Recent developments in electrochemical immunoassays and immunosensors - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 133

weight (termed a mediator) used to shuttle electrons between the redox center of a per-
oxidase enzyme (commonly HRP or glucose oxidase) and the working electrode sur-
face. Thus, a mediator must possess intrinsically fast electron transfer rates to facilitate
an enhanced heterogeneous electron transfer for peroxidases. Furthermore, a mediator
should exhibit reversible heterogeneous kinetics, a low overpotential for regeneration,
and be stable under the required range of physical conditions [45]. Recently, a mediated
electrochemical immunosensor using single walled carbon nanotube forests has been
demonstrated for detecting biotin-HRP and unlabeled biotin, which, in a competitive
immunoassay, compete for a limited number of antigen-binding sites on immobilized
anti-biotin capture antibody [50]. The mediator of choice in this system is HQ, which
is used to facilitate a coupled catalytic reduction of hydrogen peroxide by transferring
electrons between HRP and the peroxide. Detection limits of 2.5 nM and 16 M were

achieved for biotin-HRP and unlabeled biotin, respectively. In some systems, leaching
of the soluble mediators into the bulk solution may become a problem by reducing the
sensitivity of the immunosensor.
A drawback of the enzymatic signal-generating scheme described above is that it is
an indirect detection technique. Moreover, AP and other phosphatases present in bio-
logical samples such as serum and plasma could interfere in assays if not removed by
washing steps following specifi c interaction of sample analyte with its specifi c anti-
body reagent. Further, as some HRP substrates are carcinogenic [51], it is important to
select one that is safe for routine operation with an immunosensor. There has thus been
interest in developing a more direct detection technique involving electroactive labels.
In general, ferrocene derivatives are known to be fast, reversible redox compounds that
can be used as mediators in enzyme biosensors [52-55]. Among them, ferrocenecar-
boxylic acid is useful as it is a water-soluble derivative and can be easily conjugated to
IgG via EDC-modifi ed carboxylic acid terminals [56]. Upon applying an appropriate
potential, the ferrocenecarboxylic acid is oxidized rapidly without involving any inter-
mediate product. Therefore, ferrocenecarboxylic acid is a superior candidate for use
as an electroactive label conjugated to a biological component for developing a direct
signal-generating scheme at an electrochemical immunosensor. In our laboratory, we
have demonstrated ferrocenecarboxylic acid as an electroactive label, conjugated to
the signal antibody of a sandwiched immunocomplex, for the quantitative determina-
tion of the tumor marker, human chorionic gonadotrophin with a detection limit of 2.2
IU L 1 [57]. Similarly, Okochi et al. have used antibodies conjugated with ferrocen-
carboaldehyde as an electrochemical probe for detecting IgG in an on-chip fl ow immu-
noassay [58]. Recently, Hromadová et al. demonstrated the organometallic compound
(

µ

5 -cyclopentadienyl)tricarbonylmanganese (or cymantrene), as a redox label bound to
BSA [59]. Electrochemical detection was based on impedance measurements (see sec-
tion 5.5.4) of a one-electron reduction of the organometallic label.

η

5.5.3 Voltammetric immunoassays
The development of electrochemical immunosensors generally involves the immo-
bilization of an immunocomplex on a single electrode, followed by detection via the