Nanobiosensor for diclofop detection based on chemically modified afm probes (ieee sensors)
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Transcripts - Nanobiosensor for diclofop detection based on chemically modified afm probes (ieee sensors)
IEEE SENSORS JOURNAL, VOL. 14, NO. 5, MAY 2014 1467
Nanobiosensor for Diclofop Detection Based on
Chemically Modiﬁed AFM Probes
Carolina Castro Bueno, Adriano Moraes Amarante, Guedmiller S. Oliveira, Daiana Kotra Deda,
Omar Teschke, Eduardo de Faria Franca, and Fábio L. Leite
Abstract—Highly sensitive and selective functional nanobio-
breaksensors are being developed because they have signiﬁcant
applications in the sustenance and conservation of natural
resources and can be used in projects to identify degraded and
contaminated areas (of both soil and water) and as environmental
quality indicators. In the present study, a nanobiosensor was
developed based on using theoretical models (molecular docking
and molecular dynamics simulations) based on biomimicry of
the action mechanism of herbicides in plants coupled with
atomic force microscopy (AFM) tools. The herbicide molecules
were detected at very low concentrations using a unique sensor
construction: the AFM probes and the substrate were chemically
functionalized to favor covalent bonding and promote molecular
ﬂexibility, as well as to achieve reproducible and accurate results.
Computational methods were used to determine the binding
energies associated with the enzyme-herbicide interactions, which
were compared with experimental results for adhesion forces.
The theoretical results showed that the diclofop herbicide could
be assembled and attached onto the mica substrate surface and
the ACCase enzyme on the AFM probe without damaging the
diclofop molecule. The experimental results showed that using
a speciﬁc agrochemical target molecule was more efﬁcient than
using other nonspeciﬁc agrochemicals. On average, there was a
90% difference between the values of speciﬁc recognition (diclo-
fop) and nonspeciﬁc recognition (imazaquin, metsulfuron, and
glyphosate). This result validated the selectivity and speciﬁcity
of the nanobiosensor. The ﬁrst evidence of diclofop detection by
the AFM probe sensors has been presented in this paper.
Index Terms—AFM, agrochemical detection, diclofop, enzyme-
based nanobiosensor, molecular docking, molecular dynamics
FOOD production was greatly increased in the nineteenth
century by technological improvements in agricultural
Manuscript received October 15, 2013; revised December 27, 2013;
accepted January 8, 2014. Date of publication January 28, 2014; date of
current version March 18, 2014. This work was supported in part by CAPES,
in part by CNPq, in part by FAPESP under Grant 2007/05089-9, 2013/09746-
5, Proc. 2009/09120-3), CAPES, CNPq, nBioNet and in part by the Institute
of Physics Gleb Wataghin. The associate editor coordinating the review of
this paper and approving it for publication was Dr. Chang-Soo Kim.
C. Castro Bueno, G. S. Oliveira, A. M. Amarante, D. K. Deda, and
F. L. Leite are with the Nanoneurobiophysics Research Group, Department
of Physics, Chemistry and Mathematics, Federal University of São Carlos,
Sorocaba 18052-780, Brazil (e-mail: firstname.lastname@example.org;
email@example.com; firstname.lastname@example.org; daianakdn@
E. de Faria Franca is with the Chemistry Institute, Federal
University of Uberlândia, Uberlândia 18052-780, Brazil (e-mail:
O. Teschke is with the Institute of Physics Gleb Wataghin, Uni-
versidade Estadual de Campinas, Campinas 13083-970, Brazil (e-mail:
Color versions of one or more of the ﬁgures in this paper are available
online at http://ieeexplore.ieee.org.
Digital Object Identiﬁer 10.1109/JSEN.2014.2301997
techniques, such as the use of modern machinery, fertilisers,
and agrochemical molecules , . The indiscriminate use
of agrochemicals on crops resulted in environmental con-
tamination and toxic effects on human health , such as
carcinogenic effects ,  and in vivo and in vitro genotoxic
effects on mammalian cells from chromosomal aberrations .
Current agrochemical detection methods rely almost entirely
on mass spectroscopy (MS) , , magnetic solid phase
extraction (MSPE) , gas chromatography-electron cap-
ture (GC-ECD) ,  via bacterial bioluminescent
response , and high performance liquid chromatography
(HPLC) , . It is quite difﬁcult to increase the detection
limit (the sensitivity range) of these methods, and samples are
frequently reported to thermally degrade before the detection
procedure can be performed. In regard to its limits of detection
for agrochemicals, it was reported 1µg/kg (HPLC)  and
0.1–4.6 µg/L (GC-MS) , for example. These values are not
compensatory when confronted with the disadvantages of these
techniques: costly apparatus, organic solvents, and puriﬁcation
of samples prior to assay, limiting the number of samples that
can be examined.
Recently, experimental studies and theoretical models have
begun to address the challenge of establishing a causal
link between subjective computational models and activity
that can be measured in the laboratory in practice. Atomic
force microscopy (AFM)-based sensors and biosensors (called
nanobiosensors) have been fairly promising in exhibiting out-
standing performance for detecting molecules with high speci-
ﬁcity and selectivity –. Understanding the theoretical
and experimental physicochemical conditions and architec-
tures of the molecules involved in nanosensor construction
requires precise experimental management, such as to identify
the ideal solution concentrations and pH of the medium,
simulate the behavior of a smart surface, maintain system
stability, and conﬁrm that the sensory and target molecules
retain their original characteristics, such as the ability to
inhibit and to be inhibited. Thus, chemically modiﬁed AFM
cantilever/probes can be matched with speciﬁc detection meth-
ods and theoretical results for the entire process to create a
sensor that can detect a wide variety of substances at very low
concentrations. Atomic force spectroscopy (AFS) is generally
used with AFM-based nanosensors , . The AFS tool is
centred using force-distance curves (force curves) that provide
important data on the measurement of recognition events or
either speciﬁc or nonspeciﬁc bonds, which are fundamental
for creating and analyzing nanobiosensors –. Equally
essential and useful in these biosensing systems is a tool
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1468 IEEE SENSORS JOURNAL, VOL. 14, NO. 5, MAY 2014
called functionalization. This technique is used to change
the chemical properties at the surface, such that a standard
molecular design can be used to immobilize target analytes
and sensory molecules –.
In this respect, the aim of this work was to develop an
AFM-enzyme-based nanosensor to detect agrochemical
molecules, in particular, the diclofop molecule. Diclofop
methyl ester (DM)] is a post emergence herbicide used to
control wild oats and annual grasses in wheat and barley 
and belongs to the aryloxyphenoxypropionates (APPs) class
of herbicides. Diclofop related studies are important due to the
large volume used in agriculture worldwide. As an example,
it was published that the total annual usage of diclofop methyl
in China in 2006 was one to ﬁve million kilograms . In
addition, it has been informed that almost 75% of the active
ingredient on diclofop molecule may fall onto soil surfaces
upon application , and because of that, this compound
may become common in the environment . Regarding the
relevance of diclofop detection on environment, our research
group has been reported two important published papers
that addressed theoretically and experimentally data over the
relevance of this kind of analysis. Franca et al. (2011) 
reported through a combination of molecular docking,
molecular dynamics and quantum chemical calculations that
it is possible to establish a bridge between the experimental
calculated adhesion force and the theoretical simulated
results. Oliveira et al. (2013)  reported the modeling of
the proper enzyme orientation on an functionalized AFM
tip. It was showed that after 50 ns of MD simulations the
orientation assumed by the enzyme has inﬂuence on the
exposition of the enzyme binding sites. Also, in another study
from our research group, has been investigating the number
of enzymes that cover the AFM tip by using statistical
calculations of the enzyme dimensions and AFM tip area
(results not reported yet).
For sensor’s purposes, the mode of action of diclofop
chemical group is important: they inhibit the homomeric
plastidic ACCase (acetyl-CoA carboxylase) enzyme , .
This enzyme is present in almost all weed species, and
therefore, the diclofop herbicide is applied to crops in post-
emergency operations. Inhibiting the action of the ACCase
enzyme interrupts the biosynthesis of fatty acids, resulting in
plant death of grass species , .
The design and construction of the nanobiosensor are based
on the biomimicry of the natural process of host-guest interac-
tions, i.e., the nanobiosensor design is based on the diclofop
action mechanism, which consists on the speciﬁc binding of
the herbicide to the enzyme ACCase to inhibit the action
of the enzyme inside a plant cell. Thus, the assembly of
the molecules in the functionalization process is the key to
achieving selectivity and speciﬁcity. The correct arrangement
of molecules results in a unique architecture of the target
and sensory molecules, in such way that their active sites
remain available for reaction and the formation of the speciﬁc
The ACCase enzyme belongs to a multigene family and
is regulated by a complex network of developmental and
environmental signals in response to both internal and external
stimuli . This enzyme is a biotin-dependent molecule
that catalyses the irreversible carboxylation of acetyl-CoA to
malonyl-CoA through the two catalytic activities of biotin
carboxylase and carboxyl transferase. The most important
purpose of ACCase is to enable the malonyl-CoA substrate
to control lipid biosynthesis in the chloroplasts and plas-
tids of non-green tissues , . The diclofop inhibits
ACCase in several stages. First, the herbicide reduces the
electrochemical potentials across the plant cell membranes and
disrupts the transmembrane proton gradient (via electrolyte
leakage), resulting in membrane peroxidation and the subse-
quent uncontrolled release of the cell’s electrolytes inside the
plasma membrane. The plasma membrane and the subcellular
membranes are essential for grouping and supporting cellular
physiological processes; thus, damaging the plasma membrane
negatively affects the biochemical functions and the viability
of the cell . Once inside the plasma, the diclofop molecules
promote ACCase inhibition by causing plant disease symp-
toms, such as a decline in chlorophyll and carbohydrate pro-
duction, ending in chlorosis and subsequent plant death .
This action mechanism was used to design the nanobiosensor
by incorporating the architecture of biomaterials that mimic
complex biological structures. Biomimicry incorporates con-
cepts and strategies from nature into sustainable, useful, and
efﬁcient materials engineering of nanosensing applications by
using AFM and natural binding pathways.
In this context, through the use of force curves, the adhe-
sion force between two surfaces or molecules, or the force
required to break the interaction between them, can be mea-
sured , . Studies have been and are being advanced
by our research group using AFS to succeed the detection
of agrochemical through recognitions events of biomolecules,
such as enzymes and antibodies. In this study, we ﬁrst present a
brief overview of relevant theoretical studies, such as docking
calculations for different regions of the sensing element to
show cluster formation, and the use of molecular dynamics
to measure the interaction energies in a system to guarantee
that the original characteristics of molecules are retained
following their insertion into the system. These theoretical
results were used to adjust and optimize the experimental
parameters in this study to delineate a recognition mecha-
nism based on AFS principles. Finally, this work is the ﬁrst
report of the development of an enzyme-based nanobiosen-
sor and its application to diclofop detection using AFM
probe sensors in conjunction with theoretical and experimental
II. MATERIALS AND METHODS
A. Computational Procedure - Molecular System
The enzymatic model of the ACCase enzyme was taken
from the Protein Data Bank online repository, PDB ID:
1UYR and all missed residues were rebuilt according to the
Franca et al. (2011) considerations . In addition, the
APTES and diclofop molecular structures were built using
Jmol program  and the optimized geometry of both mole-
cules were scanned quantum mechanically using ORCA 2.8.0
BUENO et al.: NANOBIOSENSOR FOR DICLOFOP DETECTION 1469
program . All the undetermined force ﬁeld parameters
to run molecular dynamics (MD) simulations were extracted
from the harmonic ﬁtting curves through quantum mechanical
B. Computational Procedure - Molecular Docking
To carry out docking calculations, the enzyme and herbicide
molecules were used in the AutoDock 4.0 program . The
herbicide (diclofop) was restrained within a tridimensional
grid and was docked on a rigid ACCase enzyme using
Gasteiger-Huckel method  for partial charges considera-
tion. A pre-deﬁned 3D grid was created in several regions
along the enzyme to evaluate the clustered conﬁgurations
with favorable binding energies. Lamarckian genetic algorithm
(LGA) was applied to survey the conformational space of
the unit system to obtain a set containing a 10 LGA docked
C. Computational Procedure - Molecular
The clustered docked positions of the diclofop and
the linker-diclofop set were used to perform MD simula-
tions in order to quantify the interaction energies resulting
from van der Waals and electrostatic considerations. These
MD calculations were performed using NAMD 2.7 soft-
ware , and the results were analyzed using Tool Command
Language (TCL) scripts implanted in the VMD program .
Langevin thermostat and piston  were applied to the
system to minimize, calibrate and equilibrate the energy at
310K, and 1 atm. The NVT ensemble was used, and a cut
off of 1.4 nm was employed for the short-range electrostatic
and van der Waals interactions. The long-range contribu-
tions were modeled using the Particle Mesh Ewald (PME)
method . All force ﬁeld parameters were implemented
on the CHARMM Force Field  protocol. The simulated
systems can be observed on Table I, where the system
ACCase+diclofop, was deﬁned by E-I (enzyme-inhibitor), and
the system ACCase+APTES+diclofop was deﬁned by E-F-I
D. Experimental Procedure - Materials
The acetyl-CoA carboxylase enzyme was purchased from
CUSABIO (Hubei Province, P. R. China) as a part of the
bovine acetyl-CoA carboxylase Synthetase ELISA Kit (which
is used to perform the standard method for a speciﬁc molecular
ligand-receptor). An animal ACCase enzyme was commer-
cially available and was therefore used instead of a vegetal
Fig. 1. Schematic representation of showing the nanosensor design:
(A) cantilever; (B) AFM tip; and (C) structure of APTES-glutaraldehyde
system bound to the Si3N4+acetyl-CoA carboxylase (molecular formula:
C23H38N7O17P3S) substrate (drawing is not to scale and all bonds are not
ACCase enzyme and because of crystallographic evidence for
the similarity and mechanism of action between animals and
plants. It is also known that some aryloxyphenoxypropionate
herbicides and their analogues interfere with the maintenance
of transmembrane electrochemical gradients in animal sys-
tems; similar observations have been reported for plants .
The ACCase enzyme from the kit used in this work was in
its puriﬁed form and it was brought to room temperature
30 min before usage. The process of enzyme reconstitution
is very quick and, at ﬁrst, consists on the centrifugation of
the standard vial at 8,000 rpm for 30s. After this process,
the standard is reconstituted with 1.0 mL of sample diluent
provided by the manufacturer. Finally, it is allowed to the
standard to sit for a minimum of 15 minutes with a gentle
and uniform agitation (to avoid foaming). All the ACCase
standards were prepared fresh for each assay and used in a
four-hour period (lifetime of the enzyme). This reconstitution
solution of the ACCase enzyme from now on will be referred
as ACCase standard. Super pure-grade materials were used in
the functionalization procedure: 3-aminopropyltriethoxysilane
(APTES), triethylamine (TEA) and glutaraldehyde (as a
25% aqueous solution), which were all purchased from
Sigma-Aldrich (St. Louis, MO). The herbicides diclofop,
imazaquin, glyphosate, and metsulfuron were also purchased
from Sigma-Aldrich (St. Louis, MO). AFM cantilevers with
sharpened pyramidal tips (Si3N4 tips) were purchased from
ThermoMicroscopes Microlevers (Sunnyvale, CA).
E. Experimental Procedure - Nanobiosensor Assembly
The fabrication of the nanobiosensors was based on the
work of Etchegaray et al.  and da Silva et al. . The
AFM cantilevers were ﬁrst placed in a sterile Petri dish and
sterilised under UV light for 30 minutes. The ﬁrst step in
the functionalization of the AFM cantilevers and probes was
the silanisation of the surface with APTES [Fig. 1(a)]. This
reaction produced molecular arrays on the AFM cantilever
surface via interactions between the silicon oxide groups on
the cantilever surface and the silanol group in the silane.
The AFM tips were exposed to APTES and triethylamine
vapours for over 30 minutes to bind the APTES to the
1470 IEEE SENSORS JOURNAL, VOL. 14, NO. 5, MAY 2014
cantilever surface. The next step in the treatment was the
introduction of the bifunctional reagent glutaraldehyde [see
Fig. 1(b)], which provided a reactive termination (aldehyde
group) that could react with the amine (−NH2) groups of
the APTES [see Fig. 1(c)] on the AFM tip surface. At this
stage, the glutaraldehyde acted as a spacer to provide ﬂexibility
between the enzyme and the herbicide, thereby enhancing the
sensitivity of the sensor. The AFM cantilevers were immersed
in a 1 × 10−3 molL−1 glutaraldehyde aqueous solution and
allowed to react for 40 minutes. MilliQ water was then
used to wash off any substances that were not covalently
attached to the AFM cantilever surfaces. After the washing
process, the sensing element (i.e., the ACCase enzyme) was
induced to covalently bind to the terminal fragment of the
aforementioned glutaraldehyde molecule by immersing the
AFM cantilevers in a 4 µgL−1 ACCase enzyme solution.
The rapid washing process ensured that the MilliQ water did
not modify the enzyme structure and thereby affect the force
measurements. These samples were incubated for 35 minutes
at room temperature. At the end of the entire procedure, the
cantilevers were used for AFM/AFS measurements.
F. Experimental Procedure - Immobilization of
A similar procedure to that described in Section II.E
was used to assemble and covalently immobilize the target
molecules (the herbicides) on the substrate system (i.e., a fresh
muscovite mica layer). The ﬁrst step in the functionalization
procedure was the formation of a covalent bond between
the hydroxyl groups present on the surface of the mica
muscovite and the silanol group of the APTES molecules.
Next, the amino group of the APTES molecule was chemically
reacted with the carboxyl groups (-COOH) of the diclofop
herbicide molecule. This reaction formed a peptide bond
between the groups; the geometry of the herbicide molecules
was such that they could inhibit the enzyme, thereby
removing the need to introduce a glutaraldehyde spacer into
the target substrate system. After exposure to APTES and
TEA vapours, the substrates were immediately immersed into
the 1 mM agrochemical solutions. The target molecules were
immobilized on the surface and covered the entire area.
G. Experimental Procedure - AFM and AFS
The Atomic Force Microscope ThermoMicroscope Auto-
Probe CP was used in the AFS experiments. In addition,
according to the manufacturer, the curvature radius of the
sharpened pyramidal tip used is <20 nm. The cantilevers were
calibrated using the method described by Sader et al. ,
i.e., the system was subjected to a vibration and the resonant
frequency in air was measured. The force measures were
obtained by approximating the tip retraction rate as 10 µms−1.
The contact mode was used to measure all the force curves;
these analyses were performed in a 20 × 10−3 molL−1
sodium phosphate buffer at pH 7.0. Twenty measurements
were made for each probe, corresponding to approximately
19 statistically valid measurements. All the measurements
were obtained using two cycles at a scan rate of 0.043 nm/s
Fig. 2. Representation of the diclofop molecule and the E-I system in binding
site mode for the ACCase enzyme: (A and B) schematic and crystallographic
representation of the interactions between the diclofop and the ACCase
binding site, showing diclofop (in green) and binding mode (in magenta);
(C) diclofop docked at a binding site mode; and (D) E-I system docked at a
ACCase binding site mode.
and Si3N4 tips suitable for the contact mode with a spring
constant (k) of 0.038± 0.003 N/m (which was calculated by
averaging the values for 10 tips). The TopoMetrix SPMLab 4.0
and OriginPro 8 programs were used to perform the statistical
H. Experimental Procedure - Raman Spectroscopy
For the RAMAN spectroscopy, one drop of the ACCase
standard solution was dispensed on a silicon nitride surface
(tip material), in order to verify the mechanism of absorption
and its ﬁlm assembly on Si3N4. All the functionalization steps
were also carried out on a silicon nitride surface with the same
purpose. RAMAN spectra were obtained using a hand-held
spectrometer (FirstGuardTM/Raman Analyser V2.2.0), with a
1064 nm laser as the excitation source. The output of the laser
power was set at 300 mW, and the integration time was set at
5000 ms. The program OriginPro 8 was used for the statistical
III. RESULTS AND DISCUSSION
A. Molecular Docking and Molecular Dynamics
The herbicide diclofop inhibits ACCase enzyme by blocking
one of the two available active sites . The blockage
occurs via electrostatic and van der Waals interactions of
the diclofop to the following residues: Tyr1738A, Gly1998B,
Val2002B, Gly1734A, and Phe1956B Ala1627A (see Fig. 2).
The Phe1956B residue was removed from Fig. 2(b)–(d) in
order to make the binding site easy to view. The process
of the ACCase inhibition was reproduced according to the
docking methodology [Fig. 2(a)] and the residues were purple
coloured as can be seen in Fig. 2(b)–(d). Fig. 2(b) shows the
best conformation and position of the diclofop (in green), it
was obtained from crystallographic data, and Fig. 2(c) and (d)
show the best positions and conformations obtained from the
molecular docking calculations to the diclofop and E-I system
set. The ﬁnal docked position is almost the same from the
Molecular Dynamics simulations were carried out to deter-
mine the binding energies of the clustered docking posi-
tions. These calculations were performed considering the
ﬂuctuations and mobility of the set in aqueous solution.
BUENO et al.: NANOBIOSENSOR FOR DICLOFOP DETECTION 1471
CALCULATED INTERACTION ENERGIES OF THE
REPRESENTED SYSTEM. THE ENERGIES
WERE SCORED IN kcal/mol
Each system was initially energy-minimised and energy-
equilibrated during 1 ns.
Table II shows the electrostatic and van der Waals (vdW)
contributions to the interaction forces that kept the ligands
attached to the ACCase enzyme. When the inhibitor diclofop
occupied the binding site of the ACCase enzyme, the electro-
static interactions were the primary contribution to the total
energy. In contrast, when this inhibitor bonded to the cross-
linker, the vdW interactions were the primary contribution for
the total energy.
At the end of the inhibition process, one of the oxygen
of the carboxyl group from the diclofop was available in
solution, whereas the remaining oxygen was incorporated
into a hydrophobic pocket formed by the residues in the
reaction. Comparing the binding energies of the E-I system
and the diclofop inhibitor in Table II, it shows that the oxygen
interaction has facilitated the inhibition process in the E-I
system. This result corroborated the experimental results.
Theoretically, the diclofop herbicide could be assembled and
attached to the mica substrate surface and the ACCase enzyme
on the AFM probe without damaging the molecule’s abilities
to inhibit and be inhibited.
Therefore, the binding energy results from the simulations
showed that the diclofop retained its features of being a
speciﬁc inhibitor of the ACCase enzyme even after immobi-
lization on mica substrate. In the following section, we present
experimentally how the functionalization method works and
the results from experimental analysis, where the signal of
the AFM force curves comes from the herbicide-binding site
interaction. In this context, we have determined the proper
orientation of the enzyme on a functionalized AFM tip through
MD simulations . Also, enzyme-enzyme interaction has
been simulated at the presence of the functionalized surface
(results not reported) and we have estimated the number of
active interactions by assuming an energetically favorable
orientation of the enzymes on a surface. Therefore, it is
possible to infer that the number of enzyme binding sites
available by calculating the unbinding energy of one diclofop
molecule through steered molecular dynamics. Depending on
the number of binding sites available, it is possible to integrate
the simulated force curve in order to infer a theoretical value
to a speciﬁc substrate interacting with a target enzyme.
B. Using Raman Spectroscopy for the Analysis and
Monitoring of the Nanobiosensor Assembly
The probe functionalization process was characterized using
Raman Spectroscopy. The Raman spectrum for the ACCase
COMPARISON OF BAND ASSIGNMENTS FOR THE RAMAN SPECTRA OF THE
ACCASE AND AFTER THE FINAL FUNCTIONALIZATION STEP
was characterized by ﬁve peaks, which could be associated
with the ﬁnal Raman spectrum of the nanobiosensor (which
is labelled in light grey in Table III).
Table III shows that functionalization with the ACCase
enzyme produced similar peaks in the 450-460 cm−1 region
in the Raman spectra. These peaks could be assigned
to carbon out-of-plane bending vibrations ; in the
990-1050 cm−1 region, the peaks corresponded to the C-N
vibration / NH2 vibration : the signal increased in
this region in the nanobiosensor spectrum because of
the large number of C-N bonds in the enzyme structure.
The signals in the ﬁnal regions of the spectrum (1367
and 1462 cm−1) could be attributed to COO− stretching
vibrations / CH deformation / C-NH2 stretching and CH3
and / or CH2 deformations , . These similar values
(nanobiosensor and standard) show that the enzyme was
covalently attached to the silicon nitride surface.
C. Agrochemical Detection by Nanobiosensor
Topographical images were used to verify the absorption of
the molecules on mica. The proﬁle studies (data not shown)
provide the dimensions for the surrounding area of the ﬁlm
assembled on mica, from where can be deduce that the height
of this ﬁlms corresponds to 2.4 nm and 2.93 nm. Based
on the number of carbon-carbon bonds and considering the
average distance from 0.1 to 0.15 nm for each connection
at the molecule, it is suggested that a monolayer of APTES
form a self-assembled monolayers  of 0.5 nm of height.
Likewise, one glutaraldehyde molecule might have a height
of approximately 1 nm. The data from the three-dimensional
structure of molecules of diclofop and imazaquin, for example,
is estimated to have a height of ∼1.5 nm and 1.4 nm, respec-
tively. Added to an arrangement with APTES-glutaraldehyde
polymerization of 1.5 nm formed in the functionalization
process, corresponding to the total height of the ﬁlm.
The AFM force curves were used to quantify the inter-
actions between the AFM tip and the sample. The curves
corresponded to two force components: a curve approximation
(not shown) representing a repulsion event and a withdrawal
curve, which could have negative components when there
was a chemical interaction between the AFM tip and the
substrate. In this paper, the contact mode, which produces
1472 IEEE SENSORS JOURNAL, VOL. 14, NO. 5, MAY 2014
Fig. 3. AFS force curves recorded in a PBS solution buffer at pH 7.0 for the
developed agrochemical nanobiosensor: the blue curve represents a speciﬁc
recognition event for a single biomolecular complex formed by the interaction
between ACCase and diclofop (the speciﬁc chemical that inhibits the enzyme
action); in contrast, the other curves represent nonspeciﬁc recognition events
with low, almost null adhesion forces.
force curves as a function of distance, was used to conﬁrm the
system architecture and the speciﬁc recognition between the
enzyme and the herbicide. The force curves were performed
in an aqueous medium, in which the interactions were weak
and were thus in agreement with the results of  and
Noy et al. . Fig. 3 presents the force curves obtained for
the functionalized AFM tip and substrates modiﬁed with four
different herbicides (i.e., 3 nonspeciﬁc herbicides, imazaquin,
metsulfuron, and glyphosate, and 1 speciﬁc herbicide, diclo-
fop). A simple interpretation of the force curves shown in
Fig. 3 from the aforementioned data is that the shape of the
adhesion curve (i.e., the withdrawal curve) can be attributed
to the selectivity and speciﬁcity of the nanobiosensor for the
herbicide under consideration (diclofop). More than one set of
molecules may have participated in the recognition process.
Fig. 3 shows that the magnitude of the diclofop adhesion
forces was 85% higher (6.0±1.0nN) than those of the her-
bicide imazaquin (0.8±0.1nN) and 93% higher than those
of the herbicide metsulfuron (0.3±0.1nN). These differences
in the adhesion forces were expected because these herbi-
cides (imazaquin and metsulfuron) inhibit the action of the
enzyme acetolactate synthase (ALS) – and conse-
quently are non-speciﬁc ligands of (i.e., do not inhibit) the
enzyme ACCase. Likewise, the magnitude of the diclofop
adhesion forces was 90% higher than the herbicide glyphosate
(0.6±0.1 nN). This difference in the adhesion forces was
also expected because the herbicide glyphosate inhibits the
enzyme EPSP (5-enolpyruvyl shikimate-3-phosphate synthase)
– and consequently, is also a non-speciﬁc ligand
of (i.e., does not inhibit) the enzyme ACCase. The total
difference between the values for speciﬁc recognition (for
the diclofop inhibitor) and nonspeciﬁc recognition (for the
Fig. 4. AFS force curves recorded in a PBS solution buffer at pH 7.0 for
the control experiment for substrates without any agrochemical coating and
by saturating the tip with the complementary blocking agent (inset).
non-inhibitors imazaquin, metsulfuron and glyphosate) was
approximately 90%. This result validated the selectivity and
speciﬁcity of the nanobiosensor.
Control experiments, in which the biorecognition process
was inhibited or invalidated, were used to conﬁrm the speci-
ﬁcity of the detected speciﬁc recognition events on statistical
grounds, besides to verify and calibrate the inﬂuence of
coatings by functionalization process. The control experiments
were performed on substrates without any agrochemical coat-
ing (Fig. 4) and by saturating the tip with the complementary
blocking agent (i.e., the anti-ACCase antibody) (Fig. 4 inset).
On average, a signiﬁcant decrease of 98% of the probability
of adhesion in the force curves was observed for these control
experiments. The modulation in the vertical lines showed in
the force curves of the control experiments occurred due the
probe–sample interactions. Interactions of this nature generally
alter the overall spring constant of the system.
Since the ACCase enzyme is inhibited by diclofop in vivo,
and this inhibition depends on a speciﬁc recognition process,
the control experiments showed lower values of adhesion
force compared to the results previous discussed and occurs
essentially by van der Waals interactions or by electrostatic,
hydrophilic, and hydrophobic interactions, among others .
As a ﬁnal point, the histograms in Fig. 5 were used to
determine several speciﬁc adhesion force values. The most
important results were as follows: at a given loading rate,
the most probable adhesion force (which can be used to
standardise sensor operation on a large scale); the difference
between the adhesion forces for each group of herbicides
(speciﬁc and nonspeciﬁc); and the statistical probability of the
adhesion values that appear in the experimental measurements.
All the peaks for the different statistical distributions of the
herbicides, such as the adhesion force of the recognition event,
could be ﬁt with a Gaussian function.
Comparing this system with other biosensors deﬁned in
the literature is challenging because of the many differences
between our system and the systems in other studies, such
as the physical and chemical nature of the molecules, the
surface topography and characteristics, the charges involved
in the recognition event, the different transduction modes,
BUENO et al.: NANOBIOSENSOR FOR DICLOFOP DETECTION 1473
Fig. 5. Adhesion force histograms (n=19) for the occurrences (relative
frequency) of adhesion force values for the sensing systems at the nanoscale:
(A) ACCase/imazaquin; (B) ACCase/metsulfuron; (C) ACCase/glyphosate;
and (D) ACCase/diclofop; the histograms were ﬁtted to Gaussian functions
to identify the most probable adhesion force value to determine a standard
value for speciﬁc and nonspeciﬁc recognition events and signals.
and the functionalization method used for the nanobiosensor
active area. This is the ﬁrst report of the detection of an APP
herbicide by an AFM-tip-based nanobiosensor.
The results from this study provide important guidance
on designing highly sensitive and selective AFM-tip-based
nanosensors using biomimicry of the action mechanism of
herbicides on plants. Surface functionalization was success-
fully used to attach sensory and target molecules to the AFM
tip, which enabled the detection of small concentrations in
real time under near-physiological conditions and without
labelling. In this context, the novelty and advantage of AFM
sensors are, among other facts, the detection limits (as AFM
resolution limits) that this system can reach: the range of
pictogram and, in the best scenario, the detection of a single
molecule with a very narrow and sharpened tip, such as
a carbon nanotube tip and a controlled and standardized
The experimental results showed that the speciﬁc inter-
action was the primary contribution to the greater adhesion
force, especially for the ACCase-diclofop interaction, because
diclofop is a speciﬁc inhibitor for ACCase. The difference
between the speciﬁc and nonspeciﬁc recognition values was
90% on average. The theoretical results also provided a
direct interface with the experimental research, showing that
nanosensor molecules could be assembled and attached to the
mica substrate surface and the AFM probe without damaging
the molecules’ mode of action. Studies are currently being
carried out to determine the theoretical and experimental force
of the interactions between the herbicides and their inhibitors
and we have already begun investigating the response behavior
of the sensor for samples in which several agrochemical
groups interact in a solution. More work is needed to improve
the AFS signals, and because of that, our research group is
concentrating efforts to ﬁgure how the immobilization process
works, in order to estimate, as accurate as possible, the
forces involved on herbicide-enzyme interactions as well as
the improvements to the nanoscale detection device to monitor
residues in the environment.
The authors would like to thank Mr. L. Bonugli for
his signiﬁcant contributions to the AFM measurements,
Mr. J. R. Castro for technical assistance and the Institute of
Physics Gleb Wataghin for the experimental assays support.
Also, the authors acknowledge FAPESP (Proc. 2007/05089-
9; Proc. 2013/09746-5; Proc. 2009/09120-3), CAPES, CNPq,
FAPEMIG (CEX - APQ-02176-11) and nBioNet for their
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Carolina Castro Bueno received the Degree in
environmental engineering from Pontiﬁcia Univer-
sidade Catolica de Campinas in 2009, and the
M.S. degree in materials science - nanotechnol-
ogy and nanoscience in the area of microscopy
and atomic force spectroscopy with emphasis on
nanoscopy/nanobiosensors for monitoring environ-
mental quality in agriculture in 2013. She has
worked in the areas of nanotechnology, nanoscience
and atomic force microscopy, with emphasis on stud-
ies that involve the construction of smart surfaces
for creating nanobiosensors. Currently, she is pursuing the Doctoral degree in
environmental sciences working with Biochar development and engineering
at Universidade Estadual Paulista.
Adriano Moraes Amarante was born in Soro-
caba, Brazil. He received the Degree in bio-
physics technology from the College of Technology
São Paulo, FATEC, Sorocaba, in 2006, the B.Sc.
degree in physics from the University of São Carlos
in 2013, and the M.Sc. degree in materials science
and engineering from the University of São Carlos
in 2013. Since 2007, he has been developing several
different types of computational simulation to inter-
pret biological sensors-based to be used as probes
for agriculture and diseases detection. His research
activity includes molecular dynamics simulation and experimental-theoretical
interpretation. Since 2009, he has been a Researcher with the Federal
University of São Carlos, Brazil, and a member of the Nanoneurobiophysics
Guedmiller S. Oliveira received the B.S. and M.S.
degrees in physical chemistry from the Federal
University of Uberlândia and the Ph.D. degree in
physical chemistry from the Federal University of
São Carlos, Brazil, in 2006, 2009, and 2013, respec-
tively. Since 2007, he has been working with com-
puter simulation of the matter, providing an atomistic
point of view of the experimental procedures. In
2012, he was visiting the University of Houston,
Biology and Biochemistry Laboratory, supervised by
Prof. J. M. Briggs, where he developed a theoretical
model to interpret the immobilization of biological systems on functionalized
surfaces for nanosensors applications. His knowledge base incorporates quan-
tum mechanics theory, molecular dynamics simulation, and it combines results
from experimental and theoretical analysis through statistical thermodynamics
to better understand macromolecular phenomena. Currently, he is a Post-
Doctoral Researcher at the Federal University of São Carlos, Sorocaba.
Daiana Kotra Deda received the B.Sc. degree in
chemistry from the State University of Midwestern,
Brazil, in 2006, and the Ph.D. degree in inorganic
chemistry from the University of São Paulo, Brazil,
in 2011, for research on the development of nanoen-
capsulated photosensitizers for cancer treatment by
photodynamic therapy. Currently, she is a post-
doctoral with the Chemistry Institute, University
of São Paulo. Her main research interest is the
development and characterization of nanomaterials
and the study of their interaction with biological
systems, in vitro and in vivo toxic effects and their application as alternative
methods for treatment and diagnosis of diseases.
Omar Teschke was born in Santo Angelo, Brazil,
in 1944. He received the B.S. degree in electri-
cal engineering from Universidade Federal do Rio
Grande Sul, Porto Alegre, in 1967, the M.S. degree
in electrical engineering from Universidade Catolica
do Rio de Janeiro in 1969, under the supervision
of Prof. S. M. Rezende, and the Ph.D. degree in
electrical engineering from the University of Cali-
fornia Berkeley in 1975, under the supervision of
J. R. Whinnery. After teaching at the Universidade
Catolica do Rio de Janeiro from 1969 to 1971, he
was a Visiting Staff Member with Bell Laboratories Holmdel, NJ, USA, from
1975 to 1977. He joined the Departamento de Fisica Aplicada, Instituto de
Fisica, UNICAMP, in 1977. Currently, he is a Professor and his research
interest are interfacial processes including self-organization and self-assembly
and experimental techniques for probing the liquid state, specially interfacial
process in living species.
Eduardo de Faria Franca is a Professor of the-
oretical chemistry with the Federal University of
Uberlândia. He received the Degree from the Federal
University of Uberlândia in 2003, and the Ph.D.
degree in chemical physics from the Federal Uni-
versity of São Carlos in 2009, where he worked
with Prof. L. C. G. Freitas. As a trainee in the
Paciﬁc Northwest National Laboratory, he was with
Prof. R. D. Lins and developed a new force-ﬁeld for
the chitin and chitosan biopolymers and elucidated
new insights about these natural polymers using
molecular dynamics simulation. In 2009, he was a post-doctoral with the
polymer group: Prof. Bernhard Gross, University of São Paulo, to help
on the development of a new nanobiossensor using an AFM microscope.
His theoretical work assisted the construction of a sensible and selective
biosensor capable to detect herbicides in nanomolar concentration. He joined
the Federal University of Uberlândia as a Professor of chemistry in 2010. He
has been the author or co-author of many publications in theoretical chemistry
and crystallography since 2002. His actual projects include the development
biosensors, antitumor drugs, biofuels, and the use of biopolymer chitosan for
waste water treatment and production of artiﬁcial tissues.
Fábio L. Leite was born in Itanhaem, Brazil.
He received the B.Sc. degree in physics from
São Paulo State University, UNESP, Rio Claro, in
2000, and the M.Sc. and Ph.D. degrees in mate-
rials science and engineering from the University
of São Paulo, São Carlos, in 2002 and 2006,
respectively. Between 2007 and 2008, he was a
Post-Doctoral Researcher with the Alan Graham
MacDiarmid Institute of Innovation and Business,
Embrapa Agricultural Instrumentation (Embrapa)
with Dr. O. N. de Oliveira Jr., Dr. L. H. C. Mattoso
(Embrapa) and Alan Graham MacDiarmid, University of Pennsylvania Nobel
Prize in chemistry in 2000. His efforts in the MacDiarmid Institute focused
on conducting polymers, nanosensors and atomic force microscopy with
environmental applications. Since 2009, he has been an Assistant Prof. and
Researcher with the Federal University of São Carlos, Sorocaba, and the
Head of the Nanoneurobiophysics Research Group. He is the author of
over 40 published papers, two books, ten book chapters, and two patents.
His research interests are related to the development of nanobiosensors using
AFM and computational nanotechnological for application in studies of a
variety neurodegenerative and autoimmune diseases.