BLBS102-c45 BLBS102-Simpson March 21, 2012 14:38 Trim: 276mm X 219mm Printer Name: Yet to Come
868 Part 8: Food Safety and Food Allergensand nerve tissue, catalyses the hydrolysis of the acetylcholine, a
neurotransmitter, to yield choline and acetic acid. This enzyme is
associated with cognition in mammalian hosts. Compounds that
effect AChE activity include organophosphates and parathion,
which is routinely used as an acaricide and an insecticide. Bo-
tulinum toxin, produced by the bacterial strainClostridium bo-
tulinum, also suppresses the release mechanism of AChE (Rang
et al. 1998). The suppression of activity of this enzyme results in
the accumulation of acetylcholine in the host, which can cause
an excessive over-stimulation of the cholinergic nerves. Death
usually results from the failure of the circulatory and respira-
tory systems (Timbrell 1991). Schulze et al. (2002) developed
an amperometric AChE biosensor that was used for the detec-
tion of carbamate and organophosphate residues in a number
of fruit-containing baby foods and fruit and vegetable samples
taken from four different geographical locations. The protocol
involved the printing of thick film electrodes onto sheets of
polyvinylchloride and ‘curing’ for 30 minutes at 90◦Cpriorto
the introduction of AChE by glutaraldehyde coupling. The ac-
tivity of AChE was analysed by monitoring the formation of
thiocholine by the enzymatic hydrolysis of acetylcholine chlo-
ride. This sensor format permitted the detection of trace levels of
these analytes (lower than 5μg/kg), which included carbofuran,
carbaryl and chlorpyrifos.
Wheat is a common component of food produce that should
be monitored for the presence of pesticide residues. Del Carlo
et al. (2005) quantified the amount of a phosphothionate in-
secticide (pirimiphos-methyl) present in durum wheat using an
electrochemical biosensor. They used an AChE-inhibition as-
say and obtained a calibration curve between 25–1000 ng/mL
with a detection limit of 38 ng/mL. When real samples of du-
rum wheat were analysed, the LOD increased to 65–133 ng/mL,
presumably because of the complexity of the sample matrix.
Cell-based biosensors that are also based on the inhibition
of AChE are useful alternative formats for detecting chemi-
cal contaminants. Here, interactions between mammalian cells
and chemicals, such as pesticides, are conducive to the disrup-
tion of cell membrane permeability, which can be quantified.
To demonstrate how this assay format may be applied, Flam-
pouri et al. (2010) recently prepared neuroblastoma and fibrob-
last mammalian cells by standard cell culture methodologies and
immobilised these on a working electrode through entrapment in
sodium alginate beads. Two chemicals of interest were selected
for investigation, namely diazinon (an insecticide) and propineb
(a fungicide), and were detected at low nanomolar levels on
this platform through the monitoring of changes in membrane
potential. Of particular interest in this study was the ability of
the sensor format to subsequently differentiate between organic
(n=5) and pesticide-treated (n=8) tomato samples that had
14 detectable pesticide residues, including diazinon and thiaben-
dazole. The principle behind this particular assay was the mon-
itoring of cytosolic calcium accumulation in the neuroblastoma
cells, with pesticide residues inducing cell membrane depolar-
isation. This interesting observation demonstrates the potential
of using biosensors to differentiate between organic and non-
organic agricultural produce, which is of particular interest to
the consumer. Furthermore, with the recent trends in biosensorminiaturisation, it is likely that future sensors will permit this
differentiation to be performed ‘on-site’.
Many other examples demonstrate the use of electrochemical
biosensors for monitoring the presence of indicator molecules
that are representative of contamination. In an early example,
Voss and Galensa (2000) used an electrochemical biosensor
to monitor the presence and concentration of amino acids in
fruit juice. Amino acids were separated on a lithium cation-
exchange HPLC column, and an amperometric platform was
subsequently used to monitor the production of H 2 O 2. This assay
could detect amino acid concentrations ranging from 0.1 mg/mL
to5mg/mL(d-proline andd-methionine tol-alanine, respec-
tively) in a range of different analytical samples, including fruit
juice, wine and beer.d-alanine was detected at a concentration
of 0.5 mg/mL, and the authors suggested that the presence of this
amino acid was a result of bacterial contamination. Kriz et al.
(2002) used a SIRE (sensors based on injection of the recog-
nition element)-based biosensor to monitorl-lactate content in
baby food and tomato paste, whereby a small amount of enzyme
(lactate oxidase) was injected into an internal delivery flow sys-
tem and was held in direct spatial contact with an amperometric
transducer by a semi-permeable membrane. Measurements were
determined by the enzymatic conversion ofl-lactate to pyruvate
and H 2 O 2 , and the range of detection ofl-lactate was between
0.10 and 2.51 mM. Here, all assay measurements were com-
pared to an established spectrophotometric assay, with the con-
tributors stating that the biosensor method employed during this
analysis had a distinct advantage, as the measurement could be
performed in less than 3 minutes (as opposed to 30–35 minutes
for spectrophotometry-based analysis).
Additional examples of electrochemical sensor-based de-
tection of contaminants include the development of an im-
munosensor for the detection of 2,4-D based on electrochemical
impedance spectroscopy (Navratilov ́ ́a and Sk ́adal 2004) and,
more recently, a sensor implementing a molecularly-imprinted
conducting polymer, namely poly(3,4-ethylenedioxythiopene-
co-thiopene-acetic acid), for the detection of atrazine (Pardieu
et al. 2009). These platforms had sensitivities of 45 nmol/L and
10 −^7 mol/L for 2,4-D and atrazine, respectively, and demonstrate
the efficacy of using electrochemical detection as an alternative
to optical platforms, such as Biacore, for quality determination.
In recent years, there is a steadily increasing trend for the
development of biosensors in an array format, where multiple
analytes can be measured simultaneously on a single device.
Examples of optical platforms that permit this analysis have
been discussed earlier by Herranz et al. (2008) and Gao et al.
(2009), and many electrochemical platforms are also available.
An enzyme-based three-electrode biosensor for detecting the
presence of markers of maturity and quality in tropical fruits
was developed by Jawaheer et al. (2003). The assay was de-
veloped to quantifiably determine the presence ofβ-d-glucose,
totald-glucose, sucrose and ascorbic acid in pineapples, mangos
and papaya fruit. These markers are indicative of maturity and
quality in selected fruit products and were detected in pectin
(a natural polysaccharide present in plant cells) extracted from
these samples. A fabrication format was developed that permit-
ted the integration of the individual sensors into a multi-sensor