Recent developments in electrochemical immunoassays and immunosensors - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 125

The polymers may be used in amperometric, potentiometric, and impedimetric immu-
noassay systems [34]. Conducting polymers may provide a direct route of electron
transfer between an enzyme and the electrode surface and, where required, negate
the need for a mediator to shuttle electrons between the enzyme and the electrode.
Conducting polymers may also facilitate “reagentless” or “label-free” immunosens-
ing. A common way to immobilize antibodies involving conducting polymers is
entrapment within the polymeric chains. The antibody is usually co-immobilized with
the polymer onto the sensor surface from a monomer solution containing the anti-
body. However, entrapment may result in denaturation of the antibody, leading to a
loss of activity. Furthermore, a large proportion of the immobilized antibody will be
trapped within the polymer matrix and is thus inaccessible for binding to its antigen.
An alternative to entrapment is covalent attachment of the antibody to active groups
on a pre-immobilized conducting polymer fi lm. Recently, Darain et al. have reported
an amperometric immunosensor based on the conducting polymer poly-terthiophene
carboxylic acid (TTCA), deposited on a screen-printed carbon array (SPCA) for detect-
ing the precursor protein vitellogenin (Vtg) [35]. Electropolymerization was achieved
on the SPCA in a monomer solution of TTCA in 1:1 di(propylene glycol) methyl ether
and tri(propylene glycol) methyl ether [36]. The potential was then cycled three times
between 0.0 and 1.6 V (vs Ag|AgCl). The TTCA-coated SPCA was functionalized with
N-(3-dimethylaminopropyl)-N

-ethylcarbodiimide (EDC) after immersing the array
in a solution of EDC (10 mmol L 1 ) for 4 h at room temperature. HRP and mono-
clonal anti-Vtg antibody were then coupled to the EDC-functionalized polymer. A
competitive immunoassay was established by incubating the sensor in a solution
containing Vtg and glucose oxidase (GOx)-labeled Vtg. When glucose was added,
H 2 O 2 was formed by GOx, and subsequently reduced via the immobilized HRP. The
signal generation scheme is illustrated in Fig. 5.7. The presence of the TTCA layer
ensures that only Vtg-GOx bound by the capture antibody was involved in the enzyme
channeling, and that unbound Vtg-GOx in the bulk solution induced no signifi -
cant electrocatalytic effect. This is often termed a separation-free immunosensor.
The enzyme channeling has a catalytic effect on the amperometric signal, which
was measured at 0.3 V (vs Ag|AgCl). A detection limit (based on four times the

standard deviation of the blank) of 0.09 ng mL 1 of Vtg was achieved using this
immunosensor.

5.4.4 Self-assembled monolayers
Self-assembled monolayers (SAMs) are another attractive method for immobilizing
the capture antibody in immunoassay systems. By taking advantage of the sponta-
neous chemisorption of alkanethiols to such metals as gold or silver, highly ordered
monolayers can be assembled. Gold electrodes are most frequently used because of the
absence of stable oxide under ambient conditions. The fi rst step in the formation of an
alkanethiol SAM is the chemisorption of the sulfhydryl group of the alkanethiol to the
gold surface through the formation of a gold-sulfur bond. One well-accepted model
describing the formation of this bond suggests that it is an oxidative addition of the