Biomedical Engineering Reference
In-Depth Information
and the resulting binding can be monitored by fluorescence microscopy. The
reference solution can then be used to calculate the concentration of the analyte.
More recently, the idea has been extended by Hosokawa and co-workers to
perform a sandwich immunoassay for detecting C-reactive protein (CRP) in
human serum [
45
] (Fig.
3
c). A microfluidic device with a Y-shaped channel
configuration was used to exploit the fast lateral diffusion of small molecules.
Firstly, the channel was coated with anti-CRP followed by a blocking solution to
reduce nonspecific absorption. Human serum containing CRP was then flowed
down one of the inlet channels, where it could diffuse across the interface and bind
to the anti-CRP. A dendritic amplification method was used to increase the limit of
detection by sequentially flowing biotinylated anti-CRP, FITC-labelled streptavi-
din and biotinylated anti-streptavidin. The resulting fluorescent signal allowed
detection of 0.15 pM of CRP in only 0.5 ll of sample. A rapid diffusion-based
immunoassay for detecting theophylline (a therapeutic drug for asthma treatment)
in human serum was recently reported by Tachi et al. [
104
]. This simple design
also used a Y-shaped configuration and involved introducing the sample in one
inlet and fluorescent tracer-antibody complex in the other. Mixing occurred
downstream and the theophylline competes for binding of the antibody. Because
the tracer-antibody complex has a higher mass compared to the tracer alone, the
amount of theophylline could be monitored using fluorescence polarisation with a
relatively simple optical setup. This fluorescence polarization immunoassay
(FPIA) allowed detection of the analyte in the therapeutic range in 65 s. The
advantage of this rapid microchip-based FPIA system is that a complete assay,
from a blood sample collection to detection, can be performed within a few
minutes compared to approximately 30 min with conventional FPIA systems.
Surface immobilised immunoassays in microchips typically involve physical
adsorption or chemical binding of antibodies/antigen onto the surface, which can
be glass PDMS, poly(methyl methacrylate) (PMMA), silicon nitride, polystyrene
or cyclic polyolefin. The sample is then flowed over the surface to allow binding of
the target molecule and the rest are simply washed away downstream. The binding
activity, however, can be reduced due to an unwanted interaction with the surface.
This problem can be overcome by attachment of the antibody via linkers such as
dextran, lipids, DNA and PEG [
73
]. Wen et al. recently reported a novel technique
for immobilising antibodies on PMMA surfaces that enhances capture efficiency
and activity [
114
]. Using biotin-poly(
L
-lysine)-g-poly(ethylene glycol) (biotin-
PLL-g-PEG) as a surface linker, they were able to maximise the repulsive force
between the surface and the antibody, in this case protein A. Firstly, the PMMA
surface is activated using an oxygen plasma, then UV-induced copolymerisation is
used to graft poly(acrylic acid) to produce functional carboxyl groups (Fig.
6
).
To separate out the final immobilised antibody, a mixture of both PLL-g-PEG and
biotin-PLL-g-PEG was introduced, and the final biotinylated protein A was linked
via NeutrAvidin bridges. This method increased the efficiency and the detection
compared to using a PEI linker. This work shows a significant improvement to
on-chip surface immobilisation and could be applied to other immunoassays.
Furthermore, as immobilisation efficiencies continue to improve, there will be less
