Biosensor-Based Technologies for the Evaluation of Quality - Postharvest Biology and Technology of Fruits, Vegetables, and Flowers - page 428

the hinge region of an antibody, resulting in two identical Fab (fragment antigen binding)
fragments. The presence of the constant regions in a Fab is thought to aid in the stabilization
of the antibody variable regions, which might not function efficiently when expressed in
the monomeric single-chain fragment variable (scFv) format (Rothlisberger et al., 2005).
It was recently shown in our laboratory that the Fab antibody format is the most reliable
and sensitive format for use in small molecule competition biosensor assays. The strict
monovalency of this format can lead to a significant enhancement in assay sensitivity in
both ELISA and competition SPR analyses (Townsend et al., 2006).
The scFv is the most widely used antibody fragment. The variable regions ( V H and V H )
of the antibody are linked by a flexible peptide linker. The most frequently used linkers are
based on glycine-serine repeat structures and can be of different lengths. The length of the
linker is related to the intended valency of the molecule. When a short linker is used, the
stability and folding of the scFv do not occur properly. This is caused by the insufficient
juxtaposing of the V H and V L regions in the single chain for the monomer to function. ScFvs
selected in this format invariably form bivalent dimers as a result (Holliger et al., 1993; Kortt
et al., 1997; Atwell et al., 1999), or “diabodies,” which often have increased avidity for an
antigen over the monomeric forms typically observed when long-linker systems are used.
Long linkers (ranging from 18 to 21 amino acids) favor the production of scFv formats, which
are predominantly monomeric (Holliger et al., 1993; Perisic et al., 1994; McGuinness et al.,
1996). The incorporation of these fragments into antibody-based assay formats is important
for providing sufficient sensitivity for a vast range of diagnostic approaches that could be
applied in the food industry (Hudson and Souriau, 2003).
There have been several excellent examples of biosensor-based analysis of fruit and
vegetable products, and some of the more interesting observations are now discussed.
Minunni and Mascini (1993) used Biacore to detect traces of the herbicide atrazine in
water samples by immobilizing an atrazine conjugate onto the surface of the sensor buffer
containing a known amount of free antibody and the herbicide analyte. This competitive
assay yielded a detection limit of 50 pg/mL. Moran et al. (2002) used SPR to characterize
antibodies that were used to detect the presence of 2-(4-thiazolyl)benzimidazole, a molecule
which is used as a food preservative and an agricultural fungicide. Caldow et al. (2005)
used a Biacore Q instrument to detect the bacteriostatic antibiotic tylosin in bee's 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 2.5

μ g/kgin honey.

.

Finally, Schlecht et al (2002) used a C1 four-channel sensor chip in a study to quantifiably

detect the presence of two structurally similar organochlorine herbicides, namely, 2,4-
dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). This
was achieved by immobilizing 2,4-D analogs onto the surface of the C1 chip through a thiol-
carboxyl group reaction, and this enabled 2,4-D to be quantified down to a concentration
of 0.1 μ g/mL.

20.4 Electrochemical sensors

Electrochemical sensors have also been used extensively to detect analytes of interest in
agricultural produce. The biorecognition element in these sensors is in direct contact with a
transducer, and the resulting signal that is generated is converted from a biochemical signal
to an electrical signal. The biorecognition elements incorporated into these devices can