Biomedical Engineering Reference
In-Depth Information
antibodies, the system produced a limit of detection for
B. globigii
spores of 6.2
10
4
cfu/ml in 15 min under flow conditions.
Fluorescent sandwich immunoassays can be performed using the wave-guiding
properties of optical fibers (41). In this setup, the capture antibody is immobilized on the
outside of the optical element. The excitation source is guided through the core of the
element producing an evanescent field on the outside of the fiber. The field can excite
fluorescent-labeled probe antibodies and guide the excitation signal to the detector. The
RAPTOR fiber optic sensor uses a polystyrene optical fiber arrays and can simultaneously
test for four different analytes (42). The instrument is completely portable and automated
using a diode laser for excitation and photodiodes for detection. Using antibody cocktails
for the detection of multiple analytes and a 10-min assay time, the RAPTOR was used to
detect 98% of the target analytes with detection limits of 5
10
5
and 5
10
4
cfu/ml for
Francisella tularensis
and
B. globigii,
respectively.
A4
4 array sensor utilizing a fluorescent ELISA format has been developed (43).
Again, the sandwich immunoassay was employed. In contrast to other sensors that use
fluorescent-labeled antibodies for detection, this system utilizes signal amplification from
alkaline phosphatase-labeled secondary antibodies. Enzymatic cleavage of the substrate
produces a product that can be excited by a low-power diode laser. The system uses a
multifunctional CMOS biochip that contains a photodiode array and signal processing
integrated into the chip (44). The limit of detection for
B. globigii
was 100 spores.
The autonomous pathogen detection system is a multiplexed, liquid array system that
utilizes polystyrene beads and a flow cytometer (45). The 5.5-
m commercially available
polystyrene beads are imbedded with red and infrared dyes in precise ratios giving each
bead a unique spectral address. Antibodies specific for each target were then immobilized onto
one type of spectrally encoded bead. After the target is captured, it is sandwiched with a
green fluorescent-labeled secondary antibody. When the bead passes through the flow cyto-
meter, the red laser identifies the bead (and therefore, identifies the captured analyte) and
the green laser quantifies the amount of target captured. In theory, this system can analyze a
sample for up to 100 different analytes simultaneously. Using a 65-min assay, the system was
tested for four different analytes and produced limits of detection of 1.54
10
6
and 5
10
6
cfu/ml for
B. globigii
and
Erwinia herbicola,
respectively, with minimal cross-reactivity.
A multiarray sensor that relies on the intrinsic fluorescence of cellular components has
been designed (46). This portable system addresses several important issues: the fluores-
cence is detected from excited metabolites that are present in the cell only during cell
respiration. Therefore, only viable cells should be detected. The intrinsic fluorescence
detection concept also eliminates the need for indirect detection with a secondary
molecule such as fluorescent-labeled antibodies. This detection system is sensitive enough
to detect as few as 20 cells/ml. The system uses a 4
8 array patterned on a glass slide.
As with the detection system, antibodies are not used for cell capture. Instead, small
molecules such as the iron-containing porphyrin, hemin, are patterned onto the slide (47).
A large variety of pathogens that require iron from their hosts will attach to hemin. This
strategy can be applied for screening food or water supplies for general contamination.
For specific detection of organisms such as
Staphylococcus aureus
, small peptides identified
from phage display libraries were also immobilized on the chip. Peptides identified in this
manor have been shown to be very specific for cells and spores, although the exact
binding site is not known (48,49). The above strategy illustrates the concept of multiligand
capture of one target. Detection schemes such as this can provide more confident results
compared with systems that exclusively use a single antibody for pathogen capture.
Figure 19.3 illustrates an example of an “optimal” sensor array based on some of the
technologies discussed above: (A) the sensing surface comprised multiple ligands
(e.g., antibodies, peptides, and hemin) specific for selected-target bacteria. A fourth site,
