Biomedical Engineering Reference
In-Depth Information
The polystyrene probes are inserted after being coated with the recognition
biomolecule and dried. The probes are designed to integrate a combination
tapered sensing region with a lens for signal [25]. The coupon automatically
aligns the probe so that it is in the middle of the fluid channel [26]. In the
Raptor, the light emitted from the end of the fiber is focused onto a collec-
tion lens in the permanent portion of the biosensor, while in the BioHawk,
the light is collected normal to the fiber. The Raptor has been used to assay
for a wide variety of targets, including explosives, toxins, clinical markers,
and pathogens. It has proven to have sensitivities comparable to traditional
enzyme-linked immunoassays (ELISA) using the same recognition molecules,
but with assay times in the 10-15 min range instead of 2-4 h. Furthermore,
the assays can be performed in the presence of highly complex sample matri-
ces, including blood, urine, food homogenates, beverages, ground water, and
e
uents from air samplers. Results of such assays have been reviewed in [1,2].
11.5 Bead-Based Biosensors
The use of beads as biosensors was first made popular by Raoul Kopelman
with his PEBBLES (reviewed by Brasuel et al. in [1]). PEBBLES were small
beads originally including ion- or pH-sensitive dyes that could be injected
into cells to measure local microenvironments. Imaging was performed using
a fluorescent microscope. The Kopelman group expanded the repertoire of
PEBBLES to include the use of more specific recognition elements such as
calcium ionophores for real-time interrogation of cell function.
Building on this approach, other groups developed highly specialized
nanoparticles to measure other intracellular functions. One of the more inter-
esting particles is composed of quantum dots modified with binding protein
and a quenching dye [27]. In this system, a dye molecule is positioned on the
binding protein adjacent to the active site. The dye quenches the signal from
the quantum dot unless the binding protein binds its target. At that point, the
environment of the dye changes so that it can no longer quench the quantum
dot, and a positive signal is generated. Medintz et al. used a broad-spectrum
quenching dye coupled to genetically modified maltose-binding proteins and
demonstrated the capacity of different colored dots to generate signals in re-
sponse to different target analytes.
The next major breakthrough in bead-based sensing was the development
of coded beads that enabled simultaneous measurement of multiple analytes
using beads with different amounts of two dyes. Originally developed for com-
binatorial chemistry applications, the fluorescent coded beads enabled highly
multiplexed assays to be performed in a single fluid compartment. The bun-
dled optrode was perfect for making such measurements (see Yu and Walt
in [2]) as was flow cytometry [28]. In both the cases, beads of each code or
dye ratio are coated with a distinct biorecognition molecule. When the tar-
get binds to the bead of that code, another label is introduced using either
