4 Multiplexed Microfluidic Immunoassay Platforms
Multiplex immunoassays facilitate detection of multiple analytes from a single
sample, and are of great importance for various fields, such as medical diagnostics,
drug discovery, and other biological studies [ 86 , 87 ]. Multiplex immunoassays are
commonly performed on surface microarrays and microbead-based multiplexing. A
common characteristic of these two platforms is the reduced volume in which the
antibody-antigen recognition takes place, which is facilitated by microfluidic sam-
ple and reagent handling.
4.1 Surface Array-Based Multiplexing
Antibody-immobilized microarrays have been developed to achieve high-
throughput multiplex immunoassay using chip-based surface microarray, generated
by the immobilization of different capture antibodies on different micro compart-
ments (i.e., microspots in microarrays) [ 88 ]. Each capture antibody is identified by
the spatial encoding (x and y coordinates) on the microarray surface, and can be
analyzed using spatially resolved imaging methods such as microscopy and micro-
array scanner.
Microfluidics has mainly been employed to facilitate fluid handling for reagent
delivery to surface microarrays. Microfluidic channels have been developed to
create linear channels that connect individual rows or columns of a microarray
[ 89 ]. Microfluidic flow cells have also been coupled to microarray [ 90 ]. As men-
tioned in the previous section, microfluidics reduce the diffusion distances within
the microchannels, thereby enhancing transport of analyte and reagents onto the
array surface that leads to faster reaction rate on the surface.
Antibody microarray can be created using microfluidics. A well-known method
developed by Delamarche [ 91 ] used microfluidic networks (μFN) to pattern anti-
body on surfaces. First, antibodies were patterned on the surface of the substrate as
linear strips using the firstμFN. A secondμFN, oriented in such a way that the
channels are perpendicular to the first set of channels, was used to deliver analytes
and reagents for the immunoassays, which subsequently generated signals that can
be imaged optically (Fig.9.5). A number of immunoassays have been developed
based on this patterning method, [ 92 , 93 ] and a more complex system integrating
pneumatic valves to control the delivery of reagent solutions for simultaneous
detection of 5 different analytes from 10 samples [ 94 ]. A modification of the initial
design was reported by Ziegler et al. [ 80 ] where a stencil was used as an alternative
to microchannels for delivering and coating proteins to form microspots [ 95 ].
Using the same concept, a double layer microchannel system was developed by
Shao et al., [ 96 ] which consisted of two layers ofμFN that were perpendicular to
9 On-Chip Immunoassay for Molecular Analysis 237