Recent developments in electrochemical immunoassays and immunosensors - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 136

Nyquist plot, as shown in Fig. 5.11. A number of features can be readily identifi ed in
a Nyquist plot depending on the rate-determining processes at the immunoelectrode-
solution interface. Trace (i) in the fi gure shows a semicircle with a centre located on
the Z Re axis at higher frequencies, followed by a straight line at lower frequencies.
The semicircle feature is usually associated with the electron transfer-limited process,
while the linear portion is related to the diffusion-limited process of the system. Also,
the semicircle is offset on the Z Re axis (as ) by a value corresponding to the

magnitude of R S . For very rapid electron transfer processes, the Nyquist plot will only
reveal the linear part of the impedance spectrum, as shown by Trace (ii). In contrast, if
the electron transfer process is the rate-determining step, the impedance spectrum will
show a large semicircle without a straight line, as illustrated by Trace (iii). Note that
the diameter of the semicircle equals R ct and extrapolation of the semicircle to lower
frequencies will yield an intercept corresponding to ( R S

ω →

R ct ).
In recent years, several groups have relied on EIS to investigate antibody-antigen inter-
actions on electrochemical immunosensors [62-65]. Lasseter et al. used EIS to investi-
gate the intrinsic electrochemical response resulting from protein binding to a working
electrode [62]. Gold, silicon or glassy carbon electrodes were coated with a monolayer
of biotin that was used to bind avidin. EIS measurements revealed that avidin concentra-
tions in the nanomolar range could be detected without an auxiliary redox species, leading
to the development of label-free electrochemical immunosensing. Many of the reported
impedimetric immunosensors use a conducting polymer (e.g. polypyrrole) to immobi-
lize the capture antibody on the working electrode surface [65-67]. For example, Grant
et al. reported a label-free impedimetric immunosensor incorporating a polypyrrole
fi lm to immobilize the capture antibody [65]. In this protocol, anti-BSA antibody was
co-immobilized with the polypyrrole fi lm on the surface of SPCE. These modifi ed elec-
trodes were then incubated in solutions of different BSA concentrations. Faradaic imped-
ance was measured to construct Nyquist plots before and after binding BSA. In this case,
Z Re yielded a far smaller coeffi cient of variation than Z Im . Based on Z Re as a calibration
signal, a linear response between 0 and 75 ppm of BSA was obtained. Miao and Guan

(iii)

(ii)

(i)

R S R et Z re /

FIGURE 5.11 A Nyquist plot for an electrochemical immunosensor where (i) both the interfacial elec-
tron transfer kinetics (in the high frequency (ω) region) and the diffusion of the redox probe (in the low
frequency region) are rate determining, (ii) the diffusion of the redox probe is rate determining, and (iii) the
interfacial electron transfer kinetics are rate determining over the entire frequency range.