Mobilized lipase enzymatic biosensor for the determination of Chlorfenvinphos and Malathion in contaminated water samples: A voltammetric study

Article abstract of DOI:10.1016/j.molliq.2014.06.019

Concentration levels of organophosphorus pesticides like Chlorfenvinphos and Malathion were determined by lipase enzyme inhibition method. The developed enzymatic method was purely based on in situ generation of p-Nitrophenol by enzymatic hydrolysis of p-

Full text of DOI:10.1016/j.molliq.2014.06.019

Journal of Molecular Liquids 198 (2014) 181–186
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Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
Mobilized lipase enzymatic biosensor for the determination of
Chlorfenvinphos and Malathion in contaminated water samples:
A voltammetric study
b,
K. Gangadhara Reddy a, G. Madhavi a, , B.E. Kumara Swamy
⁎
⁎
a
Department of Chemistry, S.V.U. College of Sciences, Sri Venkateswara University, Tirupati 517502, Andhra Pradesh, India
Department of P.G. Studies and Research in Industrial Chemistry, Kuvempu University, Shankaraghatta-577 451, Shimoga, Karnataka, India
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 July 2013
Received in revised form 5 June 2014
Accepted 18 June 2014
Available online 2 July 2014
Concentration levels of organophosphorus pesticides like Chlorfenvinphos and Malathion were determined by
lipase enzyme inhibition method. The developed enzymatic method was purely based on in situ generation
of p-Nitrophenol by enzymatic hydrolysis of p-Nitrophenyl Acetate. The production of this in situ generated
p-Nitrophenol concentration was depending on the effective hydrolysis of p-Nitrophenyl Acetate by the
lipase enzyme. This electrochemically active p-Nitrophenol gave an anodic oxidation peak at a potential
of +0.024 V versus SCE. The various parameters such as effective hydrolysis of substrate, substrate concentration
and effect of pH were studied for lipase enzyme. A linear calibration for the Chlorfenvinphos and Malathion was
obtained in the concentration ranges of 100–900 μM and 100–900 mM respectively with a correlation coefficient
of 0.9894 and 0.9680 under the optimized conditions by following the incubation time of 25 min. The
electrochemical experiments were performed in 0.1 M phosphate buffer solution (pH 7.0) at room temperature.
The limit of detection and limit of quantification values were found to be 84.45 μM, 253.03 mM and 281.5 μM,
843.1 mM respectively for Chlorfenvinphos and Malathion under optimized conditions. The developed liquid
state lipase enzyme sensor could be applied for the analysis of water samples contaminated with pesticides.
The developed method gives the information about the intensity of toxicity of pesticides.
Keywords:
Lipase enzyme
Voltammetric method
Malathion
Chlorfenvinphos
p-Nitrophenyl Acetate
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
human health and environment. The problem is steadily growing and
becoming more serious, especially, in the developing countries.
Different types of organophosphorus compounds and their related
pesticides are considered to be toxic. They are widely used in agricultural
practices as pesticides and insecticides for controlling of broad spectrum
of pests and insects by contact, stomach and respiratory action [1]. The
high toxicity of organophosphorus neurotoxins and their large use in
modern agriculture practices have increased the public concerns, health
risks and the consequent contamination of water and food sources [2].
However, their wide use has resulted in their widespread distribution
in the environment which in turn shows adverse health implications,
even at trace levels in both ground and surface water. The same type of
compounds has been produced as possible nerve poisons, a further
area of application is in the military [3]. Among the existing organophos-
phorus compounds the Chlorfenvinphos and Malathion pesticides
(Table. 1) are most commonly used in the agriculture for the control of
various types of pests. The widespread global use of pesticides for crop
protection and other household uses has placed a great threat to
Many efforts have been made in finding of the pesticides. In the last
decades organochlorine insecticides (e.g. lindane, aldrin, and DDT)
were progressively replaced by organophosphorus (e.g., Dibrom,
Chlorfenvinphos and Malathion) and the derivatives of carbamic
acid (e.g. aldicarb and carbaryl) insecticides, that show low persistence
in the environment but represent a serious risk because of their high
acute toxicity. In fact, they are inhibitors of the cholinesterase
enzymes [4–6] involved in muscle contraction and impulse transmis-
sion in the nervous system. The development of efficient analytical
methods for their estimation in food and environmental samples is of
the present importance.
These small amounts of pesticide residues when present in water,
food and animal feeds create a potential hazard due to their high
mammalian toxicity. Hence, a rapid and reliable quantification of trace
levels of organophosphorus compounds is essential for monitoring of
their potential hazard to health and the environment [7]. In view of
the unavoidable routes of organophosphorus compounds into biological
systems, different traditional analytical methods and technologies have
been used for their determination such as gas and liquid chromatogra-
phy [8], gas-chromatography with mass spectrometry detection [9,10],
liquid–solid extraction followed by liquid chromatography-diode array
⁎
Corresponding authors. Tel.: +91 8282 256228; fax: +91 8282 256255.
E-mail addresses: gmkr555@yahoo.co.in (G. Madhavi), kumaraswamy21@yahoo.com
(B.E. Kumara Swamy).
http://dx.doi.org/10.1016/j.molliq.2014.06.019
0167-7322/© 2014 Elsevier B.V. All rights reserved.

182
K. Gangadhara Reddy et al. / Journal of Molecular Liquids 198 (2014) 181–186
Table 1
electrode [35], amperometric silica sol–gel immobilized mono enzymatic
acetylcholinesterase biosensor [36], amperometric nano-structured
polymer membrane containing gold nanoparticles modified acetyl-
cholinesterase biosensor [37], amperometric screen-printed cross
linking biosensor [38], covalent binding [39], direct physical adsorp-
tion onto a solid support [40], and encapsulation into a hydrogel [41]
biosensors, have been applied for continuous monitoring of pesticide
residues in the environmental samples. However the developments
of these acetylcholinesterase enzyme biosensors require costlier en-
zyme and suitable substrate. The lipase enzyme is also inhibited by
some of the organophosphorus pesticides and the indirect determi-
nation of pesticide concentration by the lipase enzyme is based on
effective inhibition of catalytic nature of lipase towards the esteric
bonds. Based on this, some of the lipase enzymatic surface acoustic
wave impedance sensor [42] and potentiometric biosensor [43]
were also developed.
The structure and molecular formulae of Chlorfenvinphos and Malathion.
Sl. No Selected organophosphorus Molecular Chemical structure
pesticide
formula
12H14Cl3O4P
01
02
Chlorfenvinphos,
C
Malathion
C10H19O6PS2
To overcome the above problem, an indirect method of mono-
enzymatic liquid state electrochemical biosensor was developed
for the determination of Chlorfenvinphos and Malathion pesticides.
The developed method is purely based on the production of the
p-Nitrophenol by p-Nitrophenyl Acetate hydrolysis by free lipase
enzyme and the pesticide affected lipase enzyme was studied by
both cyclic and differential pulse voltammetric techniques.
detection [11]. However, these methods can have very low limits of
detection (LOD) and they are highly selective and sensitive. These
methods are not applicable for field determinations, time-consuming
and require sophisticated laboratory equipments [12].
Electroanalytical (voltammetry) methods have shown remarkable
advantages in the analysis of environmental samples which are contam-
inated by toxic metals, pesticide residues, synthetic bioactive molecules
and also industrial effluents. In this context various types of modified
working electrodes play a major role since three decades. A polyvinyl
chloride (PVC) matrix based neutral carrier porphyrin (A and B)
ionophores Cu2+ selective sensor, potentiometric sensor and cop-
per (II) selective sensor based on dimethyl 4, 4(O-phenylene), bis
(3-thioallophanate) in PVC matrix have been fabricated and were
applied for the determination of copper in synthetic and real samples
[13–15]. Similarly mercury, cobalt, chromium, lead and cadmium are
equally important toxic elements in the environment, thus the determi-
nation of their levels in the environment by the applications of some of
the developed ion selective electrodes in which highly selective mercu-
ry electrode with diamine donor ligand [16], mercury selective potenti-
ometric sensor [17] and neutral carriers based polymeric membrane
electrode for the selective determination of mercury (II) [18] were
used. PVC membrane based cadmium (II), chromium (III), cobalt (II)
and lead (II) ion selective electrodes [19–22] respectively were applied
for the determination of trace levels of Cd, Cr, Co and Pb.
The continuous application of these modified working electrodes
includes the analysis of steroid and pharmaceutical samples.
Fullerene-C60 modified electrode as a sensitive voltammetric sensor
for the detection of nandrolone [23] and sensitive voltammetric
sensor for corticosteroid triamcinolone [24] steroids. The wide appli-
cations of electroanalytical techniques also include the analysis
of the pharmaceutical assay [25,26]. In situ surfactant modified
multiwalled carbon nano-tube paste electrode was applied for acet-
aminophen, aspirin & venlafaxine, desvenlafaxine by Nafion–carbon
nanotube composite glassy carbon electrode and Fullerene-C60
modified edge plane pyrolytic graphite electrode for the determina-
tion of dexamethasone in pharmaceuticals [27–29]. Gold, silver
nanoparticles modified glassy carbon paste electrode sensors were
applied for the determination of dopamine, uric acid in urine and
bold serum samples [30,31] and polygraphite electrode, fullerene
C60 coated gold electrode was applied for the determination of 2′,
3′-dideoxyadenosine, dopamine [32,33] in commercially available
pharmaceutical formulations respectively.
2. Experimental part
2.1. Reagents and solutions
All chemicals were obtained from commercial sources and
used without further purification. Lipases from Candida rugosa
(EC 3.1.1.3, type VII, ≥700/mg), p-Nitrophenyl Acetate, Chlorfenvinphos
and Malathion were purchased from Sigma-Aldrich Chemicals.
The Chlorfenvinphos and Malathion pesticide stock solutions
were prepared by dissolving in acetone (GR grade). The graphite
fine powder was procured from Sigma Aldrich and silicon oil,
sodium dihydrogen phosphate, disodium hydrogen phosphate and
acetone (GR grade) were procured from Himedia chemicals. 0.1 M
phosphate buffer was prepared by using 0.1 M disodium hydrogen
phosphate and 0.1 M sodium dihydrogen phosphate. All chemicals
were of analytical grade and the aqueous solutions were prepared
with double distilled water. The enzyme stock solutions were preserved
at −5 °C.
2.2. Apparatus
Cyclic voltammetric experiments were performed with a CH-
Instruments (USA) model no CHI610D Electrochemical work station
with a connection to a personal computer was used for the electro-
chemical measurement and treating of data. A conventional three
electrode cell was employed throughout the experiments, with a
bare carbon paste electrode (homemade cavity of 3.0 mm diameter)
as a working electrode, saturated calomel electrode (SCE) as a reference
electrode and a platinum wire as a counter electrode. All the
experiments were carried out at room temperature.
2.3. Preparation of bare carbon paste electrode
The electrochemical biosensors, developed during the last decades,
offer a new approach to organophosphorus pesticides quantification.
Based on the enzymatic inhibition method some of the electrochemical
biosensors were developed for the study of organophosphorus pesti-
cides. Cholinesterase-based biosensors were considered as one of the
best alternatives in the context of this strategy [34]. Electrochemical
sensor based on acetylene black–chitosan composite film modified
The bare carbon paste electrode was prepared by hand mixing
of 70% fine graphite powder and 30% silicon oil in an agate mortar to
produce a homogenous carbon paste. The paste was packed into the
cavity of homemade PVC (3 mm in diameter) and then smoothed on a
weighing paper. The electrical contact was provided by copper wire
connected to the paste at the end of the tube [44].

K. Gangadhara Reddy et al. / Journal of Molecular Liquids 198 (2014) 181–186
183
Fig. 1. Cyclic voltammograms of in situ generated p-Nitrophenol in 0.1 M phosphate
buffer, pH 7.0 A) substrate alone; B) substrate in the presence of 125 U of enzyme
at 50 mV/s scan rate.
Fig. 3. Differential pulse voltammograms of enzymatic hydrolysis of p-Nitrophenyl
Acetate. The inset plot shows anodic peak current (Ipa) versus pH variation.
3. Results and discussion
a non-sticky thin film on the surface of the carbon paste electrode.
For further studies these non-sticky polymeric film electrode surface
was removed by physically smoothing against a tissue paper [45].
The anodic peak current was increased with an increase of scan
rate and the obtained graph was linear with a regression coefficient
(r2) 0.9997 Fig. 2.
3.1. Cyclic voltammetric studies
The electrochemical studies of p-Nitrophenol by lipase enzymatic
hydrolysis of p-Nitrophenyl Acetate were studied in 0.1 M phosphate
buffer of pH 7.0. Fig. 1 (curve A) shows a small background current
due to the substrate alone, but in the presence of 125 U of lipase enzyme
only a recognized oxidation peak current was observed (curve B) at a
scan rate of 50 mV/s. The enzymatic hydrolysis of substrate followed
by electrooxidation of hydrolysis product of p-Nitrophenol is shown in
the Eq. (1) [45].
3.2. The effect of pH on the hydrolysis of p-Nitrophenyl Acetate
The effect of pH on the enzymatic hydrolysis of p-Nitrophenyl
Acetate to p-Nitrophenol and acetic acid in the presence of Lipase
enzyme was studied in 0.1 M phosphate buffer with pH 6.0–8.0. Fig. 3
shows various differential pulse voltammograms of hydrolysis of
p-Nitrophenyl Acetate to p-Nitrophenol with liquid state lipase
enzyme at various pH ranges and also the obtained experimental
value was close to that reported in the literature [46]. The resulting
profile showed the maximum hydrolysis of p-Nitrophenyl Acetate
by lipase at pH 7.0 which is shown in inset of Fig. 3. The potential
diagram constructed by plotting the graph of anodic peak potential
Epa vs pH of the solution shows positive potential shift with a slope of
71 mV/pH. This behavior was nearly obeying the Nernst equation for
equal no of electron and proton transfer reactions [47,48].
Hþ=OH−
p−NitrophenylAcetate þ Lipase
p−Nitrophenol þ AceticAcid
C6H4NO2OH
•
C6H4NO2 O þHþ þ e−
ð1Þ
According to Eq. (1) the generated p-Nitrophenol is oxidized at the
surface of the electrode and can give nitrophenoxy radical intermediate
and subsequently undergoes polymerization leading to the formation of
3.3. The effect of dependent parameters
The choice of appropriate substrate concentration, effect of selected
units of enzyme and enzymatic hydrolysis time are crucial for the study
of the enzymatic inhibition. The effective hydrolysis of substrate by
lipase enzyme was carried out effectively from 125 μM to 1750 μM of
the substrate. The hydrolysis of p-Nitrophenyl Acetate to p-Nitrophenol
was increased from 125 μM to 750 μM and after that the hydrolysis was
continuously decreased above 750 μM and up to 1750 μM. When the
concentration of the enzyme was gradually increased from 25 U to 250
U there was a gradual increase in the peak current response and this re-
sponse was finally saturated at 125 U of enzyme concentration where
the current values observed were almost constant. The effective hydroly-
sis of substrate was gradually increased from 5 to 25 min and the increase
was continued till a plateau level was reached and after that there was no
significant increment in the hydrolysis of the substrate up to 40 min. We
have previously developed and reported the optimized conditions for li-
pase enzyme catalytic nature and the effective hydrolysis time [49,50].
Fig. 2. The effect of scan rate on anodic peak current for p-Nitrophenol in 0.1 M phosphate
buffer of pH 7.0.

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K. Gangadhara Reddy et al. / Journal of Molecular Liquids 198 (2014) 181–186
3.4. Pesticides study
The rate of inhibition % and residual enzyme activity was determined
according to the following Eqs. (2) and (3) [36,51].
The lipase enzyme was used to carry out inhibition studies of
Chlorfenvinphos and Malathion pesticides by incubating with each
pesticide solution up to 25 min to obtain lower detection limits. Inhibi-
tor (pesticide solution) was mixed in equal ratio with 125 U of enzyme
stock solution and incubated at room temperature of 25 2 °C. To ob-
tain the inhibition plots for Chlorfenvinphos and Malathion pesticides
the percentage of inhibition method was studied. The detection was
based on the measurement of initial steady state current (Ii) response
on the hydrolysis of liquid state enzyme towards the selected concen-
tration of 750 μΜ substrate in a 0.1 M phosphate buffer solution at
pH 7.0. The equal ratio of enzyme and pesticide stock solution was
incubated up to 25 min, followed by the transfer of inhibited enzyme
into the electrochemical cell and the final steady state current (If)
response towards the hydrolysis of 750 μΜ substrate was recorded.
Inhibition I ð%Þ ¼ ½ðIi−If Þ=If ꢀ ꢁ 100
ð2Þ
ð3Þ
Residual enzyme activity ðREA%Þ ¼ ½If =Iiꢀ ꢁ 100
Quantitative analysis of individual pesticides was carried out accord-
ing to the above procedure. Chlorfenvinphos and Malathion pesticides
were known to inhibit the catalytic nature of the lipase enzyme towards
the substrate therefore a toxic reference to test the lipase enzyme
activity was chosen for these studies. Fig. 4A and D shows the differen-
tial pulse voltammograms of Chlorfenvinphos and Malathion pesticides
respectively. In Fig. 4A and D the peak ‘c’ indicates the presence of
little amount of p-Nitrophenol, a starting substance for synthesis of
Fig. 4. (A), (D) Differential pulse voltammograms of Chlorfenvinphos and Malathion pesticides in 0.1 M phosphate buffer, pH 7.0 and 750 μM substrate measurement conditions.
(B), (E) Inhibition plots of lipase enzyme by Chlorfenvinphos and Malathion pesticides after 25 min of incubation time. (C), (F) The effect of incubation time at various inhibitor
concentration on the activity of liquid phase lipase enzyme in for Chlorfenvinphos: (a) 100 μM, (b) 200 μM, (c) 350 μM, (d) 500 μM, (e) 600 μM, and (f) 900 μM and Malathion:
(a) 100 mM, (b) 200 mM, (c) 350 mM, (d) 500 mM, (e) 600 mM, (f) 800 mM, and (g) 900 mM.

K. Gangadhara Reddy et al. / Journal of Molecular Liquids 198 (2014) 181–186
185
Table 2
known that the Chlorfenvinphos is more toxic than the Malathion
as evidenced by the decrease in their current responses. The proposed
electrochemical voltammetric detection method was simple, cost
effective, less tedious, eco-friendly and can comfortably be used for
the determination of organophosphorus pesticide residues in the
environmental samples. The developed voltammetric investigation
method is useful for the investigation of intensity of the toxicity of
the pesticide substance.
The various parameters determined for Chlorfenvinphos and Malathion pesticide.
Sl. No
Parameters
Chlorfenvinphos
Malathion
1
2
3
4
5
6
7
Incubation time (min)
Response time (min)
Linear range
Correlation coefficient
Standard deviation
25
5
25
5
100 μM–900 μM
0.9896
4.5063
84.45 μM
281.5 μM
100 mM–900 mM
0.9680
6.3090
253.03 mM
843 mM
Limit of detection (LOD)
Limit of quantification (LOQ)
Acknowledgment
p-Nitrophenyl Acetate. In Fig. 4A and D the peak ‘b’ represents the
inhibition of enzyme activity in the presence of pesticide and the
effective catalysis towards substrate inhibition was completely
observed and is evidenced by the reduction in the current response.
In Fig. 4A and D the peak ‘a’ represents complete hydrolysis of the
substrate in the presence of lipase enzyme.
The authors are thankful to the University Grants Commission,
New Delhi Government of India for providing the financial assistance
through project F. No.39-744/2010 (SR).
References
Calibration plots drawn for the dependence of the percentage of in-
hibition on the concentration were linear and are shown in Fig. 4B and E,
the detection limit and the limit of quantification values were 84.45 μM,
253.03 mM and 281.5 μM and 843.1 mM for Chlorfenvinphos and
Malathion respectively. Various concentrations ranging from 100 μM
to 900 μM and 100 mM to 900 mM of both pesticides were tested in
terms of their effect on enzyme activity at different incubation times
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tion of the Chlorfenvinphos and Malathion increases, the residual
enzyme activity of the enzyme decreases with time and is shown in
Fig. 4C and F. The determination of detection limit (DL) and quantifica-
tion limit (QL) were carried out by using Formulae (4) and (5) [52,53].
The results are shown in Table. 2.
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DL ¼ 3Sb=S
ð4Þ
ð5Þ
QL ¼ 10Sb=S
Where Sb is the standard deviation, S is slope of the working curve,
DL is the detection limit, and QL is the quantification limit. Comparative
analytical figures of merits for different modified electrode sensors for
the determination of Malathion and Chlorfenvinphos are summarized
in Table. 3
4. Conclusion
In the present study, the preparation of lipase based liquid phase
biosensor was determined by the mobilization method with various
concentration levels of Chlorfenvinphos and Malathion pesticides
was carried out. Electroanalytical investigation of Chlorfenvinphos
was achieved down to 100 μM and Malathion was achieved down
to 100 mM. The detection limit and quantification limit values
of Chlorfenvinphos and Malathion were 84.45 μM, 253.03 mM,
281.5 μM, and 843.1 mM respectively. From this study it was also
Table 3
Comparative analytical values for different modified electrode sensors for the determination of Malathion and Chlorfenvinphos.
Sensor electrode material
Analytical parameters for Malathion
Linearity range (mol/L)
Detection limit (mol/L) References
1. Physical adsorption AChE/AuNPs/Chi
2. Tyrosinase enzyme immobilized in kappa carrageenan gel electrode
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0.30 × 10−9 to 1.51 × 10−6
0.01 × 10−6 to 0.1 × 10−6
10−10 to 4 × 10−8
0.06 × 10−9
5.0 × 10−9
10−10
[54]
[55]
[56]
[57]
–
1 × 10−7 to 2 × 10−4
1 × 10−3 to 9 × 10−3
2.5 × 10−6
2 × 10−3
Analytical parameters for Chlorfenvinphos
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8.595 × 10−10
[58]
[59]
[58]
-
7. Square-wave cathodic stripping voltammetry at a mercury film ultra micro electrode sensor 5.7 × 10−8 mol l−1 to 6.57 × 10−7 mol l−1 0.042 × 10−6
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8. Present work
0.1 × 10−10 to 0.1 × 10−7
1 × 10−4 to 9 × 10−4
2.108 × 10−7
8.4 × 10−4

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