Conventional, Molecular, and Biosensor-Based Detection Methods ◾ 555
wave that excites fluorophor molecules located in the antigen-antibody complex. Once excited, the
fluorophors emit light, and the light intensity is proportional to the amount of antigen present,
which is then detected by the detector. The common fluorophor molecules that are used in fiberoptic applications are Cyanine 5 and Alexa-fluor 647, and their respective excitation and emission
wavelengths do not overlap so that they could be easily detected by a fluorimeter. A semiautomated
fiber-optic device manufactured by Research International, Inc., called RAPTOR, is available,
which is built with a sample injection port, microfluidic channels, laser diodes, and a computer
interface for data analysis. The device accepts a coupon containing four fibers; thus a maximum
of four pathogens can be detected at one time. Moreover, the fibers can be reused until a positive
response is recorded.
A fiber-optic sensor has been used successfully for detection of Sta. aureus enterotoxins, Bacillus
anthracis spores, Franciscella tularensis, Bacillus globigii spores, Clo. botulinum toxin, Yersinia pestis, Brucella melitensis, and vaccinia virus. Foodborne pathogens, such as Lis. monocytogenes, Cam.
jejuni, Salmonella, and Esc. coli O157:H7, are detected from different food samples singly or in a
multiplex format. Mycotoxins, such as ochratoxin A, fumonisin B, aflatoxin B 1 , and deoxynivalenol,
are also detected in an array biosensor in multiplex format. Generally, the detection limit for bacterial cells is 10 3 –10 4 cfu/mL, and toxins are detected in the nanogram quantities (0.5–20 ng/mL).
Surface Plasmon Resonance Sensor 29,36
A surface plasmon resonance (SPR) sensor is a powerful tool that can quantify the binding
kinetics of two molecules without the need for any fluorescent tag. SPR is a phenomenon that
occurs during optical illumination of a metal surface and is used for biomolecular interaction
analysis. In this method, antibodies or receptors (receptor binding proteins, RBP) are immobilized on the surface of a thin film of a precious metal, such as gold (resonant layer), deposited
on a reflecting surface of an optically transparent waveguide. The sensing surface is located on
top of or below a high index-resonant layer and a low index-coupling layer. When light passes
through the waveguide, it causes an internal total reflection at the surface of the waveguide.
At a certain wavelength in the red or near-infrared region, the light interacts with a plasma or
cloud of electrons on the high index-metal surface, and the resulting resonance effect causes a
strong absorbance. The exact wavelength of this absorption depends on the angle of incidence,
the metal, the amount of capture molecules immobilized on the surface, and the surrounding
material. The presence of antigens interacting with the antibody causes a shift in the resonance
to longer wavelengths, and the amount of shift is proportional to the concentration of bound
molecules (Figure 42.7).
Advantages of SPR-based sensors are that they allow real-time or near real-time (below 30
minutes) detection of binding events between two molecules. The detection system is label-free,
and thus it eliminates additional reagents, assay steps, and time. The sensor can be reused for the
same analyte. In such a case, the bound analyte is desorbed by changing the pH of the solution.
It is highly sensitive and it can detect molecules in the nanogram range. Several commercial SPR
systems are available: BIACORE, Spreeta, SPR spectroscope, Optrel GbR, Reichert SR7000,
and IAsys.
SPR-based sensors have been used for detection of foodborne pathogens and toxins. In most
applications, this system shows promising results with toxins while signals with whole cells are
inconsistent, and depending on the instrumentations, some are unable to detect whole cells.
Staphylococcal enterotoxin and cells of Esc. coli O157:H7, Salmonella enterica ser. Typhimurium,
Yer. enterocolitica, and Lis. monocytogenes are detected by SPR (Figure 42.8).
wave that excites fluorophor molecules located in the antigen-antibody complex. Once excited, the
fluorophors emit light, and the light intensity is proportional to the amount of antigen present,
which is then detected by the detector. The common fluorophor molecules that are used in fiberoptic applications are Cyanine 5 and Alexa-fluor 647, and their respective excitation and emission
wavelengths do not overlap so that they could be easily detected by a fluorimeter. A semiautomated
fiber-optic device manufactured by Research International, Inc., called RAPTOR, is available,
which is built with a sample injection port, microfluidic channels, laser diodes, and a computer
interface for data analysis. The device accepts a coupon containing four fibers; thus a maximum
of four pathogens can be detected at one time. Moreover, the fibers can be reused until a positive
response is recorded.
A fiber-optic sensor has been used successfully for detection of Sta. aureus enterotoxins, Bacillus
anthracis spores, Franciscella tularensis, Bacillus globigii spores, Clo. botulinum toxin, Yersinia pestis, Brucella melitensis, and vaccinia virus. Foodborne pathogens, such as Lis. monocytogenes, Cam.
jejuni, Salmonella, and Esc. coli O157:H7, are detected from different food samples singly or in a
multiplex format. Mycotoxins, such as ochratoxin A, fumonisin B, aflatoxin B 1 , and deoxynivalenol,
are also detected in an array biosensor in multiplex format. Generally, the detection limit for bacterial cells is 10 3 –10 4 cfu/mL, and toxins are detected in the nanogram quantities (0.5–20 ng/mL).
Surface Plasmon Resonance Sensor 29,36
A surface plasmon resonance (SPR) sensor is a powerful tool that can quantify the binding
kinetics of two molecules without the need for any fluorescent tag. SPR is a phenomenon that
occurs during optical illumination of a metal surface and is used for biomolecular interaction
analysis. In this method, antibodies or receptors (receptor binding proteins, RBP) are immobilized on the surface of a thin film of a precious metal, such as gold (resonant layer), deposited
on a reflecting surface of an optically transparent waveguide. The sensing surface is located on
top of or below a high index-resonant layer and a low index-coupling layer. When light passes
through the waveguide, it causes an internal total reflection at the surface of the waveguide.
At a certain wavelength in the red or near-infrared region, the light interacts with a plasma or
cloud of electrons on the high index-metal surface, and the resulting resonance effect causes a
strong absorbance. The exact wavelength of this absorption depends on the angle of incidence,
the metal, the amount of capture molecules immobilized on the surface, and the surrounding
material. The presence of antigens interacting with the antibody causes a shift in the resonance
to longer wavelengths, and the amount of shift is proportional to the concentration of bound
molecules (Figure 42.7).
Advantages of SPR-based sensors are that they allow real-time or near real-time (below 30
minutes) detection of binding events between two molecules. The detection system is label-free,
and thus it eliminates additional reagents, assay steps, and time. The sensor can be reused for the
same analyte. In such a case, the bound analyte is desorbed by changing the pH of the solution.
It is highly sensitive and it can detect molecules in the nanogram range. Several commercial SPR
systems are available: BIACORE, Spreeta, SPR spectroscope, Optrel GbR, Reichert SR7000,
and IAsys.
SPR-based sensors have been used for detection of foodborne pathogens and toxins. In most
applications, this system shows promising results with toxins while signals with whole cells are
inconsistent, and depending on the instrumentations, some are unable to detect whole cells.
Staphylococcal enterotoxin and cells of Esc. coli O157:H7, Salmonella enterica ser. Typhimurium,
Yer. enterocolitica, and Lis. monocytogenes are detected by SPR (Figure 42.8).