Open Journal of Applied Biosensor, 2012, 1, 1-8
http://dx.doi.org/10.4236/ojab.2012.11001 Published Online May 2012 (http://www.SciRP.org/journal/ojab)
Electrochemical Biosensors for Determination of
Organophosphorus Compounds: Review
Anjum Gahlaut1, Ashish Gothwal2, Anil K. Chhillar2, Vikas Hooda2*
1Department of Biotechnology & Molecular Medicine, Pt. B. D. Sharma University of Health Sciences, Rohtak, India
2Centre for Biotechnology, Maharshi Dayanand University, Rohtak, India
Received April 2, 2012; revised May 3, 2012; accepted May 11, 2012
ABSTRACT
In last few decades there is exponential increase in use of organophosphorus (OP) compounds as pesticides and insecti-
cides leading to adverse effect on human population and live stock. There is a great need to develop portable analytical
tools that are amenable for remediation and bioremediation process monitoring, where rapid analysis of large number of
samples is essential. Determination of various organophosphorus compounds has been achieved by integrating bio-
components with different transducers. The close integration of the biological events with the generation of a signal
offers the potential for fabricating compact and easy-to-use analytical to ols of high sensitivity and specificity. With the
availability of new materials, associated with new sensing techniques has led to remarkable innovations in the design
and construction of organophosphorus biosensors. The present review describes the specifications of most of the elec-
trochemical Organophosph orus biosensors reported till d ate.
Keywords: Organophosphorus Compounds; Acetylcholinestrase; Tyrosinase; Organophosphorus Hydrolase;
Electrochemical; Biosensor
1. Introduction
Organophosphates (OPs) are usually esters, amides or
thiol derivatives of phosphoric, phosphonic, or phosphinic
acids, which have general structural formula (Figure 1)
where R1 and R2 are alkyl-, alkoxy-, alkylthio-, or ami-
do-groups. X is the acyl residue (labile fluorine-, cyano-,
substituted or branched aliphatic, aro matic, or heterocyc-
lic groups) [1,2].
Organophosphate (OP) compounds have found wide
applications as pesticides and insecticides in agriculture
and as chemical warfare agents in military practice.
Worldwide, OP compounds account for over 38% of the
total pesticides used [3]. Commonly used organophos-
phates includes parathion, malathion, methyl parathion,
chlorpyrifos, diazinon, dichlorvos, phosmet, fenitrothion,
tetrachlorvinphos and azinphos methyl. Malathion is
widely used in agriculture, residential landscaping, pub-
lic recreation areas and in public health pest control pro-
Figure 1. General structure of organophosphorus compounds.
grams such as mosquito eradication [4]. According to
World Health Organization, every year there are three
million pesticide poisonings, mostly OP-related, and
200,000 deaths worldwide that are attributed either as
self-poisoning or occupational exposure [5]. Besides
human exposure, there is also concern that these pesti-
cides could leak into ground and municipal water sup-
plies and pollute surrounding environment. Reports in
the literature have expressed concern over exposure to
non target organisms such as birds and fish, as well as
the potential for human exposure from sources such as
fresh fruits and vegetables and processed foods. These
neurotoxic compounds, which are structurally similar to
the nerve gases Soman and Sarin, irreversibly inhibit the
enzyme acetylcholine esterase, essential for the func-
tioning of the central nervous system in humans and in-
sects, resulting in the build up of the neurotransmitter
acetylcholine which interferes with muscular responses
and in vital organs produce serious symptoms and even-
tually death [6-8]. Effective methods for degradation/
disposal of these toxic compounds are needed to ensure
that human and environmental health will not be com-
promised by the continued use of OP-containing pesti-
cides. Analytical tools to properly monito r the food qual-
ity, control any treatment of water may be adopted Labo-
ratory-based methods which are commonly used for de-
*Corresponding a uthor.
C
opyright © 2012 SciRes. OJAB

A. GAHLAUT ET AL.
2
tection and measurement of OP pesticide residues in-
clude gas chromatography (GC), high-performance liq-
uid chromatography (HPLC), and capillary electrophore-
sis [9,10]. The bioanalytical methods primarily include
assays based on enzyme inhibition and immunoassay [11,
12]. Enzyme linked immunosorbant assays (ELISA) are
quite sensitive to specific compounds such as ethylpara-
thion or fenitrothion but, like most immunoassays require
multiple incubations and generate contaminated plates,
tubes, etc. In addition, the characteristics of cholineste-
rase-based assays and immunoassays for OP pesticides
are not well suited to process control monitoring applica-
tions as these are typically expensive and time-consum-
ing, further more requires trained man power. Also, labo-
ratory-based methods are not amenable to remediation
and bioremediation process monitoring where rapid ana-
lysis of large number of samples is essential. Organo-
phosphorus hydrolase (OPH) catalyzes the hydrolysis of
a wide range of OP pesticides [13]. The hydrolysis in-
volves a pH change, as well as electroactive species gen-
eration. OPH-based assays respond to OP compounds as
enzyme substrates rather than inhibitors or antigens this
is not the case with acetylcholine esterase. Consequently,
these assays can be reversible and require only the ana-
lyte of interest. However this method has disadvantages
that it employs the free enzymes which can be used once
only and the measurement is based on change in pH
which limits its sensitivity. Biosensing approach was
used to overcome problems of onsite monitoring sensi-
tivity, reliability and ability to screen large number of
samples.
2. Electrochemical Biosensor
The working of electrochemical biosensors is mainly
based on the use of a biological component/bio-receptor
element retained in direct contact with an electrochemi-
cally active transducer (electrode) to obtain an analyti-
cally useful signal by coupling biochemical and electro-
chemical interactions [14]. The principle of electroche-
mical sensors is that when an electro-active analyte is
subjected to fixed or varying potential of some prede-
fined patterns causes oxidation or reduction of analyte on
the working electrode surface, which leads to the genera-
tion of an electrochemically measurable signal by the
variation on electron fluxes. This signal can be measured
by the electrochemical detector.
2.1. Electrochemical OP Biosensor Based on
Enzyme Inhibition Process
Biosensors based on enzyme inhibition have found wide
application for detection of toxic analyte (e.g., OP pesti-
cides) which inhibit the fun ctional activity of the enzyme.
By determining the differences in enzyme activity with
or without the presence of an inhibitor form the basis of
analyte detection, according to the Equation (1):


0i0
I%AA A100

 

(1)
where A0 is the activity without an inhibitor, and Ai is
with an inhibitor. The linear range is usually comprised
between 20% and 80% of inhibition and the detection
limit is usually defined as the amount of inhibitor which
gives the decrease 20% of inhibition [15].
2.1.1. Us e of Acetylc holinesterase (Ac hE) Enz yme for
Preparation of OP Biosensors
The enzyme inhibition-based biosensors for the determi-
nation of OP pesticides is described by the following
mechanism (2) [16].
Phosphorylated AchE enzymes has lower affinity for
the substrate (Acetylcholine) called enzyme inhibition
and the degree of inhibition is proportional to the con-
centration of OP compounds in the sample. Acetylcholi-
nesterase (AchE) inhibition test, using AchE modified
amperometric transducers is based on the measure of
para-Aminophenol produced by hydrolysis of p-Amino-
phenyl acetate, or hydrogen peroxide generated as a re-
sult of the oxidation of choline produced from acetylcho-
line hydrolysis in the presence of choline oxidase. The
inhibition of AchE enzyme due to the presence of OP
compounds results in reduced reagent consumption and
products release is correspondingly detected applying
electrochemical techniques and is correlated to the OP
pesticides concentration.
AchE enzyme was used in combination with different
types of supports for the fabrication of bio-sensing de-
vices [17-34]. Table 1 summarizes the characteristics of
different AchE-based biosensors. Although, sensitive
biosensors based on AchE inhibition have few limitations:
1) since ChE is inhibited by neurotoxins which include
not only OP pesticides but also carbamate pesticides and
many other compounds, these analytical tools, are not
selective and cannot be used for quantitation of either an
individual or a class of pesticides which may be required
o monitor detoxification processes, for example, detoxi- t
 
 
22
O O
EH OR P-X RO P-E HX
Enzyme OP pesticide Phosphorylated enzyme

‖ ‖

(2)

A. GAHLAUT ET AL.
3
Table 1. Characteristics of electrochemical Acetylcholinesterase-based biosensors for OP pesticides detection.
Sr. No. Target analyte Detection technique Enzyme immobilization
technique Electrode/transducer Linearity range (M) Detection limit Ref.
1. Paraoxon Amperometry Adsorption
AuNPs, grapheme oxide
nanosheets ND 10–13 [34]
2. Chlorpyrifos oxon Amperometry Entrapment 7,7,8,8-tetracyano
quinodimethane 6 × 10–9 - 2.4 × 10–9 6 × 10–9 [39]
3. Chloropyrifos Amperometr y Covalence ZnS NPs Au 1.5 × 10–9 - 4 × 10–8 ND [37]
4. Paraoxon Amperometry Affinity MWCNT 3.6 × 10–14 - 3.6 ×10–11 5 ×10–15 [40]
5. Chlorpyrifos oxon CV Entrapment PEDOT:PSS ND 4 × 10–9 [38]
6. Chloropyrifos SWV Cross-linking SWCNT 10–11 - 10–6 10–12 [36]
7. Chloropyrifos CV Covalent binding Exfoliated graphite
nanoplatelets ND 1.58 × 10–10[35]
8. Paraoxon Amperometry Entrapment - 1.3 ×10–7 - 5 ×10–6 3.5 × 10–2 [41]
9. Paraoxon Ampe rometry Cross-linking CoPc-Prussian Blue 7.3 × 10–9 - 1.8 × 10–8 7.3 × 10–9 [42]
10. Methyl para o x o n Amperometr y Entrapment CoPc 2 × 10–9 - 4 × 10–6 2.6 × 10–9 [43]
11. Triazopho s Amperometry Adsorption MWCNT 3 × 10–8 - 7.8 × 10–6 10–8 [44]
12. Dichlorvos Amperometry Adsorption - ND 10–10 [45]
13. Dichlorvos Amperometry Entrapment CoPc ND 7 ×10–12[46]
14. Dichlorvos Amperometry Adsorption - Up to 10–16 10–17 [47]
15. Dichlorvos Amperometry Cross-linking Prussian blue 4.52 × 10–11 - 4.52 ×10–8 1 .13 × 10–11[48]
16. Trichlorfon Amperometry Adsorption TiO2 and PbO2 particles10–8 - 2 × 1 0–5 10–10 [49]
17. Monocrotophos Amperometry Adsorption AuNPs 4.5 × 10–9 - 4.5 × 1 0–6 2.7 × 10–9 [50]
18. Monocrotophos Amperometry Covalent binding AuNPs-QDs 4.5 × 10–9 - 4.5 × 10–6 1.3×10–9 [51]
19. Acephate FET Affinity CNTs ND 5.45 × 10–14[52]
20. Dimethoate Amperometry Adsorption
CNTs, zirconia NPs, Au
colloid coated Fe3O4
magnetic NPs, Prussian
blue
4.4 × 10–6 - 4.4 × 10–2 2.4 × 10–6 [29]
21. Chlorphenvinphos Amperometry - CNTs 4.90 × 10−7 - 7.46 × 10−6 1.15 × 10−7[30]
22. Malathion Amperometry Covalent binding Fe3O4NP, c-MWCNT, Au10–10 - 4 × 10–8 10–10 [32]
23. Chlorpyrifosoxon CV and amperometry Entr apment PEDOT 1 × 10−10 [33]
fication of OP pesticides. 2) These protocols involve mul-
tiple steps requiring measurement of the uninhibited ac-
tivity of ChE, followed by incubation of the sensor with
the analyte sample for 10 - 15 min (and even longer for
good sensitivity) and the measurement of the ChE again
to determine the degree of inhibition. A final step of re-
activation/regeneration, which in many cases is partial
and in some cases not possible due to irreversible inhibi-
tion, is necessary if the electrode has to be reused.
2.1.2. Tyrosine Based OP Biosensor
Tyrosinase through its cresolate activity catalyses the o-
hydroxylation of monophenol to o-diphenol, which is
further to o-quinone by its catecholase activity. Tyrosi-
nase activity is inhibited by carbamates pesticides and
atrizine that lowers the sensitivity of tyrosinase-based
biosensors. The Tyrosinase enzyme is inherently unstable
and is responsible for reducing the lifetime of the tyrosi-
nase-based biosensors. However, tyrosinase has high
optimum temperatures and there is no effect of organic
solvents on the activity of enzyme tyrosinase. Numerous
electrochemical biosensors based on the inhibition of
tyrosinase activity have been rep orte d (Table 2).
2.2. OPH Biosensor Based on Direct Catalytic
Enzymatic Reaction
In 1970s, Flavobacterium sp. ATCC 27551 and B. di-

A. GAHLAUT ET AL.
4
minuta were the first OP-degrading bacteria isolated
from soil samples [57,58]. Organophosphorus hydrolase
(OPH) has broad substrate specificity and is able to hy-
drolyze a number of OP pesticides such as paraoxon,
parathion, coumaphos, diazinon, dursban, methyl para-
thion [13]. The hydrolysis involves a pH change, as well
as electroactive species generation, thus allowing the
development of potentiometric and amperometric sensors
for OP pesticides quantification [59-65]. The change in
pH was measured using a pH electrode and there were
drawbacks of sensitivity, calibration. OPH catalyzed hy-
drolysis of parathion, methyl parathion, paraoxon, feni-
trothion, etc. yields 4-nitrophenol. The current of 4-ni-
trophenol oxidation is proportional to the OP pesticide
concentration, is recorded as a biosensor response. OP
biosensors have been successfully created using organo-
phosphorous hydrolase as the active component [66].
PTE-immobilized biosensors allow for the direct detec-
tion of Ops. However, these biosensors show lower sen-
sitivity values and higher detection limits than cholines-
terase-based biosensors. Moreover, they can only detect
some Organophosphorus (OP) compounds. The Draw-
back with such type of sensors is that the potential ap-
plied for oxidation of 4-nitrophenol lead to denaturation
of the enzyme immobilized on working electrode and
thus leads to decrease in activity and reusability. Sec-
ondly the potential may oxidize other electro active spe-
cies that may lead to generation of additional current and
false positive results. Characteristics of the other relevant
OPH based electrochemical biosensors based are su-
marised in Tabl e 3.
3. Recent Developments in the Fabrication of
Electrochemical Biosensors for OP
Pesticide s De te r m i na tion
Nanomaterials transducer modification and genetic en-
gineering of the biocomponents are the main strategies to
overcome the reported drawbacks of low sensitivity and
reusability/regeneration of working electrode. The elec-
tro-catalytical properties of the nanostructures includes -
their action as electron transfer mediators or electrical
wires, large surface to volume ratio, structural robustness,
and biocompatibility enhances the use of nano-techno-
logical approach in electrochemical biosensors develop-
ment [74]. Therefore, it gives several advantages like
electrode potential lowering, enhancement of the electron
transfer rate with no electrode surface fouling, sensitivity
increase, stability improvement, and interface function-
alization, for developing a bio-sensing system. Various
nanomaterials are used for making insoluble support for
acetylcholinesterase immobilization in electrochemical
biosensors for organophosphorus pesticides determina-
tion [75]. By the help of transducer modification with
nanomaterials, it gives opportunity to develop biosensors
with long storage stability and enables OP pesticides
detection in the nanomole-picomole range. The another
route leading to increase the biosensors sensitivity, selec-
Table 2. Characteristics of electrochemical inhibition-based biosensors using tyrosinase for OP pesticides detection.
Sr. no. Target analyte Detection
method Enzyme immobilization technique Electrode materials Linearity range (M) Detection limit (M)Ref.
1. Dichlorvos Amperometry Cross-linking + entrapment1,2-naphthoquinone-4-sulfonate (NQS)Up t o 8 × 1 0–6 6 × 10–8 [53]
3. Methyl para th i o n Amperometry Cross-linking CoPc 2.28 × 10–8 - 3.8 × 10–7 ND [54]
3. Diazinon Amperometry Cross- linking CoPc 6.24 × 10–8 - 1.64 × 1 0–7ND [54]
4. Dimethoate Amperometry Adsorption - 2 × 10–6 - 2 × 10–1 10–6 [55]
5. Paraoxon Amperometry Adsorption - 10–5 - 10–2 5 × 10–6 [55]
6. Malathion Amperometry Adsorption - 10–5 - 10–2 5 × 10–6 [55]
7. Paraoxon Amperometry Cross-linking Prussian blue 10–7 - 10–6 10–7 [56]
Table 3. Characteristics of different OPH-based electrochemical biosensors OP pesticides detection.
Sr. no. Target analyte Detection technique Immobilization methodTransducer Linearity range (M) Detection limi t (M)Ref.
1. Paraoxon Amperometr y Covalent binding SWCNTs 5 × 10–7 - 8.5 × 10–6 10–8 [67]
2. Paraoxon Amperometry Entrapment Mesoporous Carbon 2×10–7 - 8×10–6 1.2 × 10–7 [68]
3. Paraoxon Amperometry Entrapment MWCNTs Up to 4 × 10–6 15 × 10–8 [69]
4. Paraoxon Amperometry Cross-linking MWCNTs 5 × 10–7 - 2 × 10–6 0.314 × 10–6 [70]
5. Ethyl Parathion Amperometry Covalent binding - ND <3.4 × 10–9 [71]
6. Methyl Parathion Amperometry Covalent binding AuNPs-MWCNTs-QDs1.9 × 10–8 - 7.6 × 10–7 3.8 × 10–9 [72]
7. Parathion Amperometry Cross-linking CNTs 2 ×10–9 - 4 × 10–8 15 × 10–9 [73]

A. GAHLAUT ET AL. 5
tivity and stability involves the incorporation of tailor
designed biorecognition elements in the biosensing plat-
form. Increased bio-recognition element affinity for the
target analyte favoring the accessibility of the active site,
enhanced electron transfer, and oriented or more stable
immobilization can be achieved by appropriate site-di-
rected mutagenesis [76]. Genetically modified enzymes
such as AchE, are extensively used in inhibition based
biosensors for OP pesticides determ ination [33], allowin g
attaining LOD as low as 10–17 M [47].
4. Conclusion
Electrochemical biosensors have been found to be suit-
able for the monitoring of OP compounds. Signal magni-
fication and miniaturization have been achieved by the
innovation in fabrication techniques with the use of new
materials. With the discovery of new mediators, it is pos-
sible to build up an electronic interface between a redox
enzyme and transducer for improved signal transmission.
From decades variety of prototype have been success-
fully develop to monitor the conc. of OP compounds.
There is a great need for commercial exploitation of the
technology for development of portable devices that can
be used for field monitoring by untrained manpower.
5. Acknowledgements
Financial support to Centre for Biotechnology from DST
(FIST) and UGC (SAP) is greatly acknowledged.
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