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45 Biosensors for Sensitive Detection of Agricultural Contaminants, Pathogens and Food-Borne Toxins 865et al. 1999). Long linkers (ranging from 18–21 amino acids)
favour the production of scFv formats, which are predominantly
monomeric (Holliger et al. 1993, Perisic et al. 1994, McGuin-
ness et al. 1996). In summary, recombinant antibodies are an
excellent alternative to polyclonal and monoclonal antibodies
for the detection of analytes of interest, and have great potential
for application in the monitoring of quality in the food industry
(Hudson and Souriau 2003, O’Kennedy et al. 2010).Optical Immunosensors for Quality
DeterminationThere have been several excellent examples of biosensor-based
analysis for the detection of pesticides, herbicides, toxins and
bacterial cells, and some of the more pertinent observations that
employ antibody-based recognition (immunosensors) are dis-
cussed in this section. One of the earliest examples demonstrat-
ing the use of antibody-based biosensing for detecting herbicide
residues was described by Minunni and Mascini (1993). Here,
Biacore was selected as a platform to facilitate the detection
of traces (50 pg/mL) of the herbicide atrazine in water sam-
ples. Another optical biosensor was developed to detect and
quantify carbamate residues in vegetables. It was observed that
changes in the concentration of carbamate could be monitored
using chlorophenol red (Xavier et al. 2000). Moran et al. (2002)
used SPR to characterise antibodies that were subsequently
used to detect the presence of 2-(4-thiazolyl)benzimidazole,
a molecule that is used as a food preservative and an agri-
cultural fungicide. Schlecht et al. (2002) implemented a C1
four-channel sensor chip in a study to quantifiably detect
the presence of 2,4-dichlorophenoxyacetic acid (2,4-D), an
organochlorine herbicide, and monitor cross-reactivity of poly-
clonal antibodies with a structurally related analogue, namely
2,4,5-trichlorophenoxyacetic acid (2,4,5-T). This assay was per-
mitted by immobilising 2,4-D analogues onto the surface of a C1
sensor chip surface through a thiol-carboxyl group reaction, and
had a sensitivity of 0.1μg/mL. Finally, Caldow et al. (2005) used
a Biacore Q instrument to detect the bacteriostatic antibiotic, ty-
losin, in bees’ honey. This polyketide is active against most
gram-positive bacteria, mycoplasma, and certain gram-negative
bacteria. They were able to detect tylosin at the level of 2.5μg/kg
in honey, demonstrating the ability of biosensor platforms, such
as Biacore, to detect analytes in complex sample matrices. These
five key examples demonstrate early applications of using im-
munosensors for the detection of low-molecular weight ana-
lytes that have a deleterious effect on the quality of agricultural
produce.
More recently, miniaturised biosensor platforms have been de-
veloped for in situ analysis of pesticide and herbicide residues. A
portable immunosensor for the detection of 2,4-D was described
by Kim et al. (2007). Here, murine hosts were immunised with a
2,4-D-BSA conjugate, and the resultant monoclonal antibodies
were implemented for detection purposes. When tested on spiked
river water samples, this assay format had excellent sensitivity
(0.1 ppb of 2,4-D) and permitted the parallel analysis of multiple
samples. This example also demonstrates how modification of
the assay format can greatly improve sensitivity, which is a keyconsideration for the detection of analytes, such as 2,4-D, which
may reside in food or water samples in trace amounts. A sand-
wich assay format, incorporating a second biotinylated antibody,
was subsequently developed whose sensitivity was significantly
enhanced (0.1 ppt of 2,4-D).
Competitive and inhibition assay formats can be utilised effec-
tively in a number of different biosensor formats for the detection
of pesticide and herbicide residues. In some cases, this can be
applied where the direct monitoring of the interaction between
an antigen and its cognate antibody is not sufficiently sensitive.
To illustrate this, Gouzy et al. (2009) developed a Biacore-based
SPR competition assay for the detection of the herbicide iso-
proturon, as employing a direct detection assay was deemed to
be inadequate. Here, a rat-derived anti-isoproturon monoclonal
antibody was selected for biorecognition, and the competition
assay had a good LOD (0.1μg/L). Salmain et al. (2008) de-
veloped an indirect competition immunoassay format for the
detection of atrazine. The biosensor format implemented was an
IR optical platform that had nanomolar sensitivity.
Salmain et al. (2008) subsequently performed comparative
sensitivity analysis in an ELISA-based assay format, and simi-
lar observations were made. ELISA assays are routinely selected
for immunodetection purposes and, depending on the quality of
the antibody, have excellent sensitivity. However, a major draw-
back relates to the fact that analysis times are often lengthy, with
multiple incubation and washing stages required for assay com-
pletion. This is in contrast to biosensor-based assays that permit
rapid analysis and facilitate the detection of multiple analytes
on a single sensor surface, as opposed to using multiple wells.
Therefore, ELISA formats may be initially used to validate an as-
say format before transferring this to a biosensor platform. This
has been demonstrated by Herranz et al. (2008) for the eval-
uation of leporine polyclonal antibodies specific for simazine
derivatives prior to their implementation in an immunosensor
platform. The resultant assay had a LOD of 1.3 ng/L in contam-
inated water samples, and results were obtained in 30 minutes.
In the biosensor assay described by Herranz et al. (2008),
it was also possible to monitor for cross-reactivity with other
molecules, including structurally-related triazines (propazine
and atrazine), and demonstrate the absence of non-specific bind-
ing to unrelated entities, such as 2,4-D. Biosensors permit rapid
cross-reactivity analysis to be performed, where multiple ana-
lytes may be tested on a single antibody-immobilised surface.
Alternatively, where small analytes are to be detected, numer-
ous structural analogues may be immobilised on different sur-
faces (e.g. in a CM5 sensor chip, four individual flow cells are
available) and tested with a panel of antibodies. This parallel
immunosensing approach was recently demonstrated by Gao
et al. (2009) for the detection of atrazine and four additional
chemicals, namely paraverine, 17-β-estradiol, chlorampheni-
col and nonylphenol. The biorecognition elements implemented
during this analysis were either monoclonal (anti-atrazine, anti-
17-β-estradiol and anti-chloramphenicol) or polyclonal (anti-
nonylphenol and anti-paraverine) antibodies generated in murine
and leporine hosts, respectively. Optical biosensor-based plat-
forms that can sensitively detect multiple contaminants, such
as herbicide or pesticide residues, suggest the way forward for