Biosensors for pesticides - Electrochemical Sensors, Biosensors and Their Biomedical Applications - page 35

5Mercapto 2 nitrobenzoic acid (yellow)

5,5dithio2nitrobenzoic acid (Chromogen)

AChE

Acetylcholine H 2 O

Thiocholine Acetic acid

Oxidation

2 Thiocholine

2H 2e Dithio bis choline

FIGURE 2.2

Enzymatic formation of thiocholine and detection principles for pesticide.

AChE

Acetylcholine H 2 O Choline Acetic acid

COx

Betaine H 2 O 2

Choline O 2 H 2 O

FIGURE 2.3

Bienzymatic reaction for pesticide detection.

the detection of sulfometuron methyl (herbicide) [19, 46]. Immobilized ALS is more
stable than the native enzyme, but the inhibition potency is higher for the free enzyme.
Polyphenol oxidase (tyrosinase) can also be irreversibly inhibited by atrazin, diazinon,
dichlorvos, etc. Besombes et al. [47] used an electropolymerized pyrrole/tyrosinase
coating for the detection of carbamate pesticides and reported a detection limit of 2

M
for chloroisopropylphenyl carbamate. In another study a tyrosinase electrode was con-
structed by direct adsorption of the enzyme on the surface of a graphite disc electrode.
Three carbamate pesticides, ziram, diram, and zinc diethyldithiocarbamate, inhibited
the tyrosinase in a medium of reversed micella and the detection limits of this ampero-
metric biosensor were 0.074, 1.3 and 1.7

µ

M, respectively [22]. To improve the stabil-
ity and sensitivity of a single enzyme-based biosensor the modifi ed enzymes have been
employed instead of the wild type. A genetically modifi ed Drosophila melanogaster
AChE (DmAChE) allows its oriented immobilization on electrodes [7].
Another stable and versatile method for pesticide detection is the use of the second
enzyme, such as AChE and choline oxidase (COx). Multienzyme-based biosensors use
cholinesterase in conjunction with choline oxidase to measure hydrogen peroxide pro-
duction or oxygen consumption. COx oxidizes choline to betaine and hydrogen per-
oxide (Fig. 2.3). These methods couple an enzyme that is inhibited by the pesticide in
conjunction with another enzyme, which uses the product of the fi rst enzymatic reac-
tion as the substrate. Another bienzymatic amperometric biosensor has been reported
for the detection of dithiocarbamate fungicides [48, 49]. An aldehyde dehydrogenase
and diaphorase were coimmobilized in a PVA-SbQ layer and mounted on a screen-
printed electrode (SPE) with Pt sputtered carbon paste. The detection limits of bienzy-
matic amperometric biosensor for nabam [48] and zineb [49] were 8 and 9 ng mL 1 ,
respectively. Organophosphorus and carbamate pesticides can reversibly inhibit the
catalytic activity of acid phosphatase (AP); hence AP can be reused with normal wash-
ing with buffer. But amperometric detection of AP inhibition requires a bienzymatic
system (Fig. 2.4). Mazzei et al. [20] immobilized AP and GOD on a separate dialytic
membrane. The bienzyme modifi ed membrane was placed on Pt electrode using an

µ