simultaneous direct immunoassays for ovalbumin and anti-estradiol using fluores-
cence detection, completing the assay within 1 min [ 14 ]. CE-based chips have also
been integrated with electrochemical measurement [ 15 ] for sensitive detection, as
well as high throughput immunoassays in competitive mode [ 16 ]. In addition,
CE-based chip can be adapted for continuous flow, replacing used reagents and
buffer with fresh ones, thereby prolonging CE to 24 h for monitoring insulin
secretion from islet [ 17 , 18 ].
2.2 Heterogeneous Immunoassays in Microfluidic Systems
In heterogeneous immunoassays, antibodies are immobilized on the surface of a
solid support. In general, the solid support can either be the surface of the
microfluidic device or on beads typically ofμm-dimension that are embedded in
the channel or chamber within the device. Whereas antibody immobilization is a
key step because it can dramatically influences the specificity and sensitivity of the
assay, strategies for analyte delivery and washing can also affect the performance of
the assay in terms of analysis time and limit of detection.
2.2.1 Antibody Immobilization
Microfluidic substrates are commonly made from materials including glass, silicon,
silicon nitride [ 19 ], polydimethylsiloxane (PDMS)[ 20 , 21 ], polymethyl methacry-
late (PMMA) [ 22 ] and polystyrene [ 23 ]. The simplest method to immobilize
antibody onto a microfluidic substrate is by physical adsorption. While being
extremely simple, this method often results in significant reduction in antibody
activity due to steric hindrance, random and unfavourable orientation and denatur-
ation of the antibody [ 24 ]. Due to these shortcomings, more controlled and specific
immobilization strategies by bioaffinity and covalent immobilization have been
developed.
One strategy is the use of a long covalent linker to “tether” the antibody, which
keeps the antibody a defined distance away from the surface, thereby maintaining a
chemical environment around the antibody similar to the bulk solution, minimizing
problems associated with accessibility and steric effects. Indeed, different strategies
for covalent immobilization of antibodies on silicon substrates have been compared
by Yakovleva et al., [ 25 , 26 ] demonstrating that long and flexible linkers such as
polyethyleneimine (PEI) [ 27 ] and dextran [ 28 ] led to more favourable
immunoreaction than short linkers such as 3-aminopropyltriethoxysilane
(APTES). Antibody functionality can also be maximized by the use of bioaffinity
interactions. A novel antibody immobilization strategy on PMMA surface was
reported by Wen et al., [ 29 ] who used a surface linker biotin-poly(L-lysine)-
graft-poly(ethylene glycol) (biotin-PLL-g-PEG) for immobilization. The PMMA
was activated by plasma, followed by coating with poly(acrylic acid) to add
226 A. Ng