Recent developments in electrochemical immunoassays and immunosensors - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 129

accessible to bind antigen. Recently, Zhang and Meyerhoff reported an immunoassay
based upon Fab fragments immobilized on the surface of gold coated magnetic

particles for detecting C-reactive protein (CRP) [44]. The immobilization of anti-CRP
Fab fragments on the surface of the gold-plated particles was achieved by incubating

the particles in a solution of the fragments overnight at 4ºC. The Fab

-coated beads
were then incubated with different quantities of CRP. After thoroughly rinsing and
resuspending the particles, excess HRP-labeled goat anti-human CRP was introduced.
Following another washing and resuspension step, the two-site sandwich immu-
noassay was processed by introducing HRP substrate (3,3 -tetramethylbenzidine).

After terminating the enzymatic reaction with H 2 SO 4 , the absorbance of a portion of
the supernatant was measured at 490 nm. A calibration plot was constructed by plot-
ting absorbance against CRP concentration. A detection limit of 0.14 ng mL 1 was
achieved using this immobilization strategy. The results were compared to an identi-
cal system, except that Fab

,5,5

was covalently linked to uncoated magnetic particles via
a tosyl reaction with amine groups of the fragments. This assay yielded a detection
limit of 1.9 ng mL 1 . The results reveal that the self-assembled layer of Fab

fragments
on the surface of gold-plated magnetic particles provides improved orientation for
binding antigen, and is therefore an attractive mode of immobilization. Although this is
not an electrochemical immunoassay system, this type of technology could be applied
to a wide range of solid-phase immunoassays.

5.5 ELECTROCHEMICAL DETECTION TECHNIQUES

In the previous sections, we have discussed various methods for immobilizing capture
antibody on the solid phase of a system. An immunocomplex is next to be constructed
according to an adopted immunoassay format. A means of detection is then needed
to quantitatively determine the amount of analyte present. Electrochemical techniques
have often been used for detection in biosensor technology. This stems from a number
of attributes of electrochemistry including the high sensitivity of electrochemical
transducers, their compatibility with modern miniaturization/microfabrication tech-
nologies, minimal power requirements, economical cost, and independence of sam-
ple turbidity and color. In immunoassays and immunosensors, as most antibodies
and antigens are intrinsically unable to act as redox partners, an appropriate label is
often conjugated to a particular component of the immunocomplex to promote an
electrochemical reaction. The electrochemical signal produced is then used to relate
quantitatively to the amount of analyte present in a sample solution. Potentiometry,
amperometry, voltammetry, and, more recently, electrochemical impedance spectro-
scopic measurements are among the electrochemical detection techniques often used
in conjunction with immunoassay systems and immunosensors, leading to their respec-
tive categories according to the type of signal measured. The fundamental principles
of each of the four techniques are presented below, followed by discussions based
on some recent work that have specifi cally addressed problems encountered in these
areas.