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872 Part 8: Food Safety and Food Allergensbarley that permitted the detection of AFB 1 and ochratoxin A. A
more recent example of biosensor-based detection of the latter
toxin is provided by Yuan et al. (2009), who selected a Biacore Q-
based platform for their analysis. In this study, a murine-derived
monoclonal antibody was used for the detection of ochratoxin
residues in spiked cereal, beverages, juices (apple and grape)
and wine samples in a competition assay format. Sensitivity was
significantly enhanced by using gold nanoparticles.
Not all biosensor-based technologies for the evaluation of
quality use antibodies for biorecognition, and this is also appli-
cable to the detection of aflatoxins. An example is the develop-
ment of an enzyme-based SPR assay for the detection of AFB 1
and AFG1.The principle behind this assay was the ability of
these aflatoxins to bind to porcine neutrophil elastase. The effi-
cacy of using this approach was demonstrated by the excellent
sensitivity that was observed (low ppb for both analytes), which
demonstrates the potential of using enzymatic biosensors as al-
ternatives to immunosensors for detection purposes (Cuccioloni
et al. 2008).Water and Marine ToxinsMarine toxins pose a considerable threat to human health be-
cause of their potential to cause respiratory, neurological and
gastrointestinal problems, including mortalities at very low toxin
concentrations. This is primarily linked to the consumption of
contaminated shellfish meat. Hence, a number of countries have
established regulations and specific concentration limits to con-
trol the acceptable levels of these phycotoxins in seafood (FAO
2004).
Phycotoxins include a variety of toxins from different phyto-
plankton groups. They vary widely in their physical properties,
chemical structure and toxic mechanisms. The type of poison-
ing and, therefore, the phycotoxin groups are classified accord-
ing to the associated symptoms: paralytic shellfish poisoning
(PSP), neurotoxic shellfish poisoning, amnesic shellfish poison-
ing (ASP), diarrheic shellfish poisoning (DSP) and azaspiracid
shellfish poisoning associated with shellfish; and ciguatera poi-
soning associated with finfish. ASP results from contamination
with the amino acid molecule domoic acid and its derivatives.
Domoic acid is a potent kainoid, neuroexcitatory toxin pro-
duced by the marine diatomPseudo-nitzschia pungens(Pan
et al. 2001). Many species of shellfish bioaccumulate domoic
acid through the consumption of toxin-containing phytoplank-
ton. Furthermore, shellfish known to have been contaminated
with domoic acid include anchovies, crabs, mussels, clams, scal-
lops, gastropods, mackerel and oysters. It was shown that the
first gastrointestinal (nausea, vomiting, diarrhoea and abdom-
inal cramps) and neurological symptoms (memory loss) will
appear 15 minutes post-consumption of contaminated shellfish.
Vision problems may also occur, including diplopia, disconju-
gate and ophthalmoplegia. Severe intoxication will cause sig-
nificant neurological issues, including confusion and seizures,
sometimes leading to coma and death.
SPR has been applied for the detection of domoic acid in
an indirect competitive immunosensor format (Yu et al. 2005).
Domoic acid was covalently linked onto a mixed self-assembledmonolayer (SAM)-modified SPR chip and competition occurred
between free domoic acid and anti-domoic acid monoclonal an-
tibodies in solution. The detection limit was 0.1 ng/mL, which
was lower than what was initially obtained when normal col-
orimetric ELISA analysis was performed. This was due to the
lack of non-specific antibody adsorption on the SAM and the
high sensitivity of the SPR instrument. Stevens et al. (2007)
subsequently developed a portable six-channel SPR system for
domoic acid detection in clam extracts with a LOD of 3 ng/mL
and a quantitative detection range of 4–60 ng/mL. The portabil-
ity of this biosensor is a key advantage over other SPR-based
sensors, as both researchers and governmental shellfish moni-
toring programmes have highlighted the need for rapid in situ
methods for monitoring biotoxin contamination (Kroger et al. ̈
2002).
DSP results from the consumption of molluscs contaminated
with the polycyclic ether toxins okadaic acid (OA), dinophysis
toxin-1 (DTX1) and pectenotoxins (PTX). Yessotoxin (YTX) is
also classified under this grouping as it was first isolated in 1987
from scallops associated with a DSP poisoning event, although
the pharmacological activity of YTXs is different to that of DSP
toxins (Bowden 2006). OA, DTX and PTX are all produced
by dinoflagellates belonging to theDinophysisandProrocen-
trumspecies, whereas YTXs are produced byProtoceratium
reticulatum(Satake et al. 1999). The known marine organisms
susceptible to infection byDinophysisandProrocentrumin-
clude clams, mussels, oysters and scallops. The main symptom
associated with DSP poisoning is diarrhoea, but other symptoms
such as abdominal pain, nausea and vomiting may also occur.
Hospitalisation is not normally required, and no human deaths
have been documented to date.
Llamas et al. (2007) developed a unique SPR-based im-
munosensor for the detection of OA in shellfish extracts. They
utilised a polyclonal antibody generated through repeated immu-
nisation of an OA-bovine thyroglobulin (BTG) conjugate into a
rabbit host and an amine sensor chip surface functionalised with
an OA derivative, OA-N-hydroxysuccinimide. In this competi-
tion assay, 20 mussel samples, negative for OA, were spiked with
160 ng/g of OA. When compared to LC-MS, the immunosen-
sor demonstrated a slight overestimation of OA. Furthermore,
the polyclonal antibody was found to have 40% cross-reactivity
with DTX-1, but when applied to a matrix environment no cross-
reactivity was observed. Subsequently, a monoclonal antibody
was developed against an OA-BTG conjugate (Stewart et al.
2009) that showed a cross-reactivity profile for both DTX-1 and
DTX-2 that corresponded with the toxin potency of all the OA
toxins. The antibody was selected using a novel approach in
which hybridoma screening was carried out in an ELISA com-
petition format, a direct ELISA format and a SPR biosensor
format, using an OA-immobilised CM5 sensor chip.
PSP causes a significant risk to public health due to its high
mortality rate and considerable economic damage. PSPs are
water soluble, thermostable, tetrehydropurine molecules, which
are subdivided into four structural categories; carbamates,N-
sulfo-carbamoyls, decarbamoyls and deoxydecarbamoyls. The
main phycotoxins associated with PSP include the neurotoxins,
gonyatoxin (GTX) and saxitoxin (STX). The carbamate STX is