different compartments [ 20 ]. A single sample is then delivered and allowed to
interact with the antibody on the beads in all compartments. In this format, the
number of compartments that can be fabricated and fit within the device is limited,
and that a uniform distribution of the sample across all the compartments must
occur. On the other hand, in most bead-based multiplexed immunoassays, the
microbeads are encoded so that the identity of the antibody conjugated to a
particular bead can be determined by reading and decoding the signal from the
bead. The use of optically encoded microbeads does not require the precise posi-
tioning of the beads because their identity, and hence the identity of the capture
antibody, is determined at the end of the assay by imaging. The most common bead
encoding method for multiplex immunoassays is fluorescence barcoding, which is
mostly analyzed by flow cytometry, whereas in microfluidic implementations, the
beads are usually confined to a monolayer to facilitate simultaneous imaging by
microscopy. A microfluidic platform was developed by Diercks et al. [ 97 ]to
simultaneously detect four different analytes with pg/mL sensitivity from a
2.7 nL sample, using antibody-conjugated microbeads encoded by embedded
fluorophores. A microfluidic platform was developed by Sasso et al. [ 98 ] that
fully automated all step of the multiplex immunoassay using magnetic microbeads
encoded with the Luminex xMAP technology (www. luminexcorp.com). In this
system, magnetic actuation was used to transfer the beads into and out of incubation
and washing solutions, which defined the incubation time and also allowed contin-
uous flow operation to perform continuous concentration monitoring. The binding
kinetics of the assay was empirically characterized to determine the optimal
incubation times, achieving sensitivities in the range 1 pg/mL to 100 ng/mL for
detection of IL-6 and TNF-α, respectively.
Another innovative use of microbeads for multiplex immunoassays is the use of
DNA for barcoding [ 41 , 99 , 100 ]. In this method, a sandwich immunoassay was
carried out on microbeads, where the detection antibody was labeled with gold
nanoparticles that were also conjugated with double-stranded DNA (dsDNA)
barcodes. After the sandwich immunocomplex was formed at the end of the
immunoassay, the dsDNA is denatured, and the dissociated single-stranded DNA
(ssDNA) was detected by either DNA assay, PCR amplification to reach aM
sensitivity or DNA microarray for multiplex detection. This multiplex immunoas-
say combined the ease of manipulation of magnetic beads, the straightforward
signal amplification by DNA amplification, and the high multiplexing capability
of microarrays.
5 Conclusions
In this chapter, representative works on microfluidic immunoassays were catego-
rized and described according to the format of the immunoassay, fluid driving and
handling modalities, and multiplexing. Antibody-analyte interaction is accelerated
due to enhanced transport within microfluidics, resulting in more rapid analysis and
9 On-Chip Immunoassay for Molecular Analysis 239