Biosensors for pesticides - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 34

In view of the conductive and electrocatalytic features of carbon nanotubes (CNTs),
AChE and choline oxidases (COx) have been covalently coimmobilized on multiwall
carbon nanotubes (MWNTs) for the preparation of an organophosphorus pesticide
(OP) biosensor [40, 41]. Another OP biosensor has also been constructed by adsorp-
tion of AChE on MWNTs modifi ed thick fi lm [8]. More recently AChE has been cova-
lently linked with MWNTs doped glutaraldehyde cross-linked chitosan composite fi lm
[11], in which biopolymer chitosan provides biocompatible nature to the enzyme and
MWNTs improve the conductive nature of chitosan. Even though these enzyme immo-
bilization techniques have been reported in the last three decades, no method can be
commonly used for all the enzymes by retaining their complete activity.
2.3 ENZYME-BASED BIOSENSORS CONSTRUCTION
2.3.1 Pesticides measuring principles
Mainly, two principles are used in electrochemical pesticide biosensor design, either
enzyme inhibition or hydrolysis of pesticide. Among these two approaches inhibition-
based biosensors have been widely employed in analysis due to the simplicity and wide
availability of the enzymes. The direct enzymatic hydrolysis of pesticide is also extremely
attractive for biosensing, because the catalytic reaction is superior and faster than the
inhibition [27].
2.3.2 Inhibition-based biosensors
Organophosphate and carbamate pesticides are potent inhibitors of the enzyme
cholinesterase. The inhibition of cholinesterase activity by the pesticide leads to the for-
mation of stable covalent intermediates such as phosphoryl-enzyme complexes, which
makes the hydrolysis of the substrate very slow. Both organophosphorus and carbamate
pesticides can react with AChE in the same manner because the acetylation of the ser-
ine residue at the catalytic center is analogous to phosphorylation and carbamylation.
Carbamated enzyme can restore its catalytic activity more rapidly than phosphorylated
enzyme [17, 42]. Kok and Hasirci [43] reported that the total anti-cholinesterase activity
of binary pesticide mixtures was lower than the sum of the individual inhibition values.
In AChE-based biosensors acetylthiocholine is commonly used as a substrate. The
thiocholine produced during the catalytic reaction can be monitored using spectromet-
ric, amperometric [44] (Fig. 2.2) or potentiometric methods. The enzyme activity is
indirectly proportional to the pesticide concentration. La Rosa et al. [45] used 4-ami-
nophenyl acetate as the enzyme substrate for a cholinesterase sensor for pesticide deter-
mination. This system allowed the determination of esterase activities via oxidation of
the enzymatic product 4-aminophenol rather than the typical thiocholine. Sulfonylureas
are reversible inhibitors of acetolactate synthase (ALS). By taking advantage of this inhi-
bition mechanism ALS has been entrapped in photo cured polymer of polyvinyl alcohol
bearing styrylpyridinium groups (PVA-SbQ) to prepare an amperometric biosensor for