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Impedimetric label-free immunodetection of phenylurea class of herbicides

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DOI: 10.1016/j.snb.2012.06.058

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 Sensors and Actuators B 171–172 (2012) 1231–1237

Contents lists available at SciVerse ScienceDirect

Sensors and Actuators B: Chemical

journal homepage: www.elsevier.com/locate/snb

Impedimetric label-free immunodetection of phenylurea class of herbicides

Vijayender Bhalla 1 , Priyanka Sharma 1 , Satish K. Pandey, C. Raman Suri ∗
Institute of Microbial Technology (CSIR), Sector 39-A, Chandigarh 160036, India

a r t i c l e i n f o a b s t r a c t

Article history: We report label-free detection of phenylurea herbicides by impedance spectroscopy using class spe-
Received 23 April 2012 cific anti-diuron antibodies. Gold nanoparticles (∼20 nm), used as signal enhancers cum immobilization
Received in revised form 18 June 2012 matrix, were electrodeposited on carbon screen-printed electrodes (SPE) and functionalized with specific
Accepted 21 June 2012
anti-diuron antibodies for the development of bio-interface. Faradic impedance spectroscopy was per-
Available online 5 July 2012
formed and changes in electrical parameters such as resistance to charge transfer (Rct ) and capacitance
of double layer (Cdl ) were elucidated using Randles model for the equivalent circuit of the electrochemi-
Keywords:
cal cell. Rct parameter was used to draw the analytical inference and also for calculating the percentage
Impedance spectroscopy
Immunoassay
cross reactivity of diuron antibody with various common analogues. The cross-reactivity was 397% with
Randles model monuron, 64.2% with linuron and the least (22.9%) with fenuron. The diuron was detected in a label-free
Gold nanoparticles manner with a limit of detection equal to 5.46 ng mL−1 . The observed response was linear in the useful
Anti-diuron antibodies analytic range of 1–1000 ng mL−1 for the standard diuron detection in water. The obtained results showed
Herbicide diuron good promise for routine label-free monitoring of herbicides in environmental samples.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction detection are gaining importance because of the high speci-

ficity offered by the molecular receptors and high sensitivity of
Phenylurea herbicide diuron, 3-(3,4-dichlorophenyl)-1,1- the sensing platform. [13,14]. Impedance based immunochem-
dimethylurea, is a classical herbicide for weed control and one ical techniques are becoming popular because of their ability
of the prime ecological targets because of its perseverance and towards routinely automated systems enabling detection in a
stability in soil environment [1]. Due to extensive prolonged usage, fast, inexpensive label free manner without using mediators
diuron and its residues are found to be present in ground and or enzymes [15]. Impedance sensing also has the benefit that
surface water in concentrations higher than permissible limits it does not require the voltage scanning needed for anodic
[2,3]. It is a potent inhibitor of photosynthesis and has been used in stripping voltammetry and anodic oxidation, which is time con-
agriculture to inhibit the growth of weeds [4]. It reversibly binds to suming and may degrade the electrochemical interface during
exchangeable quinone (QB ) site of the D1 protein of photosystem wide potential sweeps [16]. Although impedance spectroscopy
II and thus effectively blocks the electron flow from the QA site based immunodetection is very well established, very few reports
[5]. Diuron, therefore, inhibits both the photosynthetic oxygen have previously focused on detection of a small molecule by
evolution and the reduction of the terminal acceptor, nicotinamine using capacitance/resistance changes elucidated by equivalent cir-
adenine dinucleotide phosphate, which finally turns off, the reduc- cuit modeling of the impedance data [17,18]. Electrochemical
tive pentose phosphate cycle. In addition to that, diuron has also impedance spectroscopy (EIS) suffers from poor sensitivity and
numerous detrimental effects on non-photosynthetic prokaryotic in many cases requires conjugation with nanoparticles or protein
and eukaryotic organisms [6]. The detection of this herbicide is molecules [19,15,20,21]. This paper demonstrates the feasibility
essential for safe and clean environment as it has also been shown of GNP enhanced direct label free immunodetection of a small
to have ecotoxic impacts on human health [7]. molecule as depicted in Scheme 1. The different diuron analogues
The existing methods of detection of diuron such as liquid were also evaluated for their cross reactivity with the synthesized
chromatography, gas chromatography, mass spectrometry [8–10], antibody and a comparison was drawn with previous study carried
electrophoresis [11], and immunoassay [12] suffer the drawbacks out by using the ELISA technique.
of complexity, time consuming and require costly equipments.
The antibody based electrochemical immunosensors for pesticide
2. Experimental

2.1. Materials
∗ Corresponding author. Tel.: +91 172 6665225; fax: +91 172 2690632.
E-mail address: raman@imtech.res.in (C.R. Suri). Analytical standards phenylurea herbicides (diuron, fenuron,
1
Both authors contributed equally. monuron, linuron) DCPU (3,4-dichlorophenylurea), bovine serum

0925-4005/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.snb.2012.06.058
1232 V. Bhalla et al. / Sensors and Actuators B 171–172 (2012) 1231–1237

Scheme 1. Impediometric immuodetection of herbicide diuron on GNPs modified screen-printed electrodes. Faradic impedance spectroscopy shows the changes in electrical
parameters such as resistance to charge transfer (Rct ) and capacitance of double layer (Cdl ) as elucidated using Randles model for the equivalent circuit of the electrochemical
cell.

albumin (BSA), 1-ethyl-3-(3-dimethylaminopropy) carbodiimide of formation of gold nanoparticles. The solution was allowed to boil
(EDC) and N-hydroxysuccinimide (NHS), tetrachloroauric acid, for another 10 min and stored at 4 ◦ C. The GNPs solution was diluted
sodium citrate, Tween 20 and peroxidase labeled rabbit anti-IgG 1:5 times in 0.1 M phosphate buffer, pH 7.4 and 100 ␮L was spread
were purchased from Sigma Chemical Co. (USA). Potassium fer- on the SPE surface covering the electrodes. The electrodeposition of
rocyanide K4 [Fe(CN)6 ), potassium ferricyanide K3 [Fe(CN)6 ] and all negatively charged citrate capped GNPs was performed by applying
other chemicals were of analytical grade standards and obtained +0.7 V for 1500 s. The detailed characterization of the thus formed
from local suppliers. All buffers and solutions were prepared in colloidal Au matrix on SPE is provided in our previous report [24].
ultrapure double distilled Milli-Q water. The immobilization of antibodies (100 ␮g mL−1 prepared in 0.1 M
phosphate buffer, pH 7.4) was carried out by overnight incuba-
2.2. Hapten synthesis and antibody generation tion on Au matrix at 4 ◦ C. The antibodies thus establish by Au NH2
bond between Au on GNP surface and amine groups on the lysine
DCPU, an analogue of diuron, having desirable functional groups residues of the antibody. The electrode surface loaded with anti-
for conjugation with carrier protein (BSA) was synthesized using body molecules was thoroughly rinsed with phosphate buffer (pH
green synthesis chemistry (microbial route). DCPU was used as a 7.4), supplemented with 0.001% Tween 20, to remove the weakly
hapten for antibody generation in rabbit. Different molar ratios adsorbed antibody molecules.
of protein:hapten (1:0, 1:5, 1:10, 1:20, 1:30; D0 –D4 respectively)
were prepared by using carbodiimide activation chemistry, details 2.4. Electrochemical Impedance immunoassay
mentioned in previous report [22]. All conjugates (D0 –D4 ) pre-
pared in phosphate buffer (1 mg mL−1 ) were characterized by gel The impedance measurements were performed using elec-
electrophoresis (see supplementary data). Cumulative charge and trochemical analyzer and carbon screen-printed electrodes from
particle size distribution for all the different conjugates were mea- CH instruments, USA. The SPE consisted of an Ag/AgCl reference
sured using Malvern Zetasizer and dynamic light scattering (DLS) electrode, carbon working electrode and counter electrodes. The
system. impedance spectrum was recorded at the formal potential of the
The generated anti diuron antibodies were checked for their redox couple (ferricyanide/ferrocyanide) i.e. 0.172 V (vs. screen-
affinity by isothermal titration calorimetry (ITC) experiments using printed Ag/AgCl) at an amplitude of 5 mV, with the frequency range
Microcal Auto 200 calorimeter. For this, solutions of antibody and from 1 Hz up to 100 kHz. For impedimetric measurements, 100 ␮l of
BSA–DCPU conjugate were first dialyzed separately for 48 h in electrolyte solution containing 10 mM (1:1) mixture of redox cou-
phosphate buffer. After dialysis, 5 ␮L antibody solution (0.1 ␮M) ple prepared in phosphate buffer saline (PBS) was added onto the
was injected at 5 min interval (20 times) into the isothermal cell antibody loaded sensor surface completely covering the 3 screen-
containing the conjugate solution (50 ␮M). The heat for each injec- printed electrodes. All the measurements were recorded in dark,
tion was subtracted by the heat of dilution of the injectant, which at ambient room temperature conditions using real water samples
was measured by injecting the antibody solution into the dialy- spiked with phenylurea herbicides (monuron, diuron, linuron and
sis buffer. Binding constants were estimated from the obtained fenuron). The data was analyzed using standard system software
isotherms using the calorimetric analysis program Microcal Origin available with the service software in the CH workstation.
8 supplied by the manufacturer.
3. Results and discussion
2.3. SPE surface modification
3.1. Antibody generation and characterization
The citrate capped gold nanoparticles (GNPs) were synthesized
using Frens method [23] with slight modification. Briefly 100 mL of For the generation of specific antibodies against small molecules
0.01% solution of tetrachloroauric acid (HAuCl4 ) in deionized water such as pesticides, the crucial step is to synthesize a hapten in
was taken in 250 mL Erlenmeyer flask and covered with foil. The such a way so that it can perfectly mimic the structure of the
solution was brought to boiling point on a hot plate stirrer and 4 mL compound and also containing bio-reactive groups to enable cova-
of 1% sodium citrate solution was rapidly added to the boiling solu- lent linkage with the carrier protein [25]. In the present study,
tion. The color of the solution changes to bright wine red indicative the bio-synthesized hapten i.e. DCPU was used to mimic the
 V. Bhalla et al. / Sensors and Actuators B 171–172 (2012) 1231–1237 1233

Fig. 1. Different protein:hapten conjugates prepared by reacting activated protein (BSA) in molar excess of hapten (DCPU) (a) 1:0, (b) 1:5, (c) 1:10, (d) 1:20, and (e) 1:30,
were characterized by (i) zeta (), and (ii) particle size distribution analysis by showing acceptable polydispersity index (PDI, 0.234–0.348) for all the conjugates.

target molecule diuron as containing appropriate functional group high binding affinity with Ka value equal to 4.748 × 105 with a sto-
( NH2 ) and was successfully conjugated with carrier protein. The ichiometric ratio ∼1 indicating almost 1:1 binding in the titration
hapten protein conjugate was used as an immunogen for anti- studies. The calculated Kd value using ITC indicated good affinity
bodies production and for the development of immunoassay for (∼10−6 M) of antibody for the analyte to be detected.
diuron. The synthesized hapten–protein conjugates at six differ-
ent molar ratios were characterized by gel electrophoresis (native
and SDS PAGE) (supplementary data, Fig. S1). The figure depicts
well-defined bands of BSA with gradual increments for each hap-
ten with increase in its molar ratio. It was observed that the
BSA molecules with higher conjugation density moved less far
in gel electrophoresis. This was due to the reason that negative
charges contributed by the glutamic/aspartic acid residues of the
BSA molecules are utilized during the conjugation process mak-
ing it lesser negatively charged with increase in hapten density.
The particle size distribution and zeta studies further supported
the gel electrophoresis observations by showing zeta potential
change from −32.9 to 0.09 mV as depicted in Fig. 1i and subse-
quent increase in size of the native BSA from 4.26 to 91 nm with
increase in hapten ratio (Fig. 1ii) for different conjugates. The pro-
tein size and the cumulative charge on hapten conjugation directly
affects the nature of elicited immune response. The binding of
hapten molecules with protein either disrupts or appends the nor-
mal antigenic determinants of the carrier protein replacing them
with haptenic determinants which are mainly responsible for the
formation of hapten specific antibodies [26]. The relative func-
tional specificity of the generated antibodies was measured by
standard ELISA procedures. A relatively good titer (1 × 10−6 ) of
anti-diuron antibodies was obtained. The affinity of the generated
antibodies was determined by ITC studies where the heat evolved
during the addition of the antibody to its hapten–protein conju-
gate was measured as a function of the ligand concentration at a
constant temperature. From a single titration, providing sufficient
data points were measured to directly obtain the enthalpy of bind-
ing (H = −7.972 × 106 kcal mol−1 ), entropy (S = −2.67 × 104 ), the
association constant (Ka = 4.785 × 105 M−1 ) the binding stoichiom-
etry (n = 1) and Ga equal to −16 kcal mol−1 from the experimental
titration curve (Fig. 2) by using the Origin 8 software provided Fig. 2. ITC curves for the generated antibody with hapten protein conjugate sample
solution. The antibody solution (0.1 ␮M) was injected 20 times in 5 mL increments
along with ITC. Both enthalpic and entropic contributions to the
and 3 min intervals into the hapten protein conjugate (50 ␮M) solution. The heat
Gibbs energy reflect different types of interactions underlying the for each injection was subtracted by the heat of dilution of the injectant, which was
overall process. The generated antibodies were observed to show measured by injecting the antibody solution into the dialysis buffer.
1234 V. Bhalla et al. / Sensors and Actuators B 171–172 (2012) 1231–1237

Fig. 4. Nyquist diagram (Zimaginary vs. Zreal ) for the Faradic impedance measurement
of the GNP/anti-diuron antibody electrode surface. Arrow denotes the increasing
Fig. 3. Faradic impedance spectra that correspond to GNP anti-diuron antibody concentrations of diuron 1–1000 ng mL−1 . Inset shows the equivalent circuit, which
modified SPE incubated with different concentrations of DCPU-conjugate samples, represents each component at the working electrode interface and in the solution
(a) ( ) representing the GNP anti-diuron antibody surface (b–d) representing DCPU- during the electrochemical reaction in the presence of redox couples. Rct : elec-
conjugate concentrations of ( ) 4.5 ␮g mL−1 ( ) 45 ␮g mL−1 and ( ) 450 ␮g mL−1 tron transfer impedance; Rs : solution resistor; Cdl capacitance of double layer; CPE:
respectively. constant phase element; ZW : Warburg impedance.

3.2. Direct impedance immunoassay changes in the observed impedance signal confirming the speci-
ficity of sensor response to diuron and its immediate analogues.
Impedance spectroscopy is an effective technique to probe the The Faradic EIS led to major change in resistive parameter with
features of surface-modified electrodes, in general, and detect only slight changes in Cdl . The non-Faradic measurements recorded
bimolecular interaction in a label-free manner. The complex in plain PBS buffer, for the same analytical test range, showed a
impedance is a sum of real (Z ) and imaginary components (Z ) more predominant change in Cdl equivalent to 42.6 nF for diuron.
related by the expression: The fitting of data showed a somewhat scattered data points along
the fitting line (data not depicted). Thus Faradic impedance spec-
Z ∗ (w) = Z  (w) + j · Z  (w) (4) troscopy with measurements in solution of redox probe (Fe ions)
The letter j in the above equation is the j operator. Fig. 3 that gives stable well resolved Nyquist plots and good fitting of
shows the GNP anti diuron antibody modified SPE and its reac- the data into the electrical equivalent model is the main suggested
tion towards increasing concentration of the DCPU/BSA conjugate method for detection.
(1:10). The anti-diuron antibody was immobilized on the GNP/SPE The various analytical parameters such as time stability, sensor
surface and a clear increase in semicircle half of the Nyquist plot to sensor variation and coefficient of variance of the GNP/anti-
was observed with increase in concentration of hapten–protein diuron antibody modified sensors is discussed in our previous
conjugate indicating successful anti-diuron antibody immobiliza- report [24]. The calculated limit of detection (LOD) for the present
tion on the colloidal Au base matrix. The increase in diameter diuron detection is 5.46 ng mL−1 . Faradic measurements using
of the semicircle indicates a clear increase in the charge trans- colloidal Au base matrix offers a good possibility for label-free
fer resistance (Rct ) between the redox probe and the electrodes monitoring of diuron in environmental samples. The sensitivity of
posed by the binding of hapten protein conjugate on to the sur- the present system is comparable to a previously described com-
face. The Rct and capacitance of electrical double layer (Cdl ) were petitive immunochemical assay for another herbicide i.e. atrazine
considered suitable signals for sensing the interfacial properties detection showing a sensitivity of ∼8 ng mL−1 [28]. Thus impedance
of the prepared biosensors as they show a significant variation in spectroscopy with the present GNP enhancement methodology
a concentration dependent manner. The respective changes were demonstrates a good sensing technique for routine label-free and
deduced using modified Randle’s equivalent circuit modeling [27] reagentless monitoring of herbicides in water by using the Rct
as per circuit diagram shown in inset of Fig. 4. Nyquist plot for parameter.
the Faradic impedance spectroscopy of GNP/anti-diuron antibody
modified SPE towards detection of free diuron is shown in Fig. 4. 3.3. Cross-reactivity with analogues
The fitting parameters for the data are presented in Table 1. As
can be seen in the picture the Rct increases in a linear concentra- The anti-diuron antibody was checked for its cross reactivity
tion dependent manner. It increases roughly ∼2 k in the tested with the common diuron analogues such as monuron, linuron and
herbicides concentration range between 1 ng mL−1 and 1 ␮g mL−1 . fenuron. Table 2 shows the percentage cross reactivity for diuron
The Cdl component increased simultaneously and a window 21.4 nF and its analogues. In general, members of phenylurea herbicide
was observed for the above tested concentration levels. There family are having a common chlorine substituted phenyl ring on
was insignificant change in the other components of the electri- one end which is primarily responsible for antigenicity [29], lead-
cal equivalent circuit and Rct and Cdl were the major contributing ing to broad-specific antibodies production. This type of antibody,
factors to the complex impedance as shown in Table 1. Although better called “class specific antibody” are preferred in develop-
very small in size, the hydrophobicity of the molecule could be one ing a class specific immunoassay [30]. However, the generated
major factor contributing to a larger resistive change by blocking antibodies will not show any reactivity with pesticides molecules
access of the redox probe to the surface of the electrodes. Inde- belonging to other class of pesticides e.g. organophosphate, tri-
pendent experiments performed on the GNP/anti-diuron antibody azine, carbamate, nitrophenolic, etc.
modified surface by using 2,4-dichlorophenoxyacetic acid, another The GNP/anti-diuron antibody immobilized electrodes were
herbicide not directly related to diuron, resulted in insignificant subjected to increasing concentrations of herbicides starting from
 V. Bhalla et al. / Sensors and Actuators B 171–172 (2012) 1231–1237 1235

Table 1
Major electrical parameters variation associated with biomolecular activity at the GNP modified surface. The scans were obtained in 10 mM K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] (1:1)
mixture in PBS buffer pH 7.4.

Rsol () Cdl (nF) Rct () ZW CPE (10−5 ) Error

Ab-layer 300.8 703.7 3859 0.000368 8.857 0.02555

Diuron
1 ng mL−1 299.2 702.4 4387 0.00021 8.84 0.0286
5 ng mL−1 299.9 702.1 4585 0.000195 8.834 0.03016
10 ng mL−1 299.5 703.2 4783 0.00018 8.834 0.03069
50 ng mL−1 295.8 711.8 5357 0.00013 8.824 0.03304
100 ng mL−1 296.9 720.4 5694 0.000124 8.87 0.03304
500 ng mL−1 298.5 721 5678 0.000128 8.929 0.03389
1000 ng mL−1 295.8 723.8 6314 0.000104 8.929 0.03397

Table 2
Comparison of IC50 calculated by ELISA and % cross reactivity (calculated from Rct ). with respect to polarizability by HyperChem for diuron and its analogues.

Target analyte IC50 value (ng mL−1 ) Polarizability (Å3 ) (Rct ()b % cross reactivity of
ELISAa calculated by diuron antibodyb
Hyperchem

Diuron 0.5 12.85 528 100

3-(3,4-Dichlorophenyl)-1,1-
dimethylurea)

Monouron 0.096 12.78 2100 397

3-(4-Chlorophenyl)-1,1-
dimethylurea

Linuron 0.49 17.48 339 64.2

3-(3,4-Dichlorophenyl)-1-
methoxy-1-methylurea

Fenuron 5 12.71 151 22.9

1,1-Dimethyl-3-phenylurea
a
Calculated from our previous report [22].
b
Calculated from Rct response at 1 ng mL−1 herbicide concentration.

Fig. 5. Comparison of relationship of different concentrations of ( ) diuron and its analogues: ( ) fenuron, ( ) linuron and ( ) monuron to components of Randles
equivalent circuit (a) Rct and (b) Cdl . All experiments were repeated thrice and standard deviations correspond to n = 3.
1236 V. Bhalla et al. / Sensors and Actuators B 171–172 (2012) 1231–1237

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[31] C. Kittel, Introduction to Solid State Physics, 8th ed., John Wiley & Sons, New Satish Kumar Pandey did his Master’s in Biotechnology
York, 1995. and currently pursuing his collaborative Ph.D. from Panjab
University and IMTECH, Chandigarh, India under supervi-
Biographies sion of Dr. C.R. Suri and Dr. P. Rishi. Presently he is working
on the development of an immunosensor for detection
of Salmonella enterica serovar Typhi by exploring surface
components as specific markers.
Vijayender Bhalla holds Master’s in Medical Biochem-
istry from Postgraduate Institute of Medical Education and
Research (PGIMER) and Ph.D. in Bionanotechnology from
Panjab University in Chandigarh, India. After graduating,
Vijayender joined as postdoc the group of Prof. Bruno
Samori at the University of Bologna, in Italy and later on he
moved to Montreal to join Dr. Valter Zazubovich at Con-
cordia University, Canada. Presently, he is SRA (CSIR, Pool
Scientist) at IMTECH, Chandigarh. His research is mainly C. Raman Suri received his Master’s in Electronics
focused on development of label-free immunosensors for from AMU, Aligarh, India and Ph.D. in Biotechnology
environment and healthcare applications. from Punjab University, Chandigarh, India, specializing
in immunosensor development. Currently he is working
as a Senior Scientist at Institute of Microbial Technol-
ogy, Chandigarh, India. His current activities include
hapten–protein conjugation for antibodies generation,
Priyanka Sharma got her Master’s in Environmental Sci- immunoassay development and sensor design.
ences from Panjab University, Chandigarh, India. She is
presently pursuing Ph.D. and working as Sr. Research
fellow in Biosensor lab at Institute of Microbial Tech-
nology (IMTECH), Chandigarh, India. Ms. Priyanka has
also worked as a visiting research scholar at Northwest-
ern University (USA). Her research is focused mainly on
environmental nano-biotechnology involving synthesis of
specific molecular receptors and the bioassay develop-
ment for the detection of environmental pollutants.

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