Biosensors for pesticides - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 41

2.4.1 Detection methods for pesticide immunosensors
Antibodies are not catalytically active and the direct detection of the binding of immu-
noreagents (Ag-Ab) does not provide enough sensitivity. By labeling the antigen or anti-
body, the sensitivity of immunoassay/immunosensor can be improved. The commonly
used labels are enzymes, fl uorescent compounds or electrochemically active com-
pounds. Enzymatic labels used in immunosensors are usually oxidoreductases such as
horseradish peroxidase (HRP), glucose oxidase or hydrolytic enzymes such as alkaline
phosphatase. The enzyme labeling avoids the unpleasant use of radioisotopes employed
in radioimmunoassay formats (RIA). Among the enzyme immunoassays (EIA), those
based on heterogeneous conditions are most commonly employed and are referred to
as ELISAs. For ELISAs, either Ab or Ag is immobilized on a solid phase to facili-
tate the separation of free and bound fractions. ELISAs can be grouped in three cat-
egories: direct competitive ELISA, indirect competitive ELISA and sandwich ELISA.
Immunosensors can interface with various detection systems such as electrochemical
analysis, piezoelectric detection, surface plasmon resonance analysis, etc.
2.4.2 Immunosensors for pesticides
2.4.2.1 Piezoelectric immunosensors
In piezoelectric immunosensors, the sensor surface is usually coated with an antibody
or an antigen (hapten) and the mass variation induced by the antigen-antibody binding
is correlated to the concentration of the antigen [9]. During the initial studies on pesti-
cides, piezoelectric immunosensor response was monitored after dying sensor surface
[71]. Later pesticide ligands were immobilized on sensor surface and indirect competitive
assay was carried out using high molecular antibody as a tracer [72]. Horáˇek and Skládal
[73] carried out piezoelectric measurements under wet conditions. In this study the piezo-
electric crystals were modifi ed with 2,4-dichlorophenoxyacetic acid (2,4-D) using the
self-assembled monolayer of thio compounds on the gold surface of the crystals. The bio-
sensor was placed in a fl ow-through cell and the affi nity binding of monoclonal antibodies
against 2,4-D on the modifi ed piezoelectric crystals was studied. One measurement could
be completed within 25 min with a detection limit of 0.24 ng mL 1 . In another piezoelec-
tric immunosensor, bovine serum albumin-linked atrazine was covalently immobilized on
silanized piezoelectric crystals activated using glutaraldehyde [74]. The modifi ed piezo-
electric crystal was placed in a fl ow cell and competitive immunoassay was performed
directly in a fl owing solution. These crystals could be easily regenerated using 100 mM
NaOH within 5min. Recently another piezoelectric biosensor has been reported for the
determination of atrazine employing both direct and indirect immunoassay methods [75].
The detection limit for atrazine was 0.025 and 1.5 ng mL 1 with assay times less than 10
and 25 min for indirect and direct immunoassays, respectively.
2.4.2.2 Optical immunosensors
Optical immunosensors are based on the measurement of the absorption or emission
of light induced by the immunoreactants [9]. They can also be based on evanescent