functional carboxyl groups, which form electrostatic interactions with biotin-PLL-
g-PEG. NeutrAvidin labeled-Protein A binds strongly to the biotin, effectively
linking Protein A to the surface, which captured antibody in an orientation-specific
mannerviathe Fc region of the antibody. In addition, the PEG moiety on the
PMMA surface helped reduce non-specific adsorption. This strategy resulted in a
higher analyte capture efficiency than direct covalent linkage of Protein to PMMA.
Antibody can also be directly entrapped in the microfluidic substrate material
during the fabrication step. Heyries et al. [ 30 ] spotted antibody solution on a Teflon
mould by a microarrayer, followed by pouring liquid PDMS. After curing, the
PDMS was peeled off and the antibody molecules were thereby transferred to the
PDMS. The transferred antibody showed efficient analyte capture with the overall
assay sensitivity at ng/mL sensitivity.
2.2.2 Analyte Transport and Delivery
Compared to conventional microtitre wells format, microfluidic systems offer more
efficient mass transport due to the significantly reduced diffusion distances inside
microchannels/microchambers, thus rapid analyses can be performed at small
sample volumes. However, at low analyte concentrations, the transport of analyte
can still be limited [ 31 ]. While there has been significant effort focused on a
reduction of the size of the sensing region, [ 32 ] there is less attention on the
improvement of the design of microfluidic elements. Transport can be enhanced
by further lower the dimensions of the microfluidic features, but there is obviously a
limit at which the devices can be miniaturized before the resistance to fluidic flow
becomes impractical to handle. Because diffusion is the only method for
transporting the analyte to the immobilized capture antibody, and that the replen-
ishment of the analyte at the boundary layer on the capture surface is subject to
mass transport limitations, [ 33 , 34 ] one way to improve analyte delivery to the
capture antibody is to integrate mixing functionalities in the microfluidic system.
Periodically pulsing the analyte solution in simple “forward-backward” flow within
serpentine channels led to higher analyte capture [ 35 ]. Passive mixers built with
micro-dimensioned features such as patterned grooves/herringbone ridges pat-
terned in the top of the microchannel have been shown to increase the sensitivity
of immunoassay by more than 26 % [ 36 , 37 ]. The grooves in the channel induced
mixing of the fluid, thereby encouraging the delivery of analyte to the capture
region, as well as continuously replenishing analyte molecules at the boundary
layer, preventing analyte depletion. An active mixing strategy developed by
Jennisen and Zumbrink [ 38 ] used a bubble introduced into the channel upstream
of the analyte solution, which moved over the sensing surface through the solution,
creating a vortex sheet between the fluid layer close to the surface and the bulk
solution that achieve good analyte transport to the sensor via effective mixing.
Another strategy to create enhanced transport of the analyte to the capture surface
has been developed by Hofmann et al. [ 39 ] The rapid delivery of analyte to capture
antibodies in small volume sample was achieved by a flow confinement method, in
9 On-Chip Immunoassay for Molecular Analysis 227