Biomedical Engineering Reference
In-Depth Information
intensity of incorporated dyes (e.g., BD
TM
Cytometric Bead Array), or color of
incorporated dyes (e.g., Luminex). In the assays that use particles filled with
fluorescent dyes, different dye concentrations are used to label different classes
of beads, while size-encoded classes of beads are measured with scatter signals.
The dye intensity/color and thereby the class of particle are determined in one
or more fluorescence excitation/emission channels, while the quantification of the
target molecules is performed in an additional fluorescence channel. Beads of one
class are functionalized with a specific primary antibody. Such a bead “collects”
antigens (e.g., proteins) from the solution and binds them. After incubation and
washing, a fluorescently labeled secondary antibody is added. This antibody binds
specifically to all target proteins. The amount of secondary antibody bound to the
bead is a measure for the concentration of the target molecule in the sample; it is
detected by the fluorescence intensity of the secondary antibody's label. This means
that the identifying fluorescence of the particle (class) provides a trigger and the
specificity of the system, while the fluorescence of the secondary antibody quantifies
the target analyte concentration - it provides the sensitivity. The concept of using
two antibodies is often referred to as “sandwich assay,” and it is also commonly
used in ELISA. Therefore, bead-based analyte identification and quantification is
frequently called “ELISA on the flow.”
Such multiplexed assays have been developed and commercialized, for example,
for human cytokine profiling [
52
,
53
]. In order to provide additional flexibility in
microfluidic sample preparation, magnetic beads that carry fluorescent identifiers
have also been developed and are commercially available [
54
].
A prominent example for a color-coded multiplexed bead assay is the Luminex
xMap technology [
55
] which provides up to 100 classes by using concentration
combinations of two dyes. These dyes can be excited at 635 nm, and their emission
maxima are around 660 and 710 nm. PE-fluorescence - excitable at 532 nm with
an emission maximum at 575 nm - is used for the quantification of the secondary
antibody. Therefore, the three fluorescent labels sufficiently differ in their excita-
tion/emission spectra to ensure a correct identification of the beads without cross
talk from the identifying dyes into the quantifying channel.
We have demonstrated that our microfluidic detection platform is capable of
analyzing multiplexed flow assays. We have set up an instrument with two lasers
(532 and 635 nm) and three fluorescence channels. Fluorescence light was collected
and filtered with a 535-nm-long pass filter. Subsequently, the detected light was sent
through a 648-nm dichroic mirror. The reflected portion of the light was sent through
a 585/40-nm band-pass filter onto the quantifier channel detector. Light that was
transmitted through the first dichroic mirror was directed through a 635-nm-long
pass filter to a second dichroic mirror (685 nm). This mirror splits the classifying
dye emission to two additional detectors, one filtered with a 660/32 nm and the
other filtered with a 716/40-nm band-pass filter.
Luminex xMap beads were identified, and thyroid-stimulating hormone concen-
trations were detected down to 0:1IU=ml in this setup. Figure
3.7
shows results for
Luminex calibration beads for testing the identifier (CON1) and reporter (CON2)
