Biomedical Engineering Reference
In-Depth Information
A similar study has also been conducted to determine the suitability of ascorbic
acid 2-phosphate (AAP) as an alternative substrate to 4-AP for AP under identical
conditions [48]. Although 4-APP and AAP were suitable substrates for amperometric
immunosensors, 4-APP was superior owing to its sixfold faster enzymatic reaction and
lower detection potential (approximately 200-400 mV). Notably, the lower detection
potential for the hydrolysis product of 4-APP minimizes interferences from other spe-
cies and hence improves the sensitivity of the immunosensor.
The hydrolysis products of common substrates for AP including 4-APP, 1-NP, and
phenyl phosphate (PP) can cause passivation of the surface of the sensing electrode
owing to poor diffusion characteristics, accumulation of electroinactive species, poor
redox reversibility, and polymer formation on the electrode surface [49]. Electrode
passivation will in turn result in inconsistencies in signal reproducibility and reliability,
small amperometric responses, and possibly a complete quenching of the signal prior
to a steady state current being achieved. A relatively new substrate involving hydro-
quinone diphosphate (HQDP) has been developed to minimize this fouling problem
[49]. It is based upon the hydrolysis of HQDP to hydroquinone (HQ) by AP and is
followed by the subsequent oxidation of HQ to benzoquinone (BQ) at the electrode
surface (Scheme 3).
O
P
O
O
O
O
420 mV
vs Ag/AgCI
2H
2e
O
O
OH
AP
O
O
OH
O
O
P
O
O
O
SCHEME 3
A comparison of the products of AP hydrolysis of HQDP (HQ), PP, and 1-NP using
cyclic voltammetry revealed that HQ produced well-defi ned peaks, and that the oxidation
of HQ is reversible. More importantly, no apparent passivation of the electrode surface
was observed even at high millimolar concentrations after 50 scans. Following a series
of investigations, this non-fouling nature of HQ was attributed to the non-accumulation
of its oxidation products on the electrode surface and the good diffusional properties of
HQ at the electrode-solution interface. Another positive feature of HQDP as a substrate
for AP is a tenfold greater oxidation current response of HQ compared to those obtained
in the presence of PP or 1-NP. Overall, HQDP provides a suitable and attractive alterna-
tive substrate system for AP in the development of amperometric immunosensors.
In some instances, the design of an amperometric immunosensor may be such that
the enzyme is located some distance away from the electrode surface, or the presence
of interfering substances in biological samples may require using an alternative electron
transfer pathway. This usually involves a redox-active species with a small molecular
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