the
sensitivity, selectivity, and speed of microbial pathogen and
biological toxin detection. Biosensors are detection devices
that use living organisms or biological molecules, such as
antibodies, nucleic acids, or enzymes, to recognize and
bind target analytes in the sample matrix. After binding, the
presence of the target analyte is detected by electrical signal,
a colorimetric or fluorescent indicator reaction, or some
other recognition response. The American scientist Leland
C. Clark, Jr., is generally considered to be the pioneer of
biosensors, with his development of enzyme electrodes
in 1962 [12]. Clark trapped an enzyme that reacted with
oxygen against the surface of a platinum electrode, using
a piece of dialysis membrane. He then followed activity
of the enzyme, glucose oxidase, by changes in oxygen
concentration. Because glucose oxidase is highly specific, it
reacts only with glucose, producing hydrogen peroxide and
gluconic acid. This biosensor eventually became the concept
for the basis of the commercial glucose analyzer.
Evanescent wave fiber-optic biosensors are biosensors
that utilize evanescent wave detection techniques. Electromagnetic waves propagate within an optical fiber by total
internal reflection at the exposed surface. This process
induces an evanescent electromagnetic field in any surrounding dielectric media, which decays exponentially with
distance from the surface. When fluorescent probes are used
with this system, bound fluorophore molecules immediately
adjacent to the fiber surface are strongly excited, and some
of the fluorescent signal is coupled back into the optical
fiber. The remaining fluorescent signal is scattered and
absorbed before it can pass through the sample. Nonbound
fluorophores further from the fiber surface encounter a
lower field strength and therefore are not effectively excited,
thereby providing considerable protection from bulk sample
fluorescence. The ability to examine relatively dirty sample
matrices and the absence of bulk sample fluorescence make
the evanescent wave biosensor an ideal system for the direct
detection of pathogens in complex matrix samples such as
food, water, and human clinical specimens.
Early versions of fiber-optic biosensors used silica optical
fibers. The proximal ends of the silica optical fibers were
fitted with “ST” optical connectors and the fiber’s distal
ends were tapered by computer-controlled immersion into
hydrofluoric acid after removal of several centimeters of
cladding for attachment of antibodies on the exposed core
Table 1
Typical Biosensor Assay
surface [13]. The optical waveguides were then silanized
with 3-mercaptopropyl trimethoxysilane, incubated with
-succinimidyl-4-maleimidobutyrate, and coated with
streptavidin. Capture antibodies, labeled with biotin, were
then conjugated to the streptavidin-coated waveguides
via a biotin-streptavidin bridge. More recently, 4-cm-long
injection-molded tapered polystyrene optical fibers have
been used for biosensor assays. The ends of the polystyrene
waveguides are dipped in flat black paint to provide a light
dump, and the fibers are coated with streptavidin for linkage
to biotinylated antibodies. Polystyrene optical waveguides
can be inexpensively produced in large quantities at a price
of approximately U.S.$3 each, compared with U.S.$30 each
for silica optical waveguides for use in biosensor assays.
The evanescent wave fiber-optic biosensor is ideally
suited for performing sandwich format fluoroimmunoassays
[14]. Capture antibodies immobilized on the waveguide
selectively bind specific target antigen. Following incubation with a fluorophore-labeled detection antibody to the
antigen, the fluorophore (typically cyanine-5 or Alexa Fluor
647) is excited by a 635-nm laser to generate a detectable
signal. Fluorescent molecules within 100–1000 nm of the
waveguide surface are excited within the evanescent field,
and a portion of their emission energy recouples into the
fiber. Only those labeled antigens captured by antibodies
within the evanescent wave are excited by the laser light,
and background signals from unbound particles contribute
little or nothing to the total measured fluorescent signal (see
Fig. 1 and Table 1).
This principle has been used to detect various analytes,
with a high degree of sensitivity and specificity, using an
evanescent wave fiber-optic biosensor (Analyte 2000) developed by the U.S. Naval Research Laboratory and manufactured by Research International, Monroe, WA (see Fig. 2)
[15]. This instrument uses a 635-nm diode laser to provide
the excitation light that is launched into the proximal end of
polystyrene optical waveguides and can analyze up to four
samples simultaneously using four different waveguides. A
photodiode collects and quantitates the emitted light at wavelengths above 650 nm.
Fig. 2.
The Analyte 2000.
Fig. 3. The RAPTOR.
An automated and portable version of the biosensor for
biowarfare/bioterrorism agent detection in the battlefield environment (RAPTOR, Research International) has been developed which simplifies the assay even further, increases
Table 2
Examples of Analytes Detected by Evanescent Wave Fiber-Optic Biosensors
data reproducibility, and reduces assay time to 12 min (see
Fig. 3) [16]. The RAPTOR utilizes a disposable coupon containing four polystyrene optical waveguides. The compact,
portable (5.45 kg) unit can automatically perform a user-defined, multistep assay protocol to monitor four distinct fluoroimmunoassays on a single sample occurring on the four
disposable optical waveguide sensors.
Evanescent wave fiber-optic biosensors have been used
to successfully detect various analytes, including fraction
1 antigen of Yersinia pestis [17], trinitrotoluene [18]–[20],
E. coli lipopolysaccharide endotoxin [21], pseudexin toxin
[22], Clostridium botulinum toxin A [23], staphylococcal
enterotoxin B [24], ricin [25], and PCR-amplified DNA
[26]. Other microorganisms, including Bacillus anthracis,
Francisella tularensis, Escherichia coli O157:H7, Salmonella typhimurium, Cryptosporidium parvum, and Vaccinia
virus, have been successfully detected with evanescent wave
fiber-optic biosensors (see Table 2). Some of these target analytes are potential bioterrorism agents (e.g., Yersinia pestis,
Clostridium botulinum, Bacillus anthracis, Francisella
tularensis, staphylococcal enterotoxin B, and ricin), whereas
others are associated with food-borne illness (e.g., Escherichia coli O157:H7 and Salmonella Typhimurium) and
waterborne illness (e.g., Cryptosporidium parvum). In some
instances, detection limits achieved by evanescent wave
fiber-optic biosensors are clinically significant. For example,
S. typhimurium has an infectious dose (the number of organisms necessary to cause infection) of 10 colony-forming
units [27]. Cholera toxin produces watery diarrhea when
ingested at levels as low as 10 g [28]. Biosensors can
detect the analytes at levels below the infectious dose.
Cryptosporidium parvum may have an infectious dose as
low as one oocyst [29]. The infectious dose of Escherichia
coli O157:H7 is not known, but may be as low as 1 to 100
colony-forming units [30]. In these instances, biosensors
are not yet able to detect these pathogens at detection limits
low enough to be clinically significant. Detection limits will
need to be improved through more efficient target analyte
capture on waveguides or fluorophore signal amplification.
Biosensors have been used to also directly detect E. coli
O157:H7 in seeded ground beef, apple juice, and other
complex sample matrices with minimal or no processing
[31]–[33] (see Table 3). Complex sample matrices such as
those that occur in food, unprocessed water, and human
clinical specimens present problems for detection technologies because of particulate matter, inhibitory materials, and
background interference. Such problems can be overcome
with evanescent wave fiber-optic biosensors, which flow
samples across the capture antibodies on the waveguide
surface instead of gravity immersion of samples onto capture
antibodies as occurs in immunofluorescence, slide agglutination, ELISA, and other immunodiagnostic procedures.
The use of buffers with appropriate chemicals such as the
nonionic detergent polyoxyethylenesorbitan monolaurate
Table 3
Evanescent Wave Fiber-Optic Biosensor Detection of E. coli O157:H7 in Various Sample Matrices
(Tween 20) to block capture antibody sites and prevent these
sites from binding to nonspecific background materials and
optimization of contact time between capture antibodies and
target analytes during assay can further minimze problems
that normally would be encountered with complex sample
matrices. Recovery of live target bacteria from optical
waveguides, after biosensor immunoassay, has recently been
reported [34]. Waveguides with the captured bacteria are
immersed in nutrient enrichment broth and, after several
hours of incubation, the bacteria can be recovered on agar
plates. Such recovery of live bacteria can be useful to determine organism viability, perform additional confirmatory
tests, and/or preserve evidence for criminal prosecution.
Evanescent wave fiber-optic biosensors now make it possible to detect microbial pathogens and toxins in minutes
rather than days. In addition to their sensitivity and specificity, fiber-optic biosensors avoid many of the problems associated with current rapid methods. The complex nature of
sample matrices that might contain particulate matter often
severely reduces the sensitivity of conventional bacterial detection systems. These traditional detection systems cannot
be used without extensive sample preparation to remove particulate matter and inhibitory substances commonly found
in foods, unprocessed water, or human clinical specimens.
Evanescent wave biosensors are not affected by particulate
matter in sample matrices and are therefore ideal for the rapid
detection of microbial pathogens in food, water, and clinical
Evanescent wave fiber-optic biosensors represent the next
generation of cutting-edge technology with the potential to
rapidly detect and identify microorganisms, toxins, allergens,
and other analytes of interest. Biosensors have applications
not only for food-borne contaminants, but also for waterborne contaminants as well as infectious disease pathogens.
Such modern technology for real-time or near-real-time detection of pathogenic microorganisms and toxins will play
increasingly important roles in the food and water industry
as well as in infectious diseases.
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Daniel V. Lim received the B.A. degree in
biology from Rice University, Houston, TX, in
1970 and the Ph.D. degree in microbiology from
Texas A&M University, College Station, in 1973.
is Professor of Microbiology, Department
of Biology and Center for Biological Defense,
University of South Florida, Tampa. He is
author of a textbook, Microbiology, Third
Edition (Dubuque, IA: Kendall/Hunt, 2003). His
research focuses on pathogenic/environmental
microbiology, with emphasis on virulence
mechanisms of bacterial pathogens and development of rapid procedures to
detect and identify microbial pathogens and toxins. He is well known for
his research on biosensors for rapid detection of microbial pathogens and
for his development of Lim Broth. Lim Broth is the gold standard of the
U.S. FDA and recommended by the CDC for rapid diagnosis of group B
Streptococcus, a major pathogen of newborn infants.
Dr. Lim is a Fellow of the American Academy of Microbiology and a
Member of the American Society for Microbiology Council, and he serves
on NIH and other federal study sections. He received the Florida Governor’s
Award for Outstanding Contribution in Science, the Southeastern Branch
American Society for Microbiology Margaret M. Green Outstanding
Teaching Award, and the P.R. Edwards Award for Outstanding Service and
Accomplishments in Microbiology. His research is currently supported by
more than $2.8 million annually from the Department of Defense, NIH,
U.S. EPA, and other agencies.