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45 Biosensors for Sensitive Detection of Agricultural Contaminants, Pathogens and Food-Borne Toxins 871Fungal PathogensThe sensitive detection of fungal strains is also of great impor-
tance, due to their association with crop spoilage and inherent
ability to act as human pathogens. A key consideration when
analysing fungal cells and spores by optical biosensing relates
to the fact that they are significantly larger than their bacterial
counterparts. Fungal spores are often over 40μM in diame-
ter, which complicates analysis in platforms, such as Biacore,
where system blockages may result from their direct injection.
Hence, it is possible to utilise modified assay formats, such as
a subtraction inhibition assay (SIA), for the detection of these
large fungal spores (Byrne et al. 2009). The principle behind the
SIA involves the pre-incubation of antigen with a target-specific
antibody. Unbound antibody is subsequently retained (e.g. by
centrifugation or by filtration) and enumerated by capturing on
a biosensor surface immobilised with an antibody-specific cap-
ture immunoglobulin. The observed response is inversely pro-
portional to the amount of pathogen present in the original sam-
ple or culture, and the major benefit of the SIA assay format is
that the system is not exposed to potentially harmful pathogens.
This Biacore SIA format is also applicable for the monitoring
of bacterial cells, includingL. monocytogenes(Leonard et al.
2004).
SIA-based detection of fungal pathogens was demonstrated by
Skottrup et al. (2007a), who pre-incubated a murine monoclonal
antibody with sporangia of the fungal strainPhytophthora in-
festansand used a centrifugation-based protocol for separating
free and bound antibodies. Free immunoglobulin was subse-
quently passed over a Biacore CM5 surface containing immo-
bilised goat anti-mouse polyclonal antibody, which was used to
quantitate free antibody over a pre-determined dilution series.
The LOD was 2.2× 106 sporangia/mL, and no cross-reactivity
was seen when other fungal strains, such asBotrytis cinerea
andMelampsora euphorbiaewere assayed in parallel. A similar
assay was also selected for detecting spores ofPuccinia stri-
iformis, a well-characterised plant pathogen. Here, a polyclonal
rabbit anti-mouse IgM antibody was used for the capture of a
monoclonal antibody (IgM), whose production was elicited in
a murine host immunised with whole urediniospores. The re-
sultant assay took approximately 45 minutes to complete and
permitted the detection of 3.1× 105 urediniospores/mL (Skot-
trup et al. 2007b). These two important examples demonstrate
the applicability of using biosensor-based analysis for the detec-
tion of fungal spores, which is another key consideration for the
evaluation of quality.TOXINS
MycotoxinsMycotoxins are toxic compounds that are synthesised as sec-
ondary metabolites by fungal strains. For example, the fungus
Fusarium moniliformeproduces the carcinogenic mycotoxin,
fumonisin B 1. Mullett et al. (1998) developed a rapid (approx-
imately 10 minutes) SPR method to detect this mycotoxin in
spiked samples. Here, polyclonal fumonisin B 1 -specific anti-
serum (2 mg/mL) was diluted (1:5,000, 1:10,000, 1:1,500 and1:2,000) and immobilised onto gold layers. Next, fumonisin B 1 ,
in concentrations of 0.0–100μg/mL, was subsequently passed
over each surface, prior to measuring the change in SPR. Their
assay had a detection limit of 50 ng/mL. This research group
state that the ‘lab-made’ SPR device used in their research could
be implemented as an early stage method in the screening of
large numbers of potentially contaminated food samples. Pos-
itive samples could then be analysed further by more sensitive
methods of analysis, such as liquid chromatography or elec-
trospray ionisation mass spectrometry (Hartl and Humpf 1999).
More recently, van der Gaag et al. (2003) developed a biosensor-
based assay capable of detecting fumonisin B 1 and three other
analytes, namely zearalenone, ochratoxin A and aflatoxin B 1.
The monitoring of the presence of aflatoxins, which are nat-
urally occurring mycotoxins produced by several strains ofAs-
pergillusspp., in fruit, vegetable and food produce, is also of crit-
ical significance. Produce prone to contamination includes nuts
(almonds, walnuts), cereals (rice, wheat, maize) and oilseeds
(soybean and peanuts). Aflatoxin monitoring is also important
as consumption of infected produce is implicated in carcinoma of
the liver (Bhatnagar et al. 2002, Bennett and Klich 2003). While
approximately 16 structurally diverse aflatoxins have been re-
ported, aflatoxins B 1 ,B 2 ,G 1 and G 2 and M represent the great-
est danger to human health (Keller et al. 2005). Many of these
compounds may be generated inAspergillus parasiticus, while
Aspergillus flavuscan synthesise aflatoxins B 1 and B 2 (Yu et al.
2004).
Several biosensor-based platforms have been developed to
permit the detection of trace levels of different aflatoxins (Lacy
et al. 2006). Daly et al. (2000) used a rabbit-derived polyclonal
antibody to detect AFB 1 , which was conjugated to BSA and
immobilised onto a CM5 Biacore chip. A competition assay
between free and bound AFB 1 had a linear range of detection
(3–98 ng/mL). Daly et al. (2002) subsequently generated murine
scFvs against AFB 1 by using a phage display format and incor-
porated these antibodies into a Biacore-based inhibition assay.
Dunne et al. (2005) developed a unique SPR-based inhibition
assay that incorporated monomeric and dimeric scFv antibody
fragments that could detect AFB 1. Monomeric scFvs could de-
tect between 390 and 12,000 ppb, while the dimeric scFv was
more sensitive, detecting between 190 and 24,000 ppb.
Several other research groups have developed similar rapid an-
alytical biosensor-based platforms for aflatoxin detection. Saps-
ford et al. (2006) developed an indirect competitive immunoas-
say on a fluorescence-based biosensor that permitted rapid de-
tection of AFB 1 in spiked corn (cornflakes, cornmeal) and nut
(peanuts, peanut butter) products. Mouse monoclonal antibodies
were labeled with the fluorescent dye CY5, with detectable sig-
nals inversely proportional to the concentration of AFB 1 present.
Limits of detection for nut and corn products were 0.6–1.4 ng/g
and 1.5–5.1 ng/g, respectively. Adanyi et al. (2007) also recently
described a unique protocol for permitting aflatoxin detection
by using optical wavelength lightmode spectroscopy (abbrevi-
ated to OWLS). Integrated optical wavelength sensors were se-
lected for use in this experiment with the detection range for a
competitive assay being between 0.5 and 10 ng/mL. An indi-
rect screening protocol was subsequently applied to wheat and