2.1 Homogeneous Immunoassays in Microfluidic Systems
Homogeneous microfluidic immunoassays rely on the discrimination of analyte-
bound and free antibodies for quantitative measurement. Formation of the
immunocomplex creates differences between the analyte-bound and free antibodies
in diffusion characteristics, [ 3 ] isoelectric point, [ 4 ] and electrophoretic mobilities.
In particular, gel electrophoresis separates molecules based on electrophoretic
mobility due to differences molecular weight and conformation in the case of
non-denaturing gel. Microfluidic immunoassays based on gel-electrophoresis that
separate bound and unbound analytes have recently gained interests [ 5 – 7 ]. Under
non-denaturing electrophoresis conditions, the immunocomplex remains intact in
the gel matrix, usually made of polyacrylamide [ 8 ]. Since the reagents in homoge-
neous immunoassays, especially the antibody, are not immobilized and is free in the
microfluidic channel, a concentration step is usually required to improve the
detection sensitivity. Strategies such as the use of a preconcentration membrane
to enrich the analyte have been employed. A microfluidic system integrating
analyte preconcentration, reagent loading and discrimination of analyte-bound
and non-bound antibody has been shown by Meagher et al. [ 6 ]. A protein analyte
was first loaded into large pore-size polyacrylamide gel, which then migrated under
an electric field onto a preconcentration membrane and trapped. A labeled antibody
was then loaded and concentrated in the same area. Analyte-bound and non-bound
antibodies were then separated in the separation channel containing polyacrylamide
gel with small pore size that is suitable to discriminate the two antibody species.
The assay can be carried out in less than 20 min with 10μL sample and achieved a
detection limit of 10 pM for SEB detection and metalloproteinase-8 using saliva
samples [ 9 ].
Isotachophoresis (ITP) is an electrokinetic method that has been applied in
microfluidic chip as a separation and concentration technique in microchip-based
electrophoresis that eliminates the use of a preconcentration membrane. ITP has
been used as a promising sample pretreatment for a wide variety of analytes such as
protein biomarkers, [ 10 ] and in a number of microfluidic-based immunoassays. In
particular, ITP has been used to preconcentrate the immunocomplex before the
electrophoretic separation step, resulting in enhanced sensitivity and reproducibil-
ity of the assay [ 7 , 11 ]. An example is a microchip known as the “electrokinetic
analyte transport assay” by Kawabata et al., [ 12 ] in which concentration, mixing
and incubation of the reagents and analyte were integrated on-chip using ITP prior
to electrophoretic separation, completing the entire analysis in about 2 min.
Due to its enhanced compatibility with electric force-based fluid manipulation
and the rapid and straightforward separation, capillary electrophoresis (CE) has
been used in microfluidic systems for carrying out immunoassays. Since the first
report of CE-based microfluidic chip for detection of monoclonal antibodies and a
small molecule drug, [ 13 ] the Harrison group has developed advanced CE-based
microfluidic systems comprising of six independent channels for mixing, reaction,
separation and detection within a single wafer. This CE-based chip can perform
9 On-Chip Immunoassay for Molecular Analysis 225