Recent developments in electrochemical immunoassays and immunosensors - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 121

through the system. The potential of the working electrode was set at 105 mV and a
steady state current was achieved after 5 min. Three different pathogenic microor-
ganisms were tested in this system, with the best results being for Listeria monocy-
togenes , an organism associated with the cause of the rare and potentially fatal disease
Listeriosis. For this organism, the detection limit was 10 cells mL 1 and the analyti-
cal range was between 10 and 1500 cells mL 1 . At cell concentrations greater than
1500 cells mL 1 , the amperometric signal decreased due to the “hook effect” [17-20],
which is a result of a decrease in the immunoreaction between the capture antibody
and the analyte pathogen at high concentrations.
There are also examples of non-competitive assays in the literature for analyzing
different clinically important species. For example, an immunosensor for the patho-
genic bacterium Salmonella typhi and for bacterial toxins from pathogenic Vibrio chol-
erae [21-23].
5.4 MODES OF ANTIBODY IMMOBILIZATION
The manner in which a capture antibody is immobilized on a solid phase is a criti-
cal aspect that requires careful consideration in the design of an immunoassay system,
whether it is competitive or non-competitive. A desirable feature of the chosen method
is that it results in an immobilized capture antibody that is oriented with minimal steric
hindrance to interact favorably with its target antigen. Equally important, it is highly
desirable to immobilize the antibody without a signifi cant change in its ability to bind
its antigen. Clearly, all these features have a direct bearing on the level of sensitivity
and dynamic range achievable by an immunosystem. There are several strategies for
immobilizing a capture antibody on a solid phase including covalent attachment, phys-
ical adsorption or electrostatic/physical entrapment in a polymer matrix. These com-
monly used immobilization strategies are described below.
5.4.1 Biotin-(strept)avidin interaction
Specifi c affi nity interactions for antibody immobilization have been widely used in
immunoassay systems in recent years. The (strept)avidin-biotin interaction is one such
example. This technique may be used to immobilize various types of biomolecules
such as nucleic acids, polysaccharides, and proteins, including the capture antibody in
immunoassay/immunosensor systems [24]. The technique usually involves biotinylat-
ing the capture antibody and coating a solid phase with either avidin or streptavidin.
The dissociation constants of biotin-avidin and biotin-streptavidin interactions are of
the order of 10 15 mol L 1 and are some of the largest free energies of association
yet observed for non-covalent interactions [25]. The complexes also withstand high
temperatures, pH variations, and are resistant to dissociation when exposed to chemi-
cals such as detergents and protein denaturants [26]. Equally important, the use of this
immobilization technique maintains the biological function of the immobilized anti-
body [24]. In some cases, neutravidin, which is an almost neutrally charged (pI of 6.3)