Recent developments in electrochemical immunoassays and immunosensors - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 130

5.5.1 Potentiometric immunosensors
Potentiometric immunosensors rely upon a change in potential that occurs between
an indicator and a reference electrode as a result of specifi c interaction between an
antibody and its antigen [45]. There have hitherto been few reports of immunosen-
sors relying on potentiometric detection. One of the main disadvantages of this type
of detection is the relatively small change in potential that arises from the interaction
between an antibody and its antigen. Moreover, interferences from the sample matrix
may prevent this small signal from being successfully detected. Thus such sensors
often have a compromised reliability and sensitivity. A recent example of a potentio-
metric immunosensor involves the detection of enzyme-labeled immunocomplexes
formed at the surface of a polypyrrole coated screen-printed electrode [46]. In con-
structing this electrode, an electropolymerization strategy involving cyclic voltamme-
try in the presence of aqueous pyrrole and sodium dodecyl sulfate was carried out for
at least four cycles. A constant potential was then applied to allow the polypyrrole fi lm
to settle into its fi nal state. In addition to being stable at 37ºC for at least 4 months,
these polypyrrole coated electrodes showed improved sensitivity. Capture antibody
was immobilized on the polypyrrole layer by either direct adsorption or by binding
biotinylated antibody to streptavidin coated polypyrrole. The immunoelectrode was
then incubated in a sample solution containing either hepatitis B surface antigen or the
cardiac marker troponin I. A sandwich immunoassay was completed by introducing
a signal antibody conjugated to HRP. Potentiometric measurements were conducted
in 0.01M PBS (pH 7.4). An active substrate, o -phenylenediamine dihydrochloride,
was added to initiate an enzyme turnover, and the potential was recorded after 60s.
Note that a deliberate separation of the immunoreaction from the detection step was
employed to achieve effective minimization of matrix interferences. The change in
potential was found to be proportional to the extent of the antibody-analyte reaction,
and therefore the concentration of analyte in the sample. However, the mechanism for
the change in potential, induced by the conversion of o -phenylenediamine dihydro-
chloride to 2,3-diaminophenazine by HRP, is somewhat unclear and was referred to as
a “charge-step procedure”. It was proposed that a change in pH and ionic strength of
the solution as a result of the enzymatic reaction altered the physical (porosity, density,
thickness) and electrochemical (conductivity, charge) properties of the polypyrrole
layer, resulting in the observed potential shifts. The sensitivities for hepatitis B surface
antigen and troponin I were reported to be 50 fmol L 1 and 0.4 pmol L 1 , respectively.
5.5.2 Amperometric immunosensors
In amperometry, the current produced by the oxidation or reduction of an electroac-
tive analyte species at an electrode surface is monitored under controlled potential
conditions. The magnitude of the current is then related to the quantity of analyte
present. However, as both antibody and antigen are not intrinsically electroactive, a
suitable label must be introduced to the immunocomplex to promote an electro-
chemical reaction at the immunosensors. In this respect, enzyme labels including the