Recent developments in electrochemical immunoassays and immunosensors - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 122

variation of avidin, is used to minimize any non-specifi c binding by charged species
to maintain high binding affi nity for biotin. An electrochemical immunosensor for the
detection of Mycobacterium tuberculosis , based on the immobilization of a capture
antibody using the biotin-streptavidin interaction, has recently been reported [27]. In
this system, biotinylated anti- M. tuberculosis antibody was immobilized on the sur-
face of a streptavidin-modifi ed SPCE. The electrode was initially oxidized in 0.1 mol L 1
H 2 SO 4 by applying an anodic current of 25 A for 2 min. This was to enhance the

adsorptive properties of the SPCE as a result of an increased hydrophilicity. Following
pre-treatment, streptavidin solution was applied to the electrode surface overnight. Any
remaining free sites of the SPCE were blocked with a bovine serum albumin (BSA)
solution. Next, biotinylated anti- M. tuberculosis was applied to the electrode and the
biotin-streptavidin interaction was allowed to proceed for 90 min. Incubation between
antigen M. tuberculosis and monoclonal mouse anti- M. tuberculosis was carried out
remotely, then introduced to the sensor surface for capture by the immobilized capture
antibody. The immunosensor structure was completed by introducing AP-labeled rab-
bit anti-mouse antibody (Fig. 5.5a). The substrate 3-indoxyl phosphate was then intro-
duced and converted to its Indigo product by AP. Indigo was converted to hydrosoluble
indigo carmine and the analytical signal was produced by either cyclic or square-wave
voltammetry. A detection limit of 1 ng mL 1 M. tuberculosis was achieved by this
immunoassay. The results were compared to those of a similar assay, which relied
upon the passive adsorption of monoclonal rabbit anti-mouse antibody directly onto

µ

(b)

(a)

3 IP 3 IP

AP AP AP

AP AP AP

IC IC

Electrode surface Electrode surface

AP

Streptavidin free and AP conjugate

BSA
AP

Biotinylated IgG anti- M. tuberculosis

Antigen Monoclonal anti- M. tuberculosis Rabbit IgG free and AP conjugate

FIGURE 5.5 Schematic representations of the two immunosensor formats: (a) immunosensor based on
the biotin-streptavidin interaction and (b) immunosensor based on rabbit IgG-modifi ed SPCEs. (Reprinted
from [27] with permission from Elsevier.)