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Article
Journal of
Nanoscience and Nanotechnology
Vol. 14, 6678–6684, 2014
www.aspbs.com/jnn
Nanobiosensors Exploiting Specific Interactions Between
an Enzyme and Herbicides in Atomic Force Spectroscopy
Aline C. N. da Silva1
, Daiana K. Deda1 ∗
, Carolina C. Bueno1
, Ariana S. Moraes1
, Alessandra L. Da Roz1
,
Fabio M. Yamaji1
, Rogilene A. Prado2
, Vadim Viviani2
, Osvaldo N. Oliveira, Jr3
, and Fábio L. Leite1 ∗
1
Department of Physics, Chemistry and Mathematics, Nanoneurobiophysics Research Group, Federal University of
São Carlos, P.O. Box 3031, 18052-780, Sorocaba, SP, Brazil
2
Laboratory of Biochemistry and Biotechnology of Bioluminescence, Department of Physics, Chemistry and Mathematics,
Federal University of São Carlos, P.O. Box 3031, 18052-780, Sorocaba, SP, Brazil
3
São Carlos Institute of Physics, University of São Paulo, São Carlos, P.O.Box 369, 13560-970, SP, Brazil
The development of sensitive methodologies for detecting agrochemicals has become important
in recent years due to the increasingly indiscriminate use of these substances. In this context,
nanosensors based on atomic force microscopy (AFM) tips are useful because they provide higher
sensitivity with operation at the nanometer scale. In this paper we exploit specific interactions
between AFM tips functionalized with the enzyme acetolactate synthase (ALS) to detect the ALS-
inhibitor herbicides metsulfuron-methyl and imazaquin. Using atomic force spectroscopy (AFS) we
could measure the adhesion force between tip and substrate, which was considerably higher when
the ALS-functionalized tip (nanobiosensor) was employed. The increase was approximately 250%
and 160% for metsulfuron-methyl and imazaquin, respectively, in comparison to unfunctionalized
probes. We estimated the specific enzyme-herbicide force by assuming that the measured force
comprises an adhesion force according to the Johnson–Kendall–Roberts (JKR) model, the capillary
force and the specific force. We show that the specific, biorecognition force plays a crucial role in the
higher sensitivity of the nanobiosensor, thus opening the way for the design of similarly engineered
tips for detecting herbicides and other analytes.
Keywords: Enzymes, Herbicides, Nanobiosensors, Atomic Force Microscopy, Atomic Force
Spectroscopy, Chemical Force Microscopy.
1. INTRODUCTION
The growing global demand for food has led to the use of
pesticides in ever increasing quantities,1 2
of which only an
estimated 0.1% reach their targeted pests.3 4
The remaining
99.9% translocate to other environmental areas, causing
direct damage to flora, fauna and human health due to their
highly cytotoxic and genotoxic effects.3–8
Detecting agro-
chemicals with greater efficiency, speed, and sensitivity9–14
than traditional chromatographic methods15–17
has there-
fore become important. Sensors and biosensors based on
chemically modified cantilevers may, in this context, be a
promising alternative to detection18–24
due to their excel-
lent performance in detecting analytes,25–29
including agro-
chemicals. Specific interactions, such as the “lock and
key” or “host-guest” mechanisms, for they are selective
∗
Authors to whom correspondence should be addressed.
with the binding of analytes to sensing molecules.30 31
These devices utilize a combination of biomolecule recep-
tors and a physicochemical detector, which together enable
the recognition of a specific analyte in a medium.32 33
An
essential requirement is then a well-controlled immobiliza-
tion of functional biomolecules on surfaces or nanoma-
terials, which has indeed been used in clinical diagnosis,
investigation of biomolecular interactions, environmental
monitoring, and quality control of food.20 30 34–41
Sensors based on specific interactions may employ
atomic force microscopy (AFM) as a force appara-
tus,19 42–46
where cantilevers are functionalized with sen-
sitive materials such as polymers, enzymes or antibodies.
With the interaction with target molecules that selectively
adsorb or bind by chemical affinity onto the cantilever,
selective, sensitive sensors can be produced.20 47
This is
the case of a nanobiosensor designed with microcantilevers
6678 J. Nanosci. Nanotechnol. 2014, Vol. 14, No. 9 1533-4880/2014/14/6678/007 doi:10.1166/jnn.2014.9360

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Silva et al. Nanobiosensors Exploiting Specific Interactions Between an Enzyme and Herbicides in Atomic Force Spectroscopy
functionalized with the enzyme acetolactate synthase
(ALS) for detecting metsulfuron-methyl.48
Even though
there are sensors that use the technique of covalent func-
tionalization of silicon surfaces,49
as well as the enzyme
immobilization for enhancing the recognition (as sense
element),50
the technology described in this work is the
first report about using the synergetic effect of the covalent
character of the Si C bond combined to mimetic mech-
anism of enzymatic inhibition by herbicides. This unique
structure provided by AFS is a favorable microenviron-
ment to maintain the bioactivity of an enzyme, which led
to a rapid recognition response through force curves. In
this paper, we extend the previous work to detect another
herbicide, imazaquin, and estimate the specific, biorecog-
nition force between the enzyme and the herbicide. This
is performed with a series of atomic force spectroscopy
(AFS) measurements, whose data are evaluated using the-
oretical models to calculate the adhesion and the capillary
forces. We shall show that the specific interaction is essen-
tial for the high sensitivity of the nanobiosensors.
2. METHODOLOGY
2.1. Expression of Recombinant ALS
Recombinant ALS was kindly provided by Dr. Tsutomu
Shimizu from Life Science Research Institute, Shizuwoka,
Japan. The cDNA of the ALS gene (from Oryza sativa)
was incorporated into Eco RI sites of the pGEX 2T vector
and used to transform the E. coli BL21-DE3 strain. The
colonies were grown in 500–1000 mL of LB medium con-
taining ampicillin at a temperature of 37 C until reaching
an OD600 of 0.4, then induced at 22 C for 3–4 h. The
cell suspension was centrifuged at 2,500 g for 15 min and
resuspended in 1X PBS buffer containing complete pro-
tease inhibitors (Roche), then freeze-thawed three times in
dry ice and centrifuged at 15,000 g for 15 min at 4 C.
The supernatant (crude extract) was used for cantilever
functionalization.
2.2. Chemical Functionalization of
Tips and Substrates
The nanobiosensor was fabricated according to the method
reported in Ref. [48]. The functionalization procedure
for the Si3N4 tips, cantilevers and substrates (muscovite
mica) was adapted from the method described by Wang
and collaborators.51
The tips and substrates were cleaned
by irradiation in a UV chamber (240 nm; Procleaner,
UV.PC.220, BIOForce Nanosciences, Ames, IA, USA).52
The functionalization was initiated by gaseous evapora-
tion of 3-aminopropyl triethoxysilane (APTES) in the
presence of triethylamine (both as commercial solutions),
followed by the addition of a small aliquot of a glutaralde-
hyde solution (1×10−3
M). Subsequently, 100 L of the
ALS enzyme extract (0.200 mg/mL) were added to the
probe tips, and 200 L of the ALS-inhibiting herbicides
metsulfuron-methyl and imazaquin were added to the sub-
strates (1×10−3
M). All reagents used, with the exception
of the ALS (Section 2.2), were purchased from Sigma.
2.3. Atomic Force Spectroscopy (AFS)
Force spectroscopy experiments were performed at 25 C
and a relative humidity of approximately 35%. The force
curves were obtained using an Atomic Force Micro-
scope Multimode-VS System with the PicoForce pack-
age (dedicated to force spectroscopy). The Si3N4 AFM
tips (V-shaped, model NP-10 by Veeco) employed in the
measurement of force curves possessed a nominal spring
constant of 0.12 N/m. Considerable variations can occur
between the nominal and real value of the spring constant;
each AFM tip was therefore calibrated using the thermal
noise method.53
The detection of herbicides was confirmed by examining
the force curves obtained using two types of tips: (i) tips
functionalized with the ALS enzyme and (ii) unfunctional-
ized tips. To evaluate the efficiency of the nanobiosensor,
adhesion force values were obtained on various substrates,
at different points on each substrate and using different
tips. The adhesion force values reported represent the aver-
age of 30 force curves obtained at the same point on the
substrate.
2.4. Contact Angle and Surface Energy
Contact angle analysis was performed to determine the
surface energies for calculating the work of adhesion
and the theoretical adhesion force between the AFM tips
and the substrates contaminated with herbicides. Con-
tact angle measurements were performed at 25 C using
CAM200 equipment by KSV. Due to the small size of
the tip, the system was reproduced on the macroscopic
scale using a functionalized silicon plate. Addition-
ally, measurements were performed on mica/metsulfuron-
methyl and mica/imazaquin substrates. The measurements
employed water, formamide and diiodomethane as liquids,
whose surface tensions are 72.2, 58.3 and 50.8 mJ/m2
,
respectively.
Surface energies were calculated using the Owens–
Wendt theory54
described by Eq. (1):
L 1+cos
2 d
L
= d
S +
p
L
d
L
p
S (1)
where p
S and d
S are the polar and dispersive surface
energies of the solid, respectively, and p
L and d
L are the
polar and dispersive surface energies of the liquid, respec-
tively. L represents the total surface energy ( p
L + d
L . The
surface energy ( p
L, d
L employed, in nN/m, was (51.0,
21.8), (18.0, 39.0) and (0, 50.8) for water, formamide
and diiodomethane, respectively.55
The data were plotted
for using Eq. (1), in which the linear coefficient is d
S
and the angular coefficient is p
S , from which the surface
energy of the solid material can be determined.56
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Nanobiosensors Exploiting Specific Interactions Between an Enzyme and Herbicides in Atomic Force Spectroscopy Silva et al.
2.5. Determination of the Specific Force
The specific force resulting from the interaction between
the ALS enzyme and the herbicides was determined from
the difference between the theoretical and experimental
adhesion force. The theoretical adhesion force was deter-
mined from the sum of the capillary force (determined
from contact angle measurements) and theoretical adhe-
sion force values determined using the Johnson–Kendall–
Roberts (JKR) model.57
All calculations performed, values
and equations employed are described in Section 3.2.3.
3. RESULTS AND DISCUSSION
3.1. Nanobiosensor Characterization
A representative surface topography of a silicon nitride
substrate (simulating the AFM tips) outlining the function-
alization steps is depicted in Figures 1(a)–(d). The depo-
sition of APTES (Fig. 1(b)) did not significantly affect
the surface roughness compared to the unmodified surface
(Fig. 1(a)). In both cases, a roughness of approximately
0.4 nm for a surface area of 400 m2
was observed. Fol-
lowing glutaraldehyde modification (Fig. 1(c)), the rough-
ness of the substrate increased to 1.9 nm. Enzyme coating
(Fig. 1(d)) resulted in uniform surface coverage and an
increase in roughness to 5.4 nm, indicating that the func-
tionalization was successful even after the washing steps.
3.2. Application of the Nanobiosensor to
Herbicide Detection
The nanobiosensor design and construction were based
on the biomimicry of the natural process of host-guest
Figure 1. Surface topography of the silicon nitride surface (a) uncoated
and coated with (b) APTES, (c) APTES followed by coating with glu-
taraldehyde and (d) following functionalization with the ALS enzyme.
interactions; i.e., the nanobiosensor harnessed the specific
binding interactions of the herbicides metsulfuron-methyl
and imazaquin with the enzyme ALS. As described by
Chipman,58
these agrochemicals bind to the ALS enzyme
to inhibit its action inside the plant cell.
Atomic force spectroscopy (AFS) was used to quantify
the interactions between the AFM tip and the herbicide
samples by measuring the corresponding adhesion force.
In AFS, force versus distance curves (force curves) are
used to identify recognition events that can be used for
detection,48 59 60
and to obtain insights into ways to modify
the tip with immobilization of target analytes and sens-
ing molecules.61–63
Figure 2 displays typical force curves
for the interaction between unfunctionalized and ALS-
functionalized tips and metsulfuron-methyl- or imazaquin-
contaminated substrates. Increases in the adhesion force of
approximately 250% and 160% were observed when the
nanobiosensor interacted with the herbicides metsulfuron-
methyl and imazaquin, respectively, compared to the use
of an unfunctionalized tip (16 nN). In the black curve
depicted in Figure 2, only small adhesive forces occasion-
ally appear, indicating the absence of any strong interac-
tion between the unfunctionalized tip and the herbicide.
The black curve was obtained using a substrate modified
with metsulfuron-methyl, but similar values were obtained
for imazaquin (Table I). In contrast, when the nanobiosen-
sor was employed, adhesion forces of ca. 42 and 56 nN
were observed for imazaquin (blue line) and metsulfuron-
methyl (red line), respectively. The considerable differ-
ences in adhesion force were due to the specific binding
between the ALS enzyme and the herbicides, analogously
to previous studies in which cantilevers functionalized
with specific antibodies were used to detect herbicides.52–55
Control experiments, in which the biorecognition process
was inhibited, were used to confirm the specificity of
the detected specific recognition events. This control was
achieved by saturating the tip with the complementary
blocking agent (i.e., anti-ALS antibody). The adhesion
Figure 2. Force curves for an unfunctionalized tip interacting with
metsulfuron-methyl (black line) and with a tip functionalized with the
ALS enzyme to detect the herbicides metsulfuron-methyl (red line) and
imazaquin (blue line).
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Silva et al. Nanobiosensors Exploiting Specific Interactions Between an Enzyme and Herbicides in Atomic Force Spectroscopy
Table I. Average values of the adhesion force and the coefficients of
variation using unfunctionalized and functionalized AFM tips on sub-
strates contaminated with imazaquin and metsulfuron-methyl.48
The val-
ues were obtained from force curves collected at either three different
spots on the same substrate (spots 1 to 3), at a single spot on three dif-
ferent substrates (Substrates 1 to 3), or using three different tips (tips 1
to 3).
Coefficients of variation (%)
Unfunctionalized Functionalized
Herbicide Variant tip tip
Imazaquin Spot 1 1.4 2.0
Spot 2 1.3 2.2
Spot 3 1.3 1.8
¯F
spot
adh (nN) 16 0±1 0 42 0±4 0
Substrate 1 1.3 1.3
Substrate 2 4.2 2.2
Substrate 3 1.4 2.7
¯F subs
adh (nN) 18 0±2 0 40 0±4 0
Tip 1 0.9 1.3
Tip 2 1.4 4.8
Tip 3 1.2 3.3
¯F
Tip
adh (nN) 13 9±0 8 44 0±5 0
Total average ¯F
Exp
adh (nN) 15 9±2 4 42 0±7 5
Metsulfuron Spot 1 1.1 1.4
methyl Spot 2 3.7 3.5
Spot 3 0.7 1.5
¯F
Spot
adh (nN) 16 0±2 0 57 0±4 0
Substrate 1 0.8 1.5
Substrate 2 0.9 0.9
Substrate 3 1.0 0.7
¯F Subs
adh (nN) 13 0±2 0 58 0±7 0
Tip 1 1.1 0.7
Tip 2 1.4 1.9
Tip 3 4.5 1.7
¯F
Tip
adh (nN) 14 6±0 5 66 0±3 0
Total average ¯F
Exp
adh (nN) 14 5±2 9 60 3±8 6
values were insignificant and below the value of 16 nN
for unfunctionalizated tips (see Fig. 2, in green). The pur-
pose of such experiments was to exclude any possibility
of incorrect functionalization of the tip and, consequently,
a possible interaction between herbicide with the amino or
aldehyde groups.
The distributions of adhesion forces in the analysis of
2000 force curves, collected from three different spots on a
substrate modified with imazaquin or metsulfuron-methyl
and using either unfunctionalized tips or the nanobiosen-
sor, are displayed in Figure 3. Small deviations were
observed in the measurements collected from the three
points on the substrate as can be observed from the aver-
age values of adhesion force presented in black, blue
and pink. Because the standard deviation is a measure
of the dispersion relative to an average value, and since
the measured points in our study exhibited different arith-
metic averages, the standard deviation is not a suit-
able means of comparison. Therefore, the coefficient of
variation (Eq. (2))64
was used, expressed as a percentage
and calculated for each evaluated condition on both the
imazaquin and metsulfuron-methyl substrates.
Coefficient of variation =
Standard deviation
Arithmetic mean
×100 (2)
The variability in adhesion force in Table I on the sub-
strate modified with imazaquin was ≤1.4% and ≤2.2%
for unfunctionalized tips and tips functionalized with the
ALS enzyme, respectively. For the substrate containing
the herbicide metsulfuron-methyl, variations in the adhe-
sion force were ≤3.7% and ≤3.5% for unfunctional-
ized tips and tips functionalized with the ALS enzyme,
respectively. Minor changes were also observed between
the force curves collected from three different substrates
(using the same tip) or using three different tips on the
same substrate. The average values of adhesion force for
each variable (spot, substrate and tip) and the correspond-
ing coefficients of variation are summarized in Table I. The
small changes in adhesion force demonstrate the repro-
ducibility of the tip and substrate functionalization method
and the reliability of the results. We could therefore deter-
mine the experimental adhesion force ( ¯F
Exp
adh ) from the
average among the adhesion force obtained on different
spots ( ¯F
spot
adh ), different substrates ( ¯F Subs
adh ) and using differ-
ent tips ( ¯F
Tip
adh ). These values are also presented in Table I
and were employed to determine the specific force, as
described in Section 3.2.3.
3.3. Contact Angle Measurements and
Surface Energy Calculations
Table II displays the measured contact angles and the polar
( p), dispersive ( d) and total surface energies of the solid
( s), which were calculated as specified in Section 2.4.
These values are necessary to calculate the work of adhe-
sion and the theoretical adhesion force between the AFM
tips and the substrates, as described in Section 3.2.3.
3.4. Calculation of the Specific Force
One expects the efficiency of the nanobiosensor to derive
primarily from the specific interaction between the ALS
enzyme and the herbicides. Therefore this interaction
should have an important contribution to the adhesion
force obtained experimentally. But, the adhesion force
determined from the force curves has other contribut-
ing components, including the capillary force that occurs
under ambient conditions owing to the formation of a thin
film of water on the surface under analysis. This attractive
capillary force (Fcap) is described by Eq. (3) in Table III,
where 1 and 2 are the contact angles between the water
and the plane and the water and the sphere, respectively.65
The increased capillary force for the functionalized tip in
Table III is due to the hydrophilicity of the ALS enzyme,
as indicated by the contact angle and the higher surface
energy compared to the unfunctionalized tip (Table II).
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Nanobiosensors Exploiting Specific Interactions Between an Enzyme and Herbicides in Atomic Force Spectroscopy Silva et al.
Figure 3. The histograms (fitted to Gaussian functions, continuous lines) associated with the force curves collected on three different spots of a
same substrate using unfunctionalized (a) and (b) and functionalized (c) and (d) tips and obtained from the herbicides metsulfuron-methyl (above) and
imazaquin (below). The values presented in black, blue and pink represent the average adhesion force determined from 2000 force curves obtained in
each spot analyzed. The results for metsulfuron-methyl were reproduced from Ref. [48], and are included here as a comparison with imazaquin.
The theoretical adhesion force was calculated using the
Johnson–Kendall–Roberts (JKR) model (F JKR
adh ), which is
adapted to treat adhesive interactions. JKR theory includes
an adhesive force inside the contact area and is considered
more suitable for soft samples with a low elastic modulus
and a large tip radius, as used in AFM experiments.66–68
JKR theory is described by Eq. (5), where Rt is the radius
of the tip and Wikj is the work of adhesion between two
surfaces i and j in a medium k. Radii of curvature of
20 nm and 30 nm were used for the unfunctionalized
and functionalized tips, respectively (values determined by
SEM images). The work of adhesion (Wikj) was calculated
Table II. Measurements of the contact angle and surface energies (total,
s; polar, p; dispersive, d) obtained for the tips and the substrate.
Contact angle Surface energy (mN/m)
Surface Water Formamide Diiodomethane p d s
Imazaquin 68.15 30.43 47.37 14.80 29.70 44.60
Metsulfuron- 58.55 30.78 39.14 17.20 30.80 47.90
methyl
Silicon 51.11 39.84 41.31 24.10 25.20 49.20
ALS 25.26 22.73 35.18 40.70 23.40 64.10
using Eq. (4) from the ratio between the polar ( p
) and
dispersive ( d
) surface energies of the material under study
(determined by measurements of the contact angle), in
which the tip is represented by (i) and the substrate by (j).
The results in Table III point to a higher work of adhesion
between the nanobiosensor and the herbicide-contaminated
substrates than for the unfunctionalized tip. The theoreti-
cal value for the total adhesion force (F Total
adh ) between the
AFM tips and the substrates was obtained with Eq. (6),
which represents the sum of Fcap and F JKR
adh .65
We take the difference between the experimentally
measured force of adhesion and the predicted theoret-
ical value as being the contribution from the specific
interaction between the nanobiosensor and the herbicides
metsulfuron-methyl and imazaquin (Eq. (7)). More specif-
ically, we assume that the total force comprises the adhe-
sion force, predicted theoretically with the JKR theory, the
capillary force and the specific interaction, where the latter
is not accounted for in the total theoretical force. Figure 4
displays the values of F JKR
adh , ¯F
Exp
adh and Fspec. The specific
force plays a critical role in the interaction between the
nanobiosensor and the metsulfuron-methyl-contaminated
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Silva et al. Nanobiosensors Exploiting Specific Interactions Between an Enzyme and Herbicides in Atomic Force Spectroscopy
Table III. Values for the work of adhesion (Wij), adhesion force (F JKR
adh ), capillary force (Fcap), total theoretical adhesion force (F Total
adh ), experimental
adhesion force ( ¯F
Exp
adh ) and specific force (Fspec) as calculated using JKR theory. MET = metsulfuron-methyl; IMA = imazaquin.
Nanobiosensor Unfunctionalized tip
Parameter MET IMA MET IMA Equations
Fcap (nN) 19.6 18.5 10.5 9.8 Fcap = 2 R L cos 1 +cos 2 (3)
Wij (mN/m) 101.6 95.8 95.6 70.5 Wij =
4 d
i
d
j
d
i + d
j
+
4 p
i
p
j
p
i + p
j
(4)
F JKR
adh (nN) 14.4 13.5 9.0 6.6 F JKR
adh =
3
2
RtWikj (5)
F Total
adh (nN) 34.0 32.0 19.5 16.5 F Total
adh = Fcap +F JKR
adh (6)
F
Exp
adh (nN) 60 3±8 6 42 0±7 5 14 5±2 9 15 9±2 4 See Table I
Fspec (nN) 26 3±8 6 10 0±7 5 −5 0±2 9 −0 6±2 4 Fspec = ¯F
Exp
adh −F Total
adh (7)
Figure 4. Theoretical (F JKR
adh ) and experimental adhesion force (F
Exp
adh )
and the specific force (Fspec) for the interaction between the nanobiosensor
and the herbicides metsulfuron-methyl and imazaquin.
substrate, contributing with 44% and 24% of the exper-
imentally obtained value for metsulfuron-methyl and
imazaquin, respectively. This confirms that the sensitiv-
ity of the nanobiosensor is primarily due to the presence
of specific interactions between ALS and the herbicides.
In contrast, the contribution of the specific force to the
tip-herbicide interaction for the unfunctionalized tip was
negligible, as indicated by the negative values in Table III,
ascribed to the absence of specific interactions. The higher
contribution for metsulfuron-methyl is explained by the
stronger inhibition of the ALS enzyme by herbicides
belonging to the sulfonylurea group.69
Metsulfuron-methyl and imazaquin are only two com-
pounds among a larger group of herbicides considered to
be ALS enzyme inhibitors. Our future work aims to extend
these studies to other herbicides from this group (and other
groups using other nanobiosensors) and capitalize on the
resulting variations in specific force that can be expected.
4. CONCLUSIONS
The use of cantilevers chemically modified with the
enzyme ALS proved promising for detecting the herbicides
metsulfuron-methyl and imazaquin. The adhesion force
measured with the nanobiosensor was considerably
higher than those obtained with unfunctionalized tips,
with increases of approximately 250% and 160% for
metsulfuron-methyl and imazaquin, respectively. The cAl-
culation of the total theoretical adhesion force corroborated
our experimental results and allowed the calculation of the
specific force for the interaction between the ALS enzyme
and the herbicides. The results indicated that the specific
interaction was the primary source of the greater adhe-
sion force when using the nanobiosensor, especially for the
interaction of ALS with metsulfuron-methyl. This illus-
trates the importance of surface chemical modifications
in promoting molecular recognition and, consequently, the
specific interactions which enable detecting substances
with high selectivity.
Acknowledgments: The authors acknowledge
FAPESP (Proc. 2007/05089-9, Proc. 2010/00463-2,
Proc. 2010/04599-6 and 2009/09120-3), CAPES (Proc.
23038006985201116 and Proc. 02880/09-1), CNPq (Proc.
483303/2011-9) and nBioNet network for the financial
support.
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Received: 23 May 2013. Accepted: 7 July 2013.
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